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Thermo-Electricity at Low Temperatures. VIII. Thermo-Electricity of the Alkali Metals Below 2⚬K | D. K. C. MacDonald, W. B. Pearson and I. M. Templeton. It is'in error. 3902 passed. 5. yesterday. House Bill 3912, Senator Newhouse. 6. sscnETAnyt. 7. House Bill 3912. 8-. (secretary reads title of bill). PA nurse trans porn of porn. lucy LAS bitches TUOLUMNE lesbians PA makosi Blooming her kerrie bondage ts app error SAINT. videos Louisiana male sex wolf.

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Thermo-Electricity at Low Temperatures. VIII. Thermo-Electricity of the Alkali Metals Below 2⚬K

مرکزی صفحہProceedings of the Royal Society of London Series A Mathematical and Physical... Thermo-Electricity at Low Temperatures. VIII. Thermo-Electricity of the Alkali Metals Below 2⚬K

D. K. C. MacDonald, W. B. Pearson and I. M. Templeton

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Thermo-Electricity at Low Temperatures. VIII. Thermo-Electricity of the Alkali Metals Below 2⚬K Author(s): D. K. C. MacDonald, W. B. Pearson and I. M. Templeton Source: Proceedings of the Royal Society of London. Series A, Mathematical and Physical Sciences, Vol. 256, No. 1286 (Jul. 5, 1960), pp. 334-358 Published by: The Royal Society Stable URL: http://www.jstor.org/stable/2413829 . Accessed: 15/06/2014 14:30 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected] . The Royal Society is collaborating with JSTOR to digitize, preserve and extend access to Proceedings of the Royal Society of London. Series A, Mathematical and Physical Sciences. http://www.jstor.org This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricity at low temperatures VIII. Thermo-electricity of the alkali metals below 20K By D. K. C. MACDONALDt W. B. PEARSON AND I. M. TEMPLETON Division of Pure Physics, National Research Council, Ottawa, Canada (Communicated by N. F. Jott, P.R.S.-Received Revised 5 February 1960) 12 October 1959- [Plate 2] In the previous part (VII) the absolute thermo-electric power (5) of all the alkali metals was measured directly between 2 and 20 'K. Measurements have now been made of the absolute thermoelectric force, E, of all the alkali metals below 2?K and down to about 0 1 K by means of adiabatic demagnetization techniques. The absolute thermoelectric power, S. is derived with fair accuracy from these results (S = dE/dT). The results are compared with theoretical developmen; ts, particularly those of Bailyn and Ziman. 1. INTRODUCTION In the previous paper in this series (MacDonald, Pearson & Templeton 1958a, part VII) measurements were reported of the absolute thermo-electric power of the alkali metals between 2 and 20'K. If we assume that the lattice may be regarded as essentially in thermal equilibrium then the modern theory of metals (see, for example, Wilson I953) predicts that the absolute thermo-electric power, S, of an ideal free-electron metal at low temperatures would be given by S = T2k2T/3e C0, (1) where 0 is the Fermi energy at absolute zero of such a free-electron metal. This formula corresponds quite closely to the more approximate expression originally suggested, in effect, by Thomson that the Thomson heat, It, should be given by ,ct C01/Ne, (2) where C,,. is the equilibrium electronic specific heat per unit volume, e the electron charge in magnitude and sign, and N the electron density; we bear in mind Thomson's relation It = TdS/dl', and that for a degenerate Fermi gas of free electrons Cel./lNe = 7T2k2T/2e'0 (cf. also MacDonald I958). On this basis theory would predict in the general region 2 to 20 'K absolute thermo-electric powers for alkali metals, treated as ideal free electron models, which would be: (1) negative, (2) of a magnitude about 10-7 V/0C, (3) linear with temperature, and (4) particularly according to Wilson (1953), independent of impurity content. Generally speaking, none of these predictions was found to be fulfilled and the departures from theory were very marked. Thus, for example, in caesium the t Elected F.R.S. 24 March 1960. [ 334 ] This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricity at low temperatures. VIII 335 absolute thermo-electric power was positive throughout the range 2 to 20'K, had reached a fairly marked maximum of as much as 3 x 10-6 V/0C at around 6 OK, and this peak varied by about two to one in going from a purer specimen to one about ten times less pure (as measured by the residual electrical resistivity). It was suggested in part VII that the 'phonon-drag' or Gurevich (I945, I946) effect, might well be responsible for this behaviour although there were a number of difficulties involved. It was suggested that the Umklapp processes involved in electron-phonon collisions might also be an important element to be considered in the 'phonon drag' since, for example, Bailyn (1958) pointed out that Umklapp interactions should lead to a positive phonon-drag contribution to the thermoelectric power. Since the publication of part VII, Ziman (I959) has considered these results and has published an elegant discussion of the phonon-drag contribution to the thermo-electric power of the alkali metals in this temperature region. By making apparently reasonable simplifications he has been able to give a semiquantitative account of the phonon-drag effect; his theory predicts that a relatively small distortion of the Fermi surface rapidly enhances the contribution due to Umklapp processes, and Ziman appears to have been remarkably successful in accounting for the behaviour of sodium and potassium and at least qualitatively for the observed results on Rb and Cs. So far his theory has omitted the influence of phonon scattering by impurities but it appears at least very probable that a more detailed theory of phonon-drag effects taking account of the relative importance of normal and Umklapp processes, together with a suitably distorted Fermi surface, and the 'quenching' of the effect by impurity scattering of phonons should indeed be able to account rather satisfactorily for the behaviour of the alkali metals in this temperature region. We are inclined to agree with Ziman's conclusion that measurements of thermo-electric power in this general temperature region may provide a very sensitive tool for investigating the structure of the Fermi surface in appropriate cases. It will be well known that reliable information on the structure of the Fermi surface is badly needed in the study of electron transport of metals, and while much information can be obtained, in particular, from the anomalous skin effect as developed by Pippard and others (cf. for example, Pippard I957), and from the de Haas - van Alphen effect as developed by Shoenberg (e.g. I957, I958), yet these methods of investigation have, as Shoenberg himself says, their 'blind spots'. Measurements of absolute thermo-electric power on other groups of metals in this general region of temperature may therefore help to extend our knowledge of the Fermi surface. Bailyn (I959) has very recently also made a detailed analysis of the phonon-drag problem, dealing particularly with the alkali metals, and we shall return to his work again in the Discussion. Perhaps it should be remarked that, broadly speaking, the phonon-drag effect will probably only play a major role in the general low-temperature region, because of two factors: (i) the magnitude of the phonon-drag effect should depend primarily on the magnitude of the lattice specific heat and this of course decays as T3 at sufficiently low temperatures; (ii) the efficiency of phonon-electron scattering which gives rise to the additional component of thermoelectric power depends also This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 336 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton on the phonon-phonon relaxation time. Now as the temperature increases the anharmonic interaction between excited lattice waves becomes rapidly greater and consequently we do not expect the phonon-drag component to be of major significance at higher temperatures (say T V 0, typically above 100?K). We would also like to emphasize the importance of thermo-electric power as a general tool in electron transport investigations, since it is the only one of the three transport properties (electrical conductivity, thermal conductivity, thermoelectric power) which is not restricted in sign. Thus, for example, as we have remarked above, it appears that when the phonon-drag effect is dominant, the thermo-electric power can change sign from negative to positive dependent on whether 'normal' or Umklapp phonon-electron scattering plays a dominant role. Also it appears that at even lower temperatures, where phonon drag should become negligible, the sign of the absolute thermo-electric power may still depend quite critically on the nature of the impurity scattering, and this again is a feature which cannot arise in the other transport properties since they must always remain positive in sign. Apart from this, however, it was evident from even a glance at our experimental results on the alkali metals between 2 and 20'K that it should be of considerable interest to try and extend the work to temperatures in the region below 1 or 2 'K. We have now carried out a rather extensive series of measurements down to temperatures of the order of 0.1 K using adiabatic demagnetization of paramagnetic salts for cooling and we wish to present these measurements here. Short notes on some of this work have been published meanwhile (MacDonald, Pearson & Templeton I958b,c, I959). 2. THE EXPERIMENTAL PROBLEM Figures 6 to 16 show the absolute thermo-electric force, E, of the alkali metals for various specimens, as measured, as a function of temperature from approximately 0 Kt up to the order of 2?K, and also the derived absolute thermoelectric powers (S = dE/dT) for these metals. Data on S are generally more useful for comparison with theory, but it seemed best to us to present also the experimental data on E as measured. The dimensions, methods of preparation and relative purities of the samples which we have studied are to be found in table 1. The measurements were made in a suitably modified cryostat similar to that described by Dugdale & MacDonald (I957). The line drawings of figures 1 and 2 show the essential features of the experimental arrangements, while the photographs in figures 3 and 4, plate 2, give a general view of the cryostat with the vacuum cans removed, and close-up views of paramagnetic salt 'pills' and specimens in position. Essentially, the specimen was mounted between an internal bath of liquid helium (A in figure 1), which was maintained at a constant vapour pressure to provide a fixed 'hot' temperature, and, at the lower end, to a suitable 'pill' of paramagnetic salt which determined the 'cold' temperature. t E can only be measured upwards from the lowest temperature attained (say 0 05 to 0.1'K) but a reasonableextrapolationcan then be made to absolute zero. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricityat low temperatures. VIII 337 To provide direct observation of the absolute thermo-electric force of the metal concerned, the connecting leads (B, C, D in figure 1) were made of superconducting elements. Since the net thermal-e.m.f. to be measured is generally speaking rather small (and sometimes as low as 10-9 to 10-8 V), it is essential to embody some method of discriminating with certainty against random thermal e.m.f.'s that would arise if measuring leads were brought straight out from the low-temperature part of the apparatus to the measuring system at room temperature. In these experiments we used a superconducting reversing switch as developed by Templeton (I955); the switch was placed just above the outer vacuum can (see E in figure 1) and immersed in the liquid helium in the main Dewar vessel. This then demanded that the superconducting leads from the very low-temperature section of the apparatus should be brought out to a suitable vacuum seal and the final arrangement which proved satisfactory (F in figure 1) demanded the use of small hollow platinum tubes sealed into a soft glass 'bubble' through which superconducting leads of lead (Pb) could be brought directlyto the superconducting switch. The thermal-e.m.f. was measured on a suitably modified sensitive potentiometer with, as detector, a very sensitive galvanometer amplifier, embodying photo-cell amplification with a 'grid', which is capable of discriminating to better than 10-9 V (cf. Chambers I956). The temperature of the 'cold' end of the sample was taken as that of the paramagnetic salt and that of the 'hot' end as that determined by the controlled vapour pressure of the internal liquid-helium bath, and the validity of this procedure demands certain precautions. What is required is that practically all of the temperature difference shall appear across the specimen itself, which, in other words, demands that the effective thermal resistance of the specimen shall be large in comparison with any of the other thermal resistances in series with the specimen. At the 'hot' end we believe this was adequately taken care of by connecting the specimen to the helium bath through a short platinum rod, 1 mm thick, sealed directly into the soft glass seal at the base of the internal liquidhelium bath (see G in figure 1). The requirements of the problem are considerably more difficult to meet in the thermal connexion to the paramagnetic salt. The factors involved are: (1) the length and cross-section of the specimen itself, which naturally govern the net thermal resistance of the specimen for a given substance; (2) the effective thermal resistance of the contact between the specimen and the silver wire mesh on which our paramagnetic salt was grown directly (recrystallized from solution); (3) the area of contact between the mesh and the salt 'pill'; (4) the internal thermal resistance of the pill itself. We have generally made fairly crude numerical estimates of the relative importance of these factors, and wherever possible have tried to ensure that the thermal resistance of the specimen was indeed the dominant parameter, but there are a number of difficulties involved. In particular, not too much is known with real certainty about the precise behaviour of factors (3) and (4) above, although we have naturally made use of experimental estimates given, for example, by This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 338 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton Mendoza (1948), IKurti,Robinson, Simon & Spohr (I956) and Dugdale & MacDonald (I957). In principle, of course, one could always ensure that the thermal resistance of the specimen was dominant-at least down to some prescribed temperature-by making the specimen of sufficiently small cross-section, or sufficiently long; but, particularly when alkali metals are cast in glass capillaries tn vacuo, there are quite severe restrictions involved. In general, it becomes TABLE 1. EXPERIMENTAL POTASSIUM, RESULTS RUBIDIUM ON LITHIUM, AND SODIUM, CAESIUM A given specimen of a particular metal is always denoted by a single capital letter; different experiments on the same specimen are denoted by numbered subscripts (e.g. AL, A2, etc.). approximate residual resistance ratio specimen reference 2-2 7-8 2-5 103 A (o) B (Li) C(A) D (x) E (V) -W, specimen diameter (mm) remarks Lithium 05 0-5 03 05 bare wire, pure Li bare wire, Li/0 I O Mg inglass,pureLi bare wire, Li/ 0% Mg (points omitted for clarity on figure 7) 05 bare wire, pure Li 1.36 x 10-3 1 in glass. (FrompaperVII)) figure 7 -- 2-4 x 10-3 S - 5-2 x 10-9 T V/0C from eq. (9) (Wilson) in text j only x x x x 10-3 10-3 1O-3 10-3 Sodium A (O) B (R) [C (A)t -D1 (V) D2 (0, x , +) 4.7 6-9 5-1 2-8 2-8 E1, E2 (0) F1, F2 (x) 6-3 6-0 47 5-9 CV1,G2 () H (0) J (x4) IK (+)I: JL (A)l tM (y)$ W, S 5-6 5-8 2-3 4-2 - 79 7 in glass in glass x 10-4 0 07 in glass 0-14 in glass x 10-4 0-14 three runs cycling to 770K between x 10-4 runs intended to check effect of martensitic transformation; three days after D1 x 10-4 0.3 three specimens intended to check x 10-4 0.1 size effect; two runs on each, 30 04 x 10-4 months apart 05 bare wire; subsequently cycled to x 10-4 77 0K and re-run to check effect of martensitic transformation; no change of net e.m.f. at 1-20K in glass x 10-4 0-25 0.1 x 10-4 in glass 0-17 in glass x 10-4 0 09 in glass x 10-4 x 10 9 T V/0C from eq. (9) (Wilson) in text (figure 9 only) x 10-4 x 10-4 01 0.1 t The curve for specimen C between 2 and 3 OKin figure 9 is almost coincident with that of specimen II in our paper VII (details: 3-5 x 10-4; 0-2 mm; in glass). Specimens whose references are bracketed together were filled simultaneously from a common melt. Specimens C, D, L and M were filled with very pure sodium metal given to us through the courtesy of Messrs Philips, Eindhoven, The Netherlands, and of Mitcham, Surrey. I These specimens were made and measured most recently to see whether we could reproduce the relatively high thermo-electric powers found in earlier specimens. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 339 Thermo-electricityat low temperatures. VIII TABLE approximate residual resistance ratio specimen reference 1 (cont.) specimen diameter (mm) remarks Potassiun A (V) 2*1 x B1 B2 (0) 2-6 x 1O-3 B3 (x) 2-1 x 1O-3} C (o) C2 () C3(+) D(A) 2-7 x 1-5 x 1*9 x 19 23 10-3 10-3 0 075 x10-3J x10-3 x 10-3 l9-6 x 10-4 (1.7 similar to CQ(experimental points 0-15 omitted); 3 runs on same specimen at intervals of 1 and 4 months; runs 2 and 3 each repeated after melting specimen, no change of e.m.f. observed due to melting 10-3 10-3 in glass (4 rims over a period of several months) run B, considerable scatter; generally 0-18 3 runs on same specimen at intervals of l and 3 months 007 0 25) 0-8 J from paper VTI, cf. also MacDonald et al. (I9580) -* -. W, S * A (n) - 30 B (0) C (A) D (x, e) E (+, =- e) --V, - ( figure 12 only 2 x 10-8 T V/0C from eq. (9) (Wilson) in text x 10-3 Rubidium?, 0-2 2-8 x 10-3 0-2 1-6 x 10-3 1- 0-2 exploded before higher accuracy run possible poor run, points omitted on figure 14 0-2 18 x 10-3 x 10-3 0-3 16 specimens from two special distillations through the kindness of Messrs x 10-3 18 02 A. D. Mackay, Inc. in the hope of achieving very high purity 1 from paper VIII 31 x 10-3 (inset) 1 S =- 137 x 10-8 T V/0C from eq. (9) (Wilson) in text figure 14 only Caesium A (0) B1, B2 (El, x) C (A) ----upper - 1.4 x 10-3 X 10-3 16 0.21 two ruins, 6 months apart , 0-2f 2-1 x 10-3 I 1 from paper VII lower 13 and 33 x 10-3 W, S = - 165 x 10-8 T V/0C from eq. (9) (Wilson) in text ! figure 16 only difficult to fill capillaries of much less than about 0 1 mm diam. and, apart from anything else, restrictions of experimental space limited us generally to specimens of the alkali metals of about 10 cm in overall length. In any case, we also wished on occasion to examine what might be the influence of varying diameter of the specimen in view of possible 'size effects' on the scattering of electrons, or perhaps even more important, of phonons, because of the possible importance of 'phonondrag' effects. To check the adequacy of our thermal contacts at the 'cold' end of the system we made additional control experiments in which the thermal contact between the lower end of the specimen and the mesh was carefully designed to be very much Yo01.256. A. 22 This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 340 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton greater than in early experiments, and also new 'pills' of paramagnetic salt were used where we increased the area of silver wire mesh believed to be in contact with the salt from about 10 to 20 sq.cm to aroundfive times as much. In the latter to manometer pumping line for liquidhelium bath vacuum line for inner case liquid-helium inlet valve platinum tubes -A soft glass mounting sleeve for specimen C alkali-metal specimen in glass mounted for experiment magnetic suscept- (prmary ibility indution secondary for specimen and 'pill' Icage . -B paramagnetic salt 'pill' FIGuRE 1. Outline drawing of apparatus for measurement of thermoelectric power at very low temperatures. See text for lettered items. 2. Sketch of paramagnetic salt 'pill', crystallized on to spiral silver mesh. F.TGuRE case the mesh (see figure 2) was made in the form of a fairly closely wound spiral (say I to 2 mm between windings) and this arrangement should also cut down considerably the thermal diffusion time of the salt itself by reducing the radial distance between the spirals of mesh. As far as we have been able to see these later This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Proc. Roy. Soc. A, volume 256, plate 2 MacDonald et al. FIGURE 3 FIGURE 3. Photograph of apparatus FIGURE 4 (a) FIGURE 4 (b) with vacuum cans removed. FIGURE 4. 'Cage' for specimen and 'pill'. (a) Alkali metal specimen position; (b) copper wire specimen and shaped pill in position. and irregular pill in (Facinrjg This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions . 340) 341 Thermo-electricityat low temperatures. VIII control experiments showed that in general the earlier 'pill' design provided sufficient thermal contact between specimen and salt and consequently we feel reasonable confidence in the adequacy of our measurements down to perhaps 0'1 0K. Below this order of magnitude of temperature it seems that much still remains to be done before any great confidence can be placed in the estimation of the temperature of the salt itself. - 0' b%,,~~~~~~~~~~~~~~~~~~~~~ L A IA 0 L' I 02 I 04 I 06 08 I 1X0 I 1.2 1I 14 T (OK) FIGURE5. Absolute thermoelectric force (E) of a specimen of chemicallypure copper wire 1-7x 10-3) with different paramagnetic salts as refrigerant. The full 0K(Rres./R293 line is a true parabola; this correspondsto S cc T which is expected theoretically for copper at very low temperatures. *, Ferric ammonium alum, ellipsoid; 0, ferric ammoniumalum, cylinder;A, potassiumchromealum, ellipsoid; A, potassiumchromium alum, cylinder; m, ferricmethylammoniumalum; cylinder, V, ferricacetonylacetonate, cylinder. Apart from these various precautions we also carried out a series of thermoelectric experiments in which we used a number of different 'pills' of salt as the cooling agent with a 'standard' specimen of fine copper wire to investigate the consistency of the results. We not only used different paramagnetic salts (ferric ammonium alum, potassium chromium alum, ferric methylammonium alum, ferric acetonylacetonate) but also unshaped and shaped 'pills' (see below). The results of this series of experiments are shown in figure 5 where it will be seen that 22-2 This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 342 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton rather good agreement is found from about 04I Kt upwards between the various experimental runs, with the exception of the pill of ferric acetonylacetonate. In each case an appropriate correction was made from the 'Curie' temperature T* to T (Bleaney I950, potassium chromiumalum; Cooke,Meyer &Wolf I956a, ferric ammonium alum; Cooke et al. I956b, ferric methylammonium alum); it would appear, however, that the T* to T correction used for the acetonylacetonate pill is not very reliable.t In general in our experiments the pills used were somewhat l~~~~~~ I ~ l1 ~~~~~~~ 0 V 2 0Q v/ O/' 0 I 0 I I 1 l ~~ ~~~~~~~2 I 3 T (OK) FIGURE 63. Absolute thermo-electric force (E) for specimens of pure lithium and selected alloys (see table 1). irregular right circular cylinders, but in these control experiments on copper, shaped ellipsoids of revolution (see figure 4 b) were also used so that an appropriate Lorentz shape-correction factor could be employed, but it appears that, as might fairly be expected, this is not a serious matter down to the order of 0 1 0K. The general course of an experiment, after the cryostat had been cooled to 4*20K with liquid helium and the internal liquid-helium vessel had also been filled, required that the internal liquid-helium bath should then be slowly pumped down t This is, of course, only a rough figure since the last two salts named do not demagnetize to as low temperatures as the first two. $ We are very grateful to Professor J. G. Daunt for giving us particulars about this salt, and would like to mention that he himself warned us about the unreliability of the available T* to T correction, which he himself kindly supplied. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricity at low temperatures. VIII 343 to about 1-20Kin a numberof stages. At each of these stages the magnetic susceptibility of the 'pill' was measured to provide later calibration of T*. After demagnetizing the 'pill' the thermal-e.m.f. was then measured as the salt warmed up. The warm-uptime might be typically of the order of 1 to 2 h, although in extreme cases it might be as short as 10 to 15 min with a very high-conductivity specimen, or at least as long as 3 or 4 h with a specimen of high thermal resistance. There / -15- _ V v/O/~~~~~~~~~~~ _ v +0 0-3 0 /O~V 0 T (0K) 7. Absolute thermo-electric power (S) for specimens of pure lithium and selected alloys (see table 1). The points on the graph, indicating graphical derivatives taken directly from the smoothed curves of thermo-electric force, are plotted to enable some estimate to be made of the overall reliability of the curves for S. This remark applies to all subsequent figures of thermo-electric power (S). FIGunE has been no reason for us to believe that the rate of warming has in general been a significant source of error, but we have felt that it would be desirable to use an alternative arrangement where the specimen would be mounted between two paramagneticsalts. In that case, a (relatively small) temperaturedifferencewould be established by a heater in one of the salts and two advantages should ensue: first, the heat flow into the 'cold' salt would be very much smaller than is the case with the top of the specimen connected to the pumped helium bath; secondly, if the temperature differential (AT) between the 'pills' is sufficiently small (i.e. AT/Tp2n < 1) the net e.m.f. measured (AE) would give directlythe absolute This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 344 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton thermo-electric power (S AE/AT). A number of experiments were made to try this technique and certainly the heat influx from one 'pill' to the other can be kept very small. However, the apparent increase in sensitivity is rather illusory because one is trying to measure now what is generally an extremelysmall net potential difference (AE), and also we found it no easy matter at these rather low Of~~~~~~v 0 ~ 1H 0 \ \~~~~~~~~\ C)~~~~~~~~~~~~~\ V~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - -10 \ .. 0 _ I I I , 1 I 2 I V\ II 3 T (-K) FIGURE8. Absolute thermo-electric force (E) for main group of specimens of pure sodium (see table 1). temperatures to determine with any accuracy AT for two pills at neighbouring temperatures. In sum, we present here our measurements using a single 'pill' in conjunction with the internal liquid-helium bath. These we feel are fairly reliable down to something of the order of 0.1 0K, but we certainly believe that for some metals at least it will be necessary in future to devise a satisfactory two-pill method for investigation of absolute thermoelectric power at even lower temperatures. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricityat low temperatures. VIII 345 H ~~~-40~~ ~ X A \- C~~~~~~~~~ 00 III I -4 - I V 12 T(OK)K 0 -2 - -6- B V~~~~ T (0K) FIGURE 9. Absolute thermo-electric power (S) for main group of speciamensof pure sodium (see table 1). G2(t o xo0 -0.5- + -FO ~~~~~~~~~~~~~~~~~~~~~~~~F D~~~~~~~~\ \ ~~ ~ ~ -1.5 \T 0 0.5 (a) FiGURE 1.0 G ~ ~~~~X % 0 T (OK) 3 12 (b) 10. Results of special experiments on sodium specimens (see table 1). (a) Relating to martensitic transformation; (b) relating to possible size-effect. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions ~ o-4_-~ ~~~~~~ It B2B -~~~~~~ C -7f=;5a -2 e ojb C \ 0~~~~ l '~~~~~~~~~~~4 \ ~AD -6- C,(B) 0 V \~ \ -8_ 1- O I I , 2 1 , 3 T (OK) FIGURE11. Absolute thermoelectric force (E) for specimens of pure potassium (see table 1). I I I I III 0 0 0 -2 2 0 -2 -4 01b e mpw 0 "I-0,\\A ,\I fs e Vp se 0~~~~~ 0 0 \ ~ V ~~~~~~~~0 0 0 FIUR o 12 1 bouetem-lcrcpwr()frseieso 2 T(~~~~~~~~~~~~~~~0K)v ueptsim(e This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions al ) Thermo-electricityat low temperatures. VIII l l ~ ~ ~ ~ ~ 00 l A' 347 + X lI ' ? 08 0I, ~ 0 70 ~ /.0 ~ 0e~ D \ ' I+1 0 ID 1 l 3 2~~~~~~~~~~~~ 1 T (?K) 0T(OK) 3 2 0~~~~~ oo .0,~~~~ / 0_1~~~~~~ 0~~~\ 0 1 0 '.. I ~ ~ T(0) 2 II 4> 0 W~~I, FIGtURE13. Absolute thermoelectric forcer (E) for specimens 4- 6~/ 0 , , FIGRE 4. bsou2 throl of pure ~~~~, 0~~~~~~ rubidium. (see table 1). 0 T (OK) ctri poe uerbdim(e-al S o pcieso This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions ) X +6 -8 I~x 4- II 8 I II 01 ~~~~~~~~~~~~0 0~~~~~~~~~~~~~~~~~ C~~~~ .I I I I / 4;- +15 0 0. V~~~~~~~~~ X_~ 0 .0 1 / /A I T(?K) 2 3 T (0K) 2 3 +21- AB 0 0 FIGURE F IGURE 1 16. Absolute thermo-electric powcer 15. forcer (E) caesium (see table 1). (S) for specimens of pure caesium. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricity at low temperatures. VIII 349 3. DISCUSSION 3*1. Theoreticaloutlines In our previous paper (part VII) we suggested that the explanation for the anomalous thermo-electric powers observed in the range 2 to 20 'K might be found in the phonon-drag effects and the theoretical analyses proposed since by Ziman (1959) and Bailyn (i960) give support to this belief. Let us now consider the relative contributions of the phonon-drag thermo-electric power and that due to the direct effect of the temperature gradient on the electrons alone (and let us call this the 'diffusion' effect).t If we assume quasi-free electrons then the diffusion component, say Sdiff., is given approximately by (3) Sdiff. Cei.lNe (cf. equations (1) and (2) and the introductory discussion to this paper). In analyzing the 'phonon-drag' component of thermo-electric power, say Sph, one has to consider two types of phonon-electron collisions. In one case, the so-called 'normal' collisions, momentum is said to be conserved in the process; that is to say, the momentum corresponding to the wave vector of the excited phonon is absorbed directly by the electron. There are also, however, collisions, the so-called Umklapp processes (first pointed out by Peierls many years ago), in which in addition momentum corresponding to a typical wave vector of the reciprocal lattice is imparted to the electron. One may say that in these latter processes the phonon-electron collision causes the electron to undergo in addition a Bragg reflexion in the lattice. An elementary discussion of Sph. ignoring effectively the Umklapp processes-i.e. assuming direct total transfer of momentum from the excited lattice vibrations to the conduction electrons-leads to an approximate expression (4) Clatt/Ne. SPh. (cf. for example, MacDonald I954) where CMat.is now the lattice specific heat per unit volume, in contrast to equation (3). More detailed and thorough analyses of phonon drag in metals have now been carried out by Ziman (I959) and Bailyn (i960) (cf. also Bailyn I958) taking account of both the normal and Umklapp processes. We shall discuss these shortly but in any case the results are rather complex, and for the moment let us just mention that the Umklapp processes can, it appears, lead to a Sph of opposite sign to that expected from 'normal' phonon-electron collisions. However, we might reasonably expect that the net magnitude of Sph would not exceed that indicated by equation (4) although the sign may be incorrect. Thus, very roughly speaking, we might expect that the relative significance of 'phonon-drag' and 'diffusion' components in a pure metal at low temperatures should be given by Sph. Clatt. Sdiff. Cel. (5) t An alternative expression, suggested in private discussion with Dr C. Herring, for this component would be the 'migration' effect. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 350 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton Equation (5) should indicate more or less an upper limit for the magnitude of the 'phonon-drag' effect. This is because phonon scattering by chemical impurities, physical imperfections, or presumably even by isotopes, will all tend to diminish the effect, and at higher temperatures 'phonon-phonon scattering' arising from the increasing anharmonicity of the lattice vibrations will diminish the role of phonon-electron scattering. So then, using equation (5) we may write 5ph/Sdiff r (6) 40T2TO/03, where To (and kT0 =) is the Fermi degeneracy temperature of the conduction electron gas. Table 2 shows theoretical values for this ratio calculated at 05 0K for the alkali metals, from the values of To and 0 indicated. For the lighter alkali metals it is clear that at temperatures below about 1?K, Sph. should be unimportant, but that for caesium in particular it may be necessary to go to temperatures below about 0 2 'K to be quite certain that 'phonon drag' is negligible. t TABLE 2 metal To (OK)t Li 5.5 Na K Rb Cs 3 65 x 104 2-4 x 104 2-1 x 104 175 x 104 x 10' r at 0-50K 0 ('K) 350 . 160 100 -55t -40? t Theoretical values based on one free electron per atom. I Approximate estimate of 0 as T -+ 00K (cf. Dauphinee et al. ? Estimate of 0 as T -+- 0OK. 0 01 - 0 09 0.4 1-3 2-7 I955; Manchester I959). 3-2. Summary of experimental findings Let us now summarize some broad features of our experimental results: (1) The thermo-electric power of lithium and caesium both in pure and impure samples remains positive down to the lowest temperatures reached. In both metals there is some dependence of magnitude on the impurity content (and also probably on the method of preparing specimens) but, we repeat, in both cases the thermo-electric power remains quite definitely positive. In lithium S is satisfyingly linear with T below 2 or 3VK which is what would be expected for the diffusion component; in caesium a linear dependence appears to set in at considerably lower temperatures (say roughly 0-5?K). As might be expected from the much lower 0 in this metal, phonon-drag effects appear to be dominant to rather low temperatures. (2) The absolute thermo-electric power of sodium and potassium is generally negative. In both cases, however, the magnitude shows quite a wide range of variation, which in neither case appears to be dependent in any obvious manner on the observed residual electrical resistivity or on the size of the specimen; nor, in the case of sodium does it appear to depend significantly on the martensitic t It is not unlikely that also Hanna & Sondheimer equation (5) overestimates Sph./Sdiff.. Sondheimer (I956) (cf. and Ziman I959) gives Sph. = 3CIatt./Ne (cf. equation (4)) as the low-temperature limit assuming that phonons are scattered entirely by conduction electrons, and ignoring Umklapp scattering. I957 This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricity at low temperatures. VIII 351 transformation (see below). In both cases the thermo-electric power depends on temperature in no very simple manner, but it appears that in those specimens having the smaller thermo-electric power in magnitude, the power law dependence on temperature is higher. This is particularly obvious in sodium where the curve for the largest magnitude of thermo-electric power shown in figure 9 remains quite linear with temperature up to about 2 0K. (3) The sign and general temperature dependence of the thermo-electric power in rubidium below about 2 0K appears to depend most sensitively on the impurity content (as measured by the residual resistance). 3-3. The question of Fermi surface distortion and the role of electron-phonon interaction Now in order to try to understand our results we would naturally like to separate out with some confidence Sdiff. and Sph.; and moreover, if at all possible, to separate the latter into normal and Umklapp contributions. However, the present situation from the theoretical point of view appears somewhat confusing. At sufficiently low temperatures it is true that we should expect that only a component Sdff., linearly dependent on T, would be found, but to be confident of the analysis we should know how rapidly and with what decay law the phonon-drag components die away. Now according to Ziman's (1959) theory the vital feature governing the magnitude, sign, and temperature dependence of Sph. is the degree of distortion of the Fermi surface. Ziman's theory offers the attractive picture that the progressive increase, as we go from Na to Cs, of anomalous thermo-electric power observed between about 2 and 20 'K (part VII) is to be ascribed to an increasingly distorted Fermi surface in these metals. Ziman's theory enables him to fit rather well the results for sodium using an undistorted Fermi surface and those for potassium with a mild distortion (for potassium Ziman assumes a distortion figure of 7 0O). Further distortion assumed for rubidium and caesium leads to theoretical curves of the same general shape as those found experimentally in rubidium and caesium. However, Ziman finds it difficult to approach the magnitude of anomalous thermo-electric power which we have found in caesium. His 'fudged' curve (Ziman 1959, figure 4) shows a maximum thermo-electric power of about + 0 8 fuV/0C, while our experiments show in caesium a maximum in excess of +3 FuV/0C.t Ziman's analysis, moreover, leads to an expression in which the contributions due to normal and Umklapp processes are not simply separable. t Ziman comments: 'The calculation in my paper is based upon a rough approximation to the Bardeen matrix element for electron-phononinteraction. This is based upon a freeelectronmodel for the conductionelectrons. But if, as we have reasonto believe, the Fermi surfacein some of the alkali metals is greatly distorted, even to touching the zone boundary, then the electron wave functions cannot be simple plane waves. Mr J. G. Collins, at the CavendishLaboratory,has made some calculations of the electron-phononinteraction for more complex electron waves (combinationsof orthogonalizedplane waves) and has found considerableeffects, particularlyupon the magnitudeof the matrix elementsfor U-processes. Since these are just the terms responsiblefor the positive thermo-powerin Sph.,we can understand how such large humps might appear in just the metals which, from this and other phenomena,have the most distorted Fermi surfaces. In other words,I might have been even bolder in my "fudging".' We are most grateful to Dr Ziman for correspondenceon this generalsubject. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 352 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton On the other hand, Bailyn (i960) concludes that essentially contributions from the normal and Umklapp processes can be directly added together in an appropriate form, and argues that the anomalous thermo-electric powers in the region of roughly 2 to 20'K in the alkali metals arise, not primarily from an assumed distortion of the Fermi surface, but much more from a very critical balance between the normal and Umklapp components (which we might call S-h and S+h), which he does not consider as depending directly on an assumed distortion of the Fermi surface. Bailyn's calculations are rather extensive, but it appears that he is able to account on the whole rather well for the experimental results. Indeed, he is able to make his theoretical curves in the case of rubidium and caesium achieve the same peak values as in our experiments, and the rate of decay at higher temperatures has a resemblance to that observed experimentally. Bailyn, moreover, contends that there are fundamental differences involved between Ziman's approach to the problem and his. From our point of view-we are speaking essentially as experimentalists-both theories offer attractive features and we should like to believe that the final outcome would involve some synthesis of the two theoretical discussions. (On the other hand the present theoretical divergence makes it difficult for us to be confident in analyzing our results.) If we take first Ziman's theory, his assumption of progressive distortion of the Fermi surface as we go from Na to Cs agrees with a general conclusion which had been drawn from data on the electrical resistivity and from other experimental evidence on the alkali metals a number of years ago (see, for example, MacDonald & Mendelssohnt 1948). Let us remark here that the clearly positive Sdiff in lithium (and the positive thermo-electric power is in fact maintained right up to room temperature) also suggests strongly a considerable departure from the free-electron model, which had been recognized for some considerable time. More recently also Cohen & Heine (I958) have made a broad review of the experimental data then available on the alkali metals (and also the group IB metals) and have gone on to provide some theoretical interpretation for the behaviour. In their assessment of the experimental behaviour Cohen & Heine say: 'To sum up, the experimental evidence indicates that the Fermi surface of Na is nearly isotropic, that of K somewhat distorted, that of Rb and Cs rather more so, and that of Li actually in considerable contact with the zone boundary....' Their theoretical discussion of the shape of the Fermi surface depends essentially on estimating the energy gap (Es - E.) between the first and second energy bands at the Brillouin zone face nearest to the origin in k-space. Their estimation of this energy gap involves the energy difference (Asp) in the free atom. Their theory then leads them to the conclusions: ' . . . that at the centre of the nearest zone face p-like levels are lowest for Li and Na and s-like levels are lowest for K, Rb, Cs. The magnitudes of [the energy splitting] indicate that the Fermi surface is relatively t Quoting... 'Our results seem to indicate that, except for sodium, the Fermi surfaces of the alkali metals are distorted. A critical survey of other physical data on the alkali metals0 for example, the reduced conductivity at WC, (ojIMO2)V*-appears to strengthen this assumption. Distortion of the Fermi surface in the case of lithium may possibly be due to insufficient screening of the valency electron; in the case of the higher alkalis, to the effect of electrons from the lower shells.' This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricityat low temperatures. VIII 353 undistorted in Na and K, appreciably distorted in Rb, and still more distorted in Cs. For Li... the Fermi surface actually makes contact with the nearest zone faces.' We might mention also that Cohen& Heine's theory also leads them to make predictions about the influence of impurities on the electronic energy surfaces of the parent metal and we shall mention this again later. We feel that in broad terms our experimental findings below 1 or 2?K outlined above agree quite well with these general conclusions about the Fermi surface.t Bailyn's treatment of the problem emphasizes, as we have said, the extreme sensitivity of the balance between 'normal' and 'Umklapp' phonon-electron processes in contributing to the resultant Sdiff.,and this suggests reasons to us why the large-and apparently very sensitive-variability of thermo-electric power in sodium, potassium and rubidiumis found below about 2 or 3 0K. If, for example, for potassium Sdm.at these low temperaturesis almost zero, as appearsratherprobable to us from the experimental data, then the observedthermo-electricpower down to very low temperatureswill of course be dominated by the net Sph.* If in turn, as Bailyn argues, the net Sph. depends very critically on the balance between normal and Umklapp behaviour, then presumably it would not be surprising that considerable variation would be observed from sample to sample, probably depending rather critically on the physical and chemical state of the specimen. For rubidium and caesium we might attempt a somewhat more detailed discussion as follows. We are led to suppose from the experimental evidence that in Rb Sdiff. is essentially positive which might correspond to a considerably distorted Fermi surface. In more impure samples, phonon-drag effects will then be quenched by phonon impurity scattering below about 1VK, and only the (supposed positive) diffusion component will then be observed. As impurity is removed, however, the phonondrag contribution will increase-both the (positive) Umklapp component, as was clearly observed in our previous work (MacDonald et al. I958a)-and also the negative component due to 'normal' phonon-electron collisions. We expect further that the Umklapp component will decay more rapidly (- exp {- *1Tj, where 6* is a characteristictemperature of the order of the Debye temperature 0), at very low temperatures than the 'normal' component (a (T/0)3). Thus, on this basis, the thermo-electric power in a sufficiently pure specimen should start to decay from a high (positive) Umklapp peak, should change sign as the 'normal' phonon drag dominates, and finally at 8Ufflciently low temperatures should pre- sumably become positive and linear corresponding to the (supposed positive) diffusion component. This latter and final stage is not evidenced in our experiments, but this might be because the suggested final change-over in sign does not occur until extremelylow temperatures. The issue may be complicated t One should perhaps note however, that of the absolute thermoelectric powers of the alkali metals at around room temperature, lithium it is true remains stubbornly positive but the thermo-electric power of all the remaining alkali metals is negative. For sodium, potassium and rubidium the order of magnitude is what we might expect theoretically (see, for example, Wilson 1936) while caesium has a much smaller thermo-electric power (although still negative), not more than one-tenth of that to be expected theoretically. It is of course possible that the effects of thermal expansion (which might be particularly important in Rb and Cs) would also have to be taken into account. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 354 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton by a possible sensitive dependence of Siff itself on the impurity content (see below). It should be evident that this explanation suggests a number of very interesting experiments on rubidium of both high and low purity. We hope before long to be able to obtain again supplies of rubidiummetal of sufficientlyhigh purity to extend this work. In the case of caesium we suggest that the Fermi surface is so distorted that Sdiff. is certainly positive, but also that Umklapp processes are now so frequent as practically to 'drown out' any contributionfrom 'normal' phonon-draginteractions. Thus the behaviour to be expected (and which indeed is observed),is that the thermo-electric power would decay from a positive Umklapp peakwhose magnitude would depend on the purity and which could be very large in the case of a very pure caesium specimen-down to a linear and positive diffusion component which would remain unaltered down to the lowest temperatures and be more or less independent of the purity of the sample. In orderto try to make some more quantitative assessment of our data we have 3 tried, as a crude approach, to compare our experimental results below about VK with the expression (7) S = AT+BT3+C exp (-O/T), or alternatively, for more direct comparison with the immediate experimental data in some cases (8) E =j2AT2+4BT4+ D exp (-O/T). In neither equation (7) nor (8) do we suppose that C or D is strictly a constant but are assuming that the exponential factor dominates the temperature dependence of that factor in the region concerned. In these equations the first term is intended to take care of the diffusion component and the remaining terms of the phonon drag, the first of these being the normal component and the second-in a very crude way-the contribution arising from Umklapp processes. In Ziman's analysis, as already remarked,the contributions of normal and Umklapp processes to Sph. are not readily separable. Our semi-empirical equations (7) and (8) are thus perhaps somewhat more in the spirit of Bailyn's analysis where he is able to separate directly the normal and Umklapp contributions.t Let us also point out that Bailyn's conclusion about the very delicate balance between the normal and Umklapp phonon-drag components leads him to say (Bailyn i960) that 'the dwindling away [of the phonon-drag component as T-- 0] is as much caused by the changing relative importance of these large terms as to the effect that each becomes smaller (our italics). In fact, there is very little cause for expecting a T3 dependence .., in the range dominated by phonon drag. Of course, if T gets low enough, Sg (our Sph.) will vary as T3, but this will take place, it would seem, in the region where phonon drag may be neglected altogether.' Be that then t Bailyn (private communication) suggests that at very low temperatures one would expect for the phonon-drag component BT3 + C(O*/T+ 3) exp (-O*1T), where C is now essentially a constant. Sph. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 355 Thermo-electricity at low temperatures. VIII as it may, a summary of the comparisons of equations (7) and (8) with our data is given in table 3 but we would not suggest that there is any great significancein the results, particularly in view of the doubt about the theory itself which exists at present. We have also included in table 3 estimates by Bailyn (private communication) of the two characteristic temperatures (denoted by 0I and 0,I) which are involved in his theoretical discussion of the problem (cf. Bailyn i960). If we take table 3 at all seriously, we see that a 0* is indicated for each metal which is somewherearound 0 I to 0-2 of the Debye SD and that the estimated Umklapp component of phonon drag is much more important in rubidium and caesium than in sodium, for example. Indeed, in sodium most of the experimental TABLE3 01 metal Na K Rb Cs A (V/deg2) B (V/deg4) -0 05 to -30 +0 5 to -10 +2-0 to -0-2 +5 0 -002 to -0-08 -0.15 to -030 0 to -0-6 -2.3 C (V/deg) - 500 to 5000 800 D (V) + 10 to + 20 +48 500 - 0On 0* (OK) (OK) (OK) 8 to 20 , 21 - 16 -6 33 24 15 11 44 18 11 8.5 exp (-0*/T), Note 1. Parameters to fit 108S = AT+BT3+C 108E = WAT 1+ BT + D exp (-0*/T), where S is measured in volts/0C and E in volts. Note 2. 0, and 01, are taken from Bailyn's (i960) theoretical discussion (see text for further details). or results at very low temperatures can be fitted quite satisfactorily by ignoring altogether the third term in equations (7) and (8). There is also found a considerable variability in the magnitude of Sdiff in sodium, potassium and rubidium which shows up quite clearly in the experimental results themselves for sodium and potassium. If we turn, for example, to Wilson (I936) he suggests that at sufficiently low temperatures (T < 0) Sdiff should be independentof impurity content and have the same value as in a very pure metal. The value then predicted is Sdiff =.22 (9 a) T/3eI0 rr2k/T\ (9b) 3e To where 0 is the Fermi energy for the metal concerned at absolute zero, and To is the corresponding electron degeneracy temperature (see, for example, table 2). For comparison the values predicted by equation (9) are indicated on our experimental graphs of observed thermo-electric power. 3-4. The effect of alloying and of martensitic transformations It is evident then from our results that considerable variability exists in the value of Sdff. in a given metal, but this is not particularly surprising since we have already remarked several times elsewhere on the dependence of Sdiff on impurity. As a matter of interest we show in figures 6 and 7 some of the results of experiments that we have been making more recently on a series of dilute alloys 1VOL.256. 23 This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions A. 356 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton of magnesium in lithium, which show up very clearly the quite rapid and regular dependence in this metal of Sdiff on magnesium content. We are extending the scope of these experiments at present and hope to report more fully later on this work. To emphasize further the effects of impurities we might also mention that we have known for some years that the thermo-electric power in copper at low temperatures was very sensitive to the presence of certain impurities and moreover more recently (Gold, MacDonald, Pearson & Templeton i960) we have found that in extremely pure copper (having a residual resistance of about 4 x 10-4) a very remarkable sensitivity of the low-temperature thermo-electric power still exists. Finally, one particular point must be mentioned. In sodium one might be tempted to suggest that a phonon-scattering mechanism responsible for the variation in thermo-electric power would arise from variability in the fraction of the martensitic transformation from b.c.c. structure to h.c.p. structure which occurs in sodium spontaneously around 35?K on cooling (Barrett 1948, I955, 1956; Barrett & Trautz 1948). Although Barrett originally believed that the amount transformed was rather small (about 5 %0), more recent work in these laboratories (Martin i960; Dugdale & Gugan i960) indicates that something nearer 50 %is commonly met with (see also, Hull & Rosenberg I959). In the b.c.c. phase one expects that the Fermi surface does not touch the nearest Brillouin zone boundary, but in the h.c.p. phase it presumably must intersect the first zone It boundary perpendicular to the hexagonal axis (cf. Dugdale & Gugan i960). would therefore seem very reasonable that the transformation might affect quite strongly the net magnitude of Sph.; in the fraction that had transformed to the h.c.p. structure, Sph. might now show a considerable Umklapp (positive) component thus diminishing the net (negative) magnitude of Sphl. Since, furthermore, the fraction which transforms may be quite variable from specimen to specimen, and the transformation is found to have practically no influence on the observed residual electrical resistance (Dugdale & Gugan i960), it might seem that the variable martensitic transformation could offer a fair explanation for the observed variability of the thermo-electric power at low temperatures in sodium with no corresponding variation in residual electrical resistance. Two experiments were therefore made specifically to test whether the martensitic transformation was a significant factor in the observed thermo-electric power. An experimental run was made below 4 ?K down to the lowest temperatures; the apparatus was then warmed up to liquid-nitrogen temperatures (- 77?K) (but not significantly higher) and then cooled again to 4?K for a further experimental run. According to Martin, and to Dugdale & Gugan, this procedure should inhibit markedly the fraction transformed on the second cooling. The experiments were were made both on a specimen cast in vacuo in the usual way, and also on a bare extruded wire of sodium (- 1 mm in diameter) wound on a Teflon former similar to that used by Dugdale & Gugan. In neither case was there any significant difference in the observed thermo-electric power at low temperatures after the intermediate warming cycle (cf. figure 10a and table 1). It thus appears improbable that the martensitic transformation can be an important factor in the low-temperature thermo-electric power of sodium. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 357 Thermo-electricityat low temperatures. VIII 4. CONCLUSION To sum up, in the two 'extreme' alkali metals, lithium and caesium, the thermoelectric power appears to remain stubbornly positive down to the lowest temperatures measured. This is at least consistent with the assumption of a highly distorted Fermi surface in both of these metals. In sodium and potassium, the thermo-electric power is generally negative and, particularly in sodium, would appear not inconsistent with the assumption that the Fermi surface is reasonably free from distortion. There are still many aspects, however, of our experimental findings which remain to be understood more fully. It appears very clear to us from these results that we would do well to make experiments at even lower temperatures, particularly in such metals as potassium, rubidium and caesium of the alkali metal group, and that there are many questions about the influence of alloying which it would be attractive to examine experimentally. Cohen & Heine's (I958) theory would suggest, for example, that the addition of rubidium to potassium as a parent metal would increase the distortion of the Fermi surface, while the addition of sodium might be expected to reduce any distortion present. In general terms too we feel that there is much yet for us to understand about the influence of very small amounts of impurity on the thermo-electric power at very low temperatures. We are most grateful to Dr M. Bailyn, Dr J. S. Dugdale and Dr A. V. Gold for discussions, to Dr J. M. Ziman for correspondence, and to Drs Bailyn, Dugdale and Ziman also for helpful comments on the manuscript. We thank Dr A. H. Cooke, Dr B. B. Goodman, and Dr N. KUrti, F.R.S., for their kind and helpful correspondence on questions of very low-temperature techniques, and Dr Cooke particularly for helping us to obtain supplies of ferric methylammonium alum, and we thank Dr Howard 0. McMahon of Arthur D. Little, Inc. for supplying us with some ferric acetonylacetonate. The interest of Mr Paul Nisley, Messrs A. D. Mackay, Inc. in our problem of obtaining sufficiently pure rubidium and caesium metal has been much appreciated. We wish to thank Mr D. J. Huntley for much help in some of these experiments and in preparing data. Finally, our thanks are due to Messrs F. W. Richardson and J. Broome for their tireless production of liquid helium, and to Miss Joan Spong for assistance in taking readings and in numerical computation of results. REFERENCES Bailyn, M. I958 Phys. Rev. 112, 1587. Bailyn, M. i960 Phil. Mag. (submitted for publication). Barrett, C. S. I948 Amer. MHn. 33, 749. Barrett, C. S. I955 J. Inst. Metals, 84, 43. Barrett, C. S. I956 Acta Cryst. 9, 671. Barrett, C. S. & Trautz, 0. R. I948 Trans. Amer. Inst. Min. Engrs, 175, 579. Bleaney, B. I950 Proc. Roy. Soo. A, 204, 216. Chambers, R. G. I956 Proc. Roy. Soc. A, 238, 344. Cohen, M. H. & Heine, V. I958 Advanc. Phys. (Phil. Mag. 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I954 Physica, 20, 996. MacDonald, D. K. C. I958 Z. Phys. Chem. 16, 310. MacDonald, D. K. C. & Mendelssohn, K. I948 Nature, Lond. 161, 972. MacDonald, D. K. C., Pearson, W. B. & Templeton, I. M. 1958a Proc. Roy. Soc. A, 248, 107 (part VII). MacDonald, D. K. C., Pearson, W. B. & Templeton, I. M. I958b Phil. Mag. 3, 657. MacDonald, D. K. C., Pearson, W. B. & Templeton, I. M. 1958C Phil. Mag. 3, 917. MacDonald, D. K. C., Pearson, W. B. & Templeton, I. M. I959 Phil. Mag. 4, 380. Manchester, F. D. I959 Canad. J. Phys. 37, 525. Martin, D. L. I960 Proc. Roy. Soc. A, 254, 433. Mendoza, E. I948 'Les phenomenes cryomagnetiques', Hommage National a Paul Langevin et Jean Perrin. Paris: College de France. Pippard, A. B. i957 Phil. Trans. A, 250, 325. Shoenberg, D. I957 Progr. Low Temp. Phys. vol. 2 (ed. Gorter), p. 226. Amsterdam: North Holland Publishing Co. Shoenberg, D. I958 Low Temp. Phys. and Chem., Proc. 5th Int. Conf. (Madison, WVis., U.S.A.), p. 450. Sondheimer, E. H. I956 Canad. J. Phys. 34, 1246. Templeton, I. M. I955 J. Sci. Instrum. 32, 172. Wilson, A. H. I936 (2nd ed. I953) Theory of metals. Cambridge University Press. Ziman, J. M. I959 Phil. Mag. 4, 371. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions

SCAMP5 Plays a Critical Role in Synaptic Vesicle Endocytosis during High Neuronal Activity

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Haiyan Zhao,1,2Yoonju Kim,1,2,3Joohyun Park,1,2,3Daehun Park,1,2Sang-Eun Lee,1,2Iree Chang,1and Sunghoe Changcorresponding author1,2,3,4

Haiyan Zhao

1Department of Physiology and Biomedical Sciences,

2Biomembrane Plasticity Research Center,

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Yoonju Kim

1Department of Physiology and Biomedical Sciences,

2Biomembrane Plasticity Research Center,

3Neuroscience Research Institute, Medical Research Center, and

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Joohyun Park

1Department of Physiology and Biomedical Sciences,

2Biomembrane Plasticity Research Center,

3Neuroscience Research Institute, Medical Research Center, and

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Daehun Park

1Department of Physiology and Biomedical Sciences,

2Biomembrane Plasticity Research Center,

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Sang-Eun Lee

1Department of Physiology and Biomedical Sciences,

2Biomembrane Plasticity Research Center,

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Iree Chang

1Department of Physiology and Biomedical Sciences,

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Sunghoe Chang

1Department of Physiology and Biomedical Sciences,

2Biomembrane Plasticity Research Center,

3Neuroscience Research Institute, Medical Research Center, and

4Bio-Max Institute, Seoul National University College of Medicine, Seoul, South Korea 110-799

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Author informationArticle notesCopyright and License informationDisclaimer

1Department of Physiology and Biomedical Sciences,

2Biomembrane Plasticity Research Center,

3Neuroscience Research Institute, Medical Research Center, and

4Bio-Max Institute, Seoul National University College of Medicine, Seoul, South Korea 110-799

corresponding authorCorresponding author.

Correspondence should be addressed to Sunghoe Chang, PhD, Department of Physiology, Seoul National University College of Medicine, # 309 Biomedical Science Bldg., 28 Yeongeon-dong, Jongno-gu, Seoul 110-799, South Korea., [email protected]

Contributed by

Author contributions: H.Z., Y.K., and S.C. designed research; H.Z., Y.K., J.P., D.P., S.-E.L., and I.C. performed research; H.Z., Y.K., J.P., D.P., S.-E.L., I.C., and S.C. analyzed data; H.Z., Y.K., I.C., and S.C. wrote the paper.

I. Chang's present address: Leibniz-Institut für Altersforschung, Fritz-Lipmann-Institut e.V. (FLI), Beutenbergstraβe 11, 07745 Jena, Germany.

Received 2014 May 28; Revised 2014 Jun 16; Accepted 2014 Jun 20.

Copyright © 2014 the authors 0270-6474/14/3410085-11$15.00/0

Abstract

Secretory carrier membrane protein 5 (SCAMP5), a recently identified candidate gene for autism, is brain specific and highly abundant in synaptic vesicles (SVs), but its function is currently unknown. Here, we found that knockdown (KD) of endogenous SCAMP5 by SCAMP5-specific shRNAs in cultured rat hippocampal neurons resulted in a reduction in total vesicle pool size as well as in recycling pool size, but the recycling/resting pool ratio was significantly increased. SCAMP5 KD slowed endocytosis after stimulation, but impaired it severely during strong stimulation. We also found that KD dramatically lowered the threshold of activity at which SV endocytosis became unable to compensate for the ongoing exocytosis occurring during a stimulus. Reintroducing shRNA-resistant SCAMP5 reversed these endocytic defects. Therefore, our results suggest that SCAMP5 functions during high neuronal activity when a heavy load is imposed on endocytosis. Our data also raise the possibility that the reduction in expression of SCAMP5 in autistic patients may be related to the synaptic dysfunction observed in autism.

Keywords: neuronal activity, recycling pool, resting pool, SCAMP5, synaptic vesicle endocytosis

Introduction

Secretory carrier membrane proteins (SCAMPs) are secretory vesicle components found in exocrine glands. Of the five currently known SCAMPs (SCAMPs 1–5), SCAMPs 1–3 share a common domain structure comprising a cytoplasmic N-terminal domain with multiple endocytic NPF repeats, 4 highly conserved transmembrane regions, and a short cytoplasmic C-terminal tail. SCAMPs 4 and 5 lack the N-terminal NPF repeats and were thus thought not to function in endocytosis (Fernández-Chacón and Südhof, 2000).

SCAMPs 1–4 are expressed ubiquitously, whereas SCAMP 5 is known to be brain specific. SCAMPs 1 and 5 are highly abundant in synaptic vesicles (SVs; Fernández-Chacón and Südhof, 2000). SCAMPs 1–3 were shown to play a role in fusion pore formation at the plasma membrane during dense-core vesicle (DCV) secretion in PC12 cells and also in trafficking events in the trans-Golgi network (TGN) and endosomal recycling compartment, suggesting that their basic role is in vesicular trafficking (Wu and Castle, 1998; Fernández-Chacón et al., 1999; Guo et al., 2002; Liu et al., 2002; Lin et al., 2005; Liu et al., 2005; Liao et al., 2007; Liao et al., 2008; Zhang and Castle, 2011).

Although the high levels of SCAMP1 expression in SVs suggested that it played a role in synaptic physiology, analysis of SCAMP1 knock-out mice showed that this protein was not essential for synaptic functions (Fernández-Chacón et al., 1999), raising the possibility that other, brain-specific SCAMPs such as SCAMP5 might be active. SCAMP5 is expressed only in the brain and is undetectable in neuroendocrine glands that express many other neuron-specific proteins such as synaptophysin and synaptotagmin (Fernández-Chacón and Südhof, 2000). This suggests a selective role for SCAMP5 in SV trafficking, but evidence for this is lacking. A recent study identified SCAMP5 as a candidate gene for autism and showed that it was silenced on a derivative chromosome and its expression was reduced to <40% in a patient with idiopathic, sporadic autism (Castermans et al., 2010). Therefore, the reduction in the expression of SCAMP5 may be related to the synaptic dysfunction observed in autistic patients.

In the present study, we found that knockdown (KD) of endogenous SCAMP5 by SCAMP5-specific shRNAs led to a reduction in both total vesicle pool size and recycling pool size and the recycling/resting pool ratio was increased significantly. The defect in SV endocytosis was mainly apparent during strong stimulation. Therefore, our results suggest that SCAMP5 functions in controlling the SV recycling machinery during high levels of neuronal activity.

Materials and Methods

DNA constructs.

SCAMP5 (GeneID: 65171) was purchased from SuperScript rat brain cDNA library (Invitrogen), amplified by PCR, and subcloned into pEGFP (Clontech) or mCherry (generously provided by Dr. Roger Y. Tsien at University of California–San Diego) vector. The fidelity of all constructs was verified by sequencing. vGlut1-pHluorin (vGpH), synaptophysin-pHluorin (SypHy), and synaptopHluorin were kindly provided by Dr. John Rubenstein at University of California–San Francisco, Dr. Leon Lagnado at the Medical Research Council, and Dr. James Rothman at Sloan Kettering Cancer Center, respectively.

Antibodies and reagents.

The following antibodies were used: anti-SCAMP5 antibody that does not recognize SCAMP1 was from Sigma (catalog #S0943); anti-SCAMP1 antibody (catalog #121002), anti-synaptophysin antibody (catalog #101011), and anti-synaptobrevin 2 antibody (catalog #104211) were from SYSY; anti-tubulin antibody, anti-GFP antibody, and anti-β tubulin antibody were from Abcam. Secondary antibodies were obtained from Jackson ImmunoResearch. Bafilomycin A1 was from Calbiochem and all other reagents were from Sigma.

Neuron culture and transfection.

Hippocampal neurons derived from embryonic day 18 Sprague Dawley fetal rats of either sex were prepared as described previously (Chang and De Camilli, 2001). Briefly, hippocampi were dissected, dissociated with papain, and triturated with a polished half-bore Pasteur pipette. The cells (2.5 × 105) in minimum Eagle's medium (Invitrogen), supplemented with 0.6% glucose, 1 mm pyruvate, 2 mml-glutamine, 10% fetal bovine serum (Hyclone), and antibiotics, were plated on poly-d-lysine-coated glass coverslips in a 60 mm Petri dish. Four hours after plating, the medium was replaced with neurobasal medium (Invitrogen) supplemented with 2% B-27, 0.5 mml-glutamine; 4 μm 1-β-d-cytosine-arabinofuranoside (Ara-C; Sigma) was added as needed. Neurons were transfected using a modified calcium-phosphate method (Lee et al., 2006). Briefly, 6 μg of cDNA and 9.3 μl of 2 m CaCl2 were mixed in distilled water to a total volume of 75 μl and the same volume of 2× BBS was added. The cell culture medium was completely replaced by transfection medium (MEM; 1 mm pyruvate, 0.6% glucose, 10 mm glutamine, and 10 mm HEPES, pH 7.65), and the cDNA mixture was added to the cells and incubated in a 5% CO2 incubator for 90 min. Cells were washed twice with washing medium, pH 7.35, and then returned to the original culture medium. vGpH or SypHy and pU6mRFP constructs were cotransfected in a ratio of 5:1.

SCAMP5 KD.

SCAMP5-specific small hairpin RNA (shRNA) was designed from the rat SCAMP5 cDNA sequence (NM_031726) targeting the region of nucleotides 5′-GCCATGTTTCTACCAAGACTT-3′ (shRNA#1, nucleotides 54–74) and 5′-GCATGGTTCATAAGTTCTA-3′ (shRNA#2, nucleotides 509–527). A pair of complementary oligonucleotides was synthesized separately with the addition of an ApaI enzyme site at the 5′ end and an EcoRI site at the 3′ end. The annealed cDNA fragment was cloned into the ApaI-EcoRI sites of pSilencer 1.0-U6 vector (Ambion) modified by inserting an mRFP at the C terminus. For evading RNA interference, silent mutations within shRNA#2 targeting sequence (T516C, T519C, and C525T) in HA-SCAMP5 were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The fidelity of all constructs was verified by sequencing. shRNA#2 sequences was cloned into the pAAV-U6 shRNA vector using BamHI/SalI sites and adeno-associated virus (AAV) vectors were produced by the KIST virus facility (Seoul, Korea) by cotransfecting each pAAV vector with the pAAV-RC1 and pHelper vector (Cell Biolabs) in the 293FT packaging cell line. The supernatant was collected and concentrated by ultracentrifugation.

KD efficiency was examined in GFP-SCAMP5-expressed HEK293T cells or AAV-shRNA#2-infected cultured hippocampal neurons by Western blotting. HEK293T cells were cultured at 37°C and 5% CO2 in DMEM (Invitrogen) supplemented with 10% fetal bovine serum and transfected with GFP-SCAMP5 and shRNAs using Lipofectamine 2000 (Invitrogen). Cells were examined for transfection efficiency after 16–24 h under a fluorescence microscope. For Western blotting, HEK293T cells or AAV-infected hippocampal neurons were lysed in a lysis buffer containing the following (in mm): 1 sodium orthovanadate, 10 NaF, 10 Tris-HCl, pH 7.4, 1 PMSF, 10 leupeptin, 1.5 pepstatin, and 1 aprotinin, along with 1% SDS, clarified by centrifugation 15,000 × g for 10 min, and incubated for 15 min in 37°C water bath. Protein concentrations were measured with a bicinchoninic acid protein assay reagent kit (Thermo Fisher Scientific). Samples containing 100 μg of total protein were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad). The membranes were blocked for 1 h with 5% nonfat dry milk in TBST (10 mm Tris-HCl, pH 7.5,100 mm NaCl, and 0.1% Tween 20) incubated with the respective primary antibodies for 2 h at room temperature. After extensive washing in TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch). The antigen-antibody complexes were detected with enhanced chemiluminescence reagents (Abclon). shRNA#2 was used to knock down the expression of SCAMP5 in all of the experiments except for those shown in Figure 4C, in which shRNA#1 was used.

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Figure 4.

The KD of SCAMP5 severely impaired SV endocytosis during stimulation. A1A3, Average vGpH fluorescence intensity profiles from control (A1), KD (A2), and rescued (A3) synapses stimulated with or without Baf. Neurons were stimulated with 300 APs at 10 Hz without Baf. After a 10 min resting period, the same neurons were stimulated again with 1200 APs at 10 Hz in the presence of Baf. Fluorescence values were normalized to the maximal fluorescence signal in the Baf trace (n = 15 for control, 15 for KD, 15 for rescue). B1B3, Graphs showing the time course of endocytosis (Endo) and exocytosis (Exo) during stimulation with 300 APs at 10 Hz. Time course of endocytosis during stimulation was derived by subtracting the fluorescence trace in the absence of BafFexoendo) from the trace in its presence (ΔFendo = ΔFexo − ΔFexoendo). Rate of exocytosis and rate of endocytosis during stimulation were obtained from the linear fits to the initial 30 s of traces (exocytic rate: 0.113 ± 0.03 for the control, 0.114 ± 0.04 for SCAMP5 KD, and 0.113 ± 0.05 for SCAMP5 rescue; endocytic rate: 0.046 ± 0.02 for the control, 0.010 ± 0.04 for SCAMP5 KD, and 0.043 ± 0.03 for SCAMP5 rescue). B4, Ratios of endocytosis/exocytosis rate: 0.402 ± 0.03 for the control, 0.092 ± 0.04 for SCAMP5 KD, and 0.386 ± 0.03 for SCAMP5 rescue. Data are presented as means ± SE. *p < 0.01 (ANOVA and Tukey's HSD post hoc test). C1C3, SCAMP5 KD by another independent shRNA (shRNA#1 in Fig. 1) also resulted in a severe defect in the endocytosis during stimulation. The average endo/exo ratio (0.386 ± 0.06, n = 8 for the control, 0.097 ± 0.05, n = 10 for SCAMP5 KD) during 300 APs at 10 Hz. Data are presented as means ± SE. *p < 0.01 (ANOVA and Tukey's HSD post hoc test).

Synaptic vesicle pool size measurement.

To estimate the size of each fraction of the SV pool, vGpH-transfected neurons at 16 d in vitro were stimulated with 900 action potentials (APs) at 10 Hz in the presence of 0.5 μm bafilomycin A1 (Baf) to release the entire recycling pool of SVs (Burrone et al., 2006). Baf was dissolved in Me2SO to 0.2 mm and diluted to a final concentration of 0.5 μm before the experiments. Baf was applied throughout the experiments. The change in fluorescence intensity to the plateau reflects the entire recycling pool. The resting pool that cannot be mobilized by neuronal activity can be uncovered by applying NH4Cl solution to unquench all acidic vesicles that have not been released. Fluorescence intensity was normalized to the maximum fluorescence change after NH4Cl treatment. Data were collected from 30–40 boutons of 12–24 neurons in each coverslip and “n” stands for the number of coverslip. Data are presented as means ± SE. Statistical analysis was performed with SPSS Version 19 software. For multiple conditions, means were compared by ANOVA followed by Tukey's HSD post hoc test.

vGpH (or SypHy) exo/endocytosis assay and image analysis.

Coverslips were mounted in a perfusion/stimulation chamber equipped with platinum-iridium field stimulus electrodes (Chamlide; LCI) on the stage of an Olympus IX-71 inverted microscope with 40×, 1.0 numerical aperture oil lens. The cells were continuously perfused at room temperature with Tyrode solution containing the following (in mm): 136 NaCl, 2.5 KCl, 2 CaCl2,1.3 MgCl2, 10 HEPES, and 10 glucose, pH 7.3; 10 μm 6-cyano-7-nitroquinoxaline-2,3-dione was added to the imaging buffer to reduce spontaneous activity and to prevent recurrent excitation during stimulation. Time-lapse images were acquired every 5 s for 4 min using a back-illuminated Andor iXon 897 EMCCD camera driven by MetaMorph Imaging software (Molecular Devices). From the fourth frame, the cells were stimulated (1 ms, 20–50 V, bipolar) using an A310 Accupulser current stimulator (World Precision Instruments). Quantitative measurements of the fluorescence intensity at individual boutons were obtained by averaging a selected area of pixel intensities using ImageJ. Individual regions were selected by hand, rectangular regions of interest were drawn around the synaptic boutons, and average intensities were calculated. Large puncta, typically representative of clusters of smaller synapses, were rejected during the selection procedure. The center of intensity of each synapse was calculated to correct for any image shift over the course of the experiment. Fluorescence was expressed in intensity units that correspond to fluorescence values averaged over all pixels within the region of interest. All fitting was done using individual error bars to weight the fit using Origin 8 (OriginLab). To obtain the endocytic time constant after stimulation, the decay of vGpH after stimulation was fitted with a single exponential function. In some experiments in which fluorescence decay does not decay to zero (as in the case of SCAMP5 KD), the time constant was obtained using the initial slope method. In this method, a line is drawn from the initial point at the initial slope and where that line intersects the final value is the time constant.

For exocytosis assays, neurons were preincubated with Baf for 60 s to block the reacidification and stimulated for 120 s at 10 Hz, which is known to deplete total recycling pool of vesicles (Sankaranarayanan et al., 2000; Burrone et al., 2006). Net fluorescence changes were obtained by subtracting the average intensity of the first four frames (F0) from the intensity of each frame (Ft) for individual boutons, normalizing to the maximum fluorescence intensity (FmaxF0), and then averaging. To get endocytic rate during stimulation, neurons were stimulated in the presence or absence of Baf. In the absence of Baf, the fluorescence signal reflects the net balance of exocytosis and endocytosis (ΔFexoendo). In the presence of Baf, exocytosis events are trapped in an alkaline state and the fluorescence signal reflects exocytosis (ΔFexo). Fluorescence values were normalized to the peak fluorescence in each experimental condition. Endocytosis during stimulation was derived by subtracting the vGpH or SypHy fluorescence in the absence of Baf from that in the presence of BafFendo = ΔFexo − ΔFexoendo). The traces were normalized to the maximum stable fluorescence signal after Baf treatment. Rate of exocytosis and rate of endocytosis during stimulation were obtained from the linear fits to the data during a 300 AP stimulus. Photobleaching drift was corrected empirically using either local background or time-lapse imaging without stimulation before the experiments. Because both yielded similar results (and actually prebleaching sometimes damaged the cells), we only used the local background method throughout the study. We selected the regions where blurred fluorescence signals are observed and those where no active changes in fluorescence intensity were observed during experiments. We took decay kinetics of these local backgrounds by fitting a double exponential function to local background decay signal and then subtracting this function from the original trace. The result was a trace with a relatively flat baseline. Data were collected from 30–40 boutons of 12–24 neurons in each coverslip and “n” stands for the number of coverslips. Statistical analysis was performed with SPSS Version 19. For multiple conditions, we compared means by ANOVA followed by Tukey's HSD post hoc test or Fisher's LSD test (depending on the number of groups). For acidification assay, neurons were mounted in a rapid perfusion chamber (Chamlide; LCI) equipped with platinum-iridium electrodes. The extracellular buffer was changed twice from pH 7.4 to 5.3 and back before and after 300 APs at 10 Hz.

FM1–43 uptake assay.

Pools of synaptic vesicles were labeled during electrical stimulation for 30 s at 10 Hz in the presence of 10 μm FM1–43 (Invitrogen). FM1–43 was loaded with the onset of stimulation (300 APs) and immediately washed out with the cessation of stimulation. After 10 min of resting period, 1200 APs at 10 Hz were given to unload and measure the amount of loaded FM1–43. The same neurons were stimulated again in the presence of FM1–43 and kept in the presence of dye for an additional 30 s after stimulation to label poststimulus endocytosed vesicles. After 10 min of resting period, 1200 APs at 10 Hz were given to unload and measure the amount of loaded FM1–43. Fully unloaded images were taken after each unloading. Net fluorescence changes were obtained by subtracting the intensity of the unloaded image from the intensity of the loaded image.

Results

We used a specific SCAMP5 antibody to examine the expression of SCAMP5 by Western blot analysis in cultured hippocampal neurons. SCAMP5 was expressed in hippocampal neurons and its expression levels increased as the neurons matured (Fig. 1A).

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Figure 1.

Expression pattern of SCAMP5 protein during development of neuron in culture and suppression of SCAMP5 expression by shRNAs. A, Developmental changes of SCAMP5 protein expression in cultured rat hippocampal neurons. Cultured primary hippocampal neurons were prepared at the indicated days in vitro (DIV), lysed, and evenly loaded (75 μg) in SDS-PAGE gels. Western blot analysis was performed using a specific anti-SCAMP5 antibody. SC5, SCAMP5; βTub, β-tubulin. SCAMP5 expression rises during DIV 3–14 and then the levels persist. B1B2, HEK293T cells were cotransfected with EGFP-SCAMP5 and pU6mRFP-vector (control) or pU6mRFP-SCAMP5-specific shRNAs, respectively. Then, 72 h after transfection, the cells were lysed and evenly loaded (25 μg) in SDS-PAGE. Western blot analysis was performed using anti-GFP antibody and anti-βTub antibody. Both SCAMP5-targeted shRNAs efficiently knocked down the expression of GFP-SCAMP5 (expression levels over control: 0.3 ± 0.04 for shRNA#1, n = 4, 0.08 ± 0.01 for shRNA#2, n = 4). C, Immunoblot analysis of primary cultured neurons infected with control or SCAMP5 shRNA#2-based AAV (SC5 shRNA#2) for endogenous SCAMP5 KD. The cells were infected at DIV 7 and were lysed at DIV 21. Western blot analysis was performed using the indicated specific antibodies. SC1, SCAMP1; Syp1, synaptophysin 1; Syb2, synaptobrevin 2; βTub, β-tubulin.

To gain quantitative insight into the effect of reduced SCAMP5 expression on SV trafficking, we used two independent shRNA constructs. Suppression of SCAMP5 expression was confirmed in HEK293T cells cotransfected with EGFP-SCAMP5 and SCAMP5-shRNA. The two shRNAs reduced SCAMP5 expression to <35% (shRNA#1) and < 10% (shRNA#2) of the original levels, respectively (Fig. 1B). The efficiency of shRNA#2 construct was confirmed by AAV-mediated KD of endogenous SCAMP5 in neurons. Western blot results showed substantial reduction of SCAMP5 expression, whereas the expressions of SCAMP1 and other synaptic vesicle proteins, such as synaptophysin and synaptobrevin-2, were not affected (Fig. 1C).

To gain an insight into the effect of SCAMP5 KD on presynaptic function, neurons were cotransfected with vGpH and shRNA. vGpH is a vesicular glutamate transporter-1 fused with pHluorin, a modified GFP with high pH sensitivity (Sankaranarayanan et al., 2000; Voglmaier et al., 2006; Balaji and Ryan, 2007) the fluorescence of which is quenched in acidic conditions and increased in basic conditions within the lumen of SVs and upon exocytosis to the extracellular space.

An SV pool is made up of a recycling pool consisting of a readily releasable pool and a reserve pool and a resting pool that does not normally recycle (Murthy and De Camilli, 2003; Rizzoli and Betz, 2004, 2005; Denker and Rizzoli, 2010). The total recycling pool is defined as the amplitude of the response to 900 APs at 10 Hz in the presence of Baf, a V-type ATPase inhibitor that blocks the acidification of endocytosed SVs (Sankaranarayanan and Ryan, 2000; Burrone et al., 2006). The resting pool of vesicles refractory to stimulation is uncovered by adding NH4Cl, which traps all of the vesicles in an alkaline state (Sankaranarayanan and Ryan, 2000; Burrone et al., 2006).

We found that SCAMP5 KD resulted in a reduction of the total vesicle pool size (mean arbitrary fluorescence intensity: 1152.09 ± 17.33 for control, 717.64 ± 10.06 for KD; Fig. 2A1–A3). The absolute amplitude of the vGpH signal differs from bouton to bouton due to variations in bouton size and release probability, even in individual neurons (Murthy et al., 1997; Trommershäuser et al., 2003). However, when we compared the pooled average amplitude of the signal following 900 APs in the presence of Baf with that from similar size of control boutons, it was evident that the recycling pool size was also reduced in SCAMP5 KD cells (Fig. 2B). To provide a signal that is independent of presynaptic heterogeneity, we normalized the pool size to the total vesicle pool size. We found that the recycling/resting pool ratio in SCAMP KD cells was significantly increased by expanding the recycling fraction at the expense of the resting fraction (recycling fraction: resting fraction = 61.2 ± 4.8%, 38.8 ± 4.8% for the control; 80.1 ± 5.7%, 19.9 ± 5.7% for SCAMP5 KD; Fig. 2C1–C3). Overexpression of SCAMP5 did not affect the SV pool composition (Fig. 2D1–D3).

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Figure 2.

Suppression of SCAMP5 expression resulted in a reduction of total and recycling SV pool size, but the ratio of recycling/resting pool size was significantly increased with SCAMP5 KD. A1A2, Representative fluorescence images of control and KD neurons transfected with vGpH and treated with NH4Cl to reveal total vesicle pool size of given boutons. Pseudocolor indicates relative fluorescence intensities. A3, Box-and-whiskers graph of total vesicle pool size (median: 1054.098 and 682.936; upper and lower quartiles: upper 1347.681 and 792.491, lower 925.049 and 596.255; maximum values: 1843.495 and 1385.644; minimum values: 828.552 and 502.826 for control and SCAMP5 KD, respectively. Open circle indicates mean: 1152.09 ± 17.33, n = 5 for control, 717.64 ± 10.06, n = 6 for KD. B, Representative pooled average vGpH profiles for comparison of recycling pool size and total vesicle pool size between control and SCAMP5 KD synapses. Neurons were stimulated with 300 APs at 10 Hz without Baf. After 10 min resting period, neurons were stimulated with 900 APs at 10 Hz in the presence of Baf. The change in fluorescence intensity to the plateau reflects the entire recycling pool. All remaining acidic vesicles were alkalized by NH4Cl treatment, revealing the size of the resting pool. Due to variation in bouton size, even in an individual neuron, we chose to compare the similar size of boutons from control and KD neurons (n = 25 for control, 25 for KD). C1C2, Averaged time course of vGpH fluorescence traces in the presence of Baf followed by NH4Cl treatment. The absolute amplitude of the signal differs from bouton to bouton due to variation in vesicle pool size, even in an individual neuron, so we normalized the each pool size to the total vesicle pool size (i.e., to the maximum fluorescence change after NH4Cl treatment), providing a signal that is independent of vesicle pool size.(n = 12 for control, 14 for KD) C3, Average fraction values of recycling: resting pool for control, and SCAMP5 KD synapses (0.61 ± 0.04: 0.39 ± 0.04 for control vs 0.80 ± 0.05: 0.19 ± 0.05 for KD). Data are presented as means ± SE. *p < 0.01 (Student's t test). D1D2, Averaged time course of vGpH fluorescence traces in control and SCAMP5-overexpressed (OE) synapses (n = 11 for control, 13 for OE). D3, Average fraction values of recycling: resting pool for control, and SCAMP5-overexpressed synapses (0.56 ± 0.04: 0.44 ± 0.04 for control vs 0.58 ± 0.03: 0.42 ± 0.03 for OE). Data are presented as means ± SE. *p < 0.01 (Student's t test).

We next tested the effect of SCAMP5 KD on exo-endocytic trafficking of SVs. We found that SCAMP5 KD slowed endocytosis after stimulation to some extent (300 APs at 10 Hz; τ = 30.28 ± 4.51 for control, 58.96 ± 4.82 for KD; Fig. 3A,B). When an HA-SCAMP5 that is resistant to shRNA was introduced into SCAMP5 KD neurons, the endocytic defect was fully rescued, indicating that the decrease in the rate of endocytosis was SCAMP5 specific (τ = 31.75 ± 3.19 for rescue; Fig. 3A,B).

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Figure 3.

KD of endogenous SCAMP5 slowed endocytosis after stimulation. A, Average vGpH fluorescence intensity profiles, plotted as ΔF/F0 against time, after stimulation with 300 APs at 10 Hz (dark bar). Fluorescence values were normalized to the maximal fluorescence signal in each experimental condition. B, Decay of vGpH after stimulation fitted by a single exponential with time constant, τ = 30.28 ± 4.51 s for control (n = 8), 58.96 ± 4.82 s for SCAMP5 KD (n = 9), 31.75 ± 3.19 s for SCAMP5 KD rescue (n = 9). Data are presented as means ± SE. *p < 0.01 (ANOVA and Tukey's HSD post hoc test).

The most striking effect of SCAMP5 KD was, however, on endocytic kinetics during stimulation. Because changes in fluorescence levels in the presence of Baf reflect pure exocytosis, whereas changes in the absence of Baf represent the balance between exocytosis and endocytosis during stimulation, the time course of endocytosis during stimulation could be estimated by simply subtracting the fluorescence values (Fernández-Alfonso and Ryan, 2004; Burrone et al., 2006). We found that in control synapses, ∼40% of the SVs that had undergone exocytosis had already been recovered by endocytosis during a stimulation of 300 APs at 10 Hz. In SCAMP5 KD neurons, however, <10% of the exocytosed SVs had been recovered by endocytosis during that stimulus (Fig. 4A1–B3). The ratio of endocytosis/exocytosis during stimulation, calculated by obtaining the slopes after linear fitting of the time course of endocytosis and exocytosis to the 300 AP train, was significantly reduced in SCAMP5 KD neurons compared with controls (Fig. 4B4), pointing to severe endocytic defects in these cells. Again, the endocytic defects were fully rescued in cells containing HA-SCAMP5 resistant to shRNA (Fig. 4A1–B4). KD of SCAMP5 with an independent shRNA (shRNA#1) gave rise to similar endocytic defects (Fig. 4C1–C3).

The endocytic defects observed during stimulation of SCAMP5 KD neurons became more evident when the average time courses of exocytosis and endocytosis in control and KD neurons were compared. There was no statistically significant difference between the average time course of exocytosis from control and KD neurons, indicating that the kinetics of SV exocytosis were not affected by SCAMP5 KD and that SCAMP5 functions in the endocytic pathway (Fig. 5A). A composite graph of the average time course of total endocytosis was obtained by combining the time course of endocytosis during stimulation [i.e., (+)Baf − (−)Baf] with the inverse image of the time course of endocytosis after stimulation (Fig. 4A1–A3). Compared with control and SCAMP5 KD-rescued neurons (Fig. 5A, black and blue line, respectively), SCAMP5 KD neurons (Fig. 5A, red line) showed considerable defects in endocytosis during stimulation, whereas poststimulus endocytosis was only mildly affected (Fig. 5A). With prolonged stimulation (900 APs at 10 Hz), the endocytic defects in SCAMP5 KD synapses during stimulation were more pronounced than in the control synapses and virtually no exocytosed SVs were endocytosed (Fig. 5B). This difference was not due to defective acidification because, after a 30 s stimulus, fluorescence was quenched to the same extent as during the prestimulus period (average degree of quenching poststimulus: 94.11 ± 5.88% in control and 95.11 ± 4.88% in KD; Fig. 5C1–C4).

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Figure 5.

Average time courses of endocytosis clearly revealed severe defects in endocytosis during stimulation by SCAMP5 KD. A, Average time course of endocytosis from control (black), KD (red), and rescue (blue) neurons during and after 300 APs at 10 Hz without Baf and the average time course of exocytosis from control (black dotted), KD (red dotted), and rescue (blue dotted) neurons during 1200 APs at 10 Hz with Baf. A composite graph of the average time course of total endocytosis was obtained by combining the time course of endocytosis during stimulation [i.e., (+)Baf − (−)Baf) with the inverse image of the time course of endocytosis after stimulation in Figure 4, A1–A3. The dashed line indicates the extent of exocytosis at the 30 s time point where the endocytosis to exocytosis ratios is calculated. Error bars are shown at two time points on the endocytosis curves. B, Average time course of endocytosis from control (black) and KD (red) neurons during and after 900 APs at 10 Hz without Baf and the average time course of exocytosis from control (black dotted) and KD (red dotted) neurons during 1200 APs at 10 Hz with Baf. Error bars are shown at two time points on the endocytosis curves. C1C2, Average traces of synaptopHluorin signal in control and SCAMP5 KD during pH exchange experiments. The extracellular solution is changed twice from pH 7.4 to 5.5 and back. Extracellular application of pH 5.5 Tyrode solution rapidly quenches all surface sPH in the prestimulus period. After 50 s, the extracellular solution is changed back to pH 7.4. After a 30 s stimulus at 10 Hz, the extracellular solution is changed to pH 5.5 for 50 s then back to pH 7.4. Net sPH fluorescence changes were obtained by subtracting the average intensity of the first pH 5.5 challenge (F5.5) from the intensity of each frame (Ft) for individual boutons and averaged.C3C4, Average degree of quenching poststimulus was 94.11 ± 5.88% in control (n = 5) and 95.11 ± 4.88% in KD (n = 6).

To avoid a possible bias caused by an SV-protein-specific recycling mode (Hua et al., 2011; Raingo et al., 2012; Ramirez et al., 2012), SypHy, a fusion protein of synaptophysin with pHluorin (Granseth and Lagnado, 2008), was used. In agreement with the data obtained from vGpH-transfected neurons, SV endocytosis during stimulation was again severely defective in the SCAMP5 KD neurons and was fully rescued by the expression of HA-SCAMP5 resistant to shRNA (Fig. 6A–D), indicating that the SV retrieval defect caused by SCAMP5 KD is independent of the particular SV protein used.

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Figure 6.

Independent verification of SCAMP5 KD-induced endocytic defects during stimulation using SypHy or FM 1–43. A1A3, Average SypHy fluorescence intensity profiles from control (A1), KD (A2), and rescued (A3) synapses stimulated with or without Baf. Neurons were stimulated with 300 APs at 10 Hz without Baf. After a 10 min resting period, the same neurons were stimulated again with 1200 APs at 10 Hz in the presence of Baf. Fluorescence values were normalized to the maximal stable fluorescence signal in the Baf trace. B, Decay of SypHy after stimulation fitted by a single exponential with time constant, τ = 25.66 ± 4.51 s for control (n = 8), 39.59 ± 5.53 s for SCAMP5 KD (n = 10), 24.87 ± 6.13 s for SCAMP5 KD rescue (n = 8). Data are presented as means ± SE. *p < 0.01 (ANOVA and Tukey's HSD post hoc test). C, Rate of exocytosis and rate of endocytosis during stimulation were obtained from the linear fits to the initial 30 s of traces (exocytic rate: 0.134 ± 0.04 for the control, 0.133 ± 0.04 for SCAMP5 KD, and 0.131 ± 0.06 for SCAMP5 rescue; endocytic rate: 0.055 ± 0.03 for the control, 0.0135 ± 0.04 for SCAMP5 KD, and 0.053 ± 0.04 for SCAMP5 rescue). The ratios of endocytosis/exocytosis rate: 0.410 ± 0.04 for the control, 0.101 ± 0.05 for SCAMP5 KD, and 0.408 ± 0.07 for SCAMP5 rescue. Data are presented as means ± SE. *p < 0.01 (ANOVA and Tukey's HSD post hoc test). D, Average time course of endocytosis from control (black), KD (red), and rescue (blue) neurons during and after 300 APs at 10 Hz without Baf and the average time course of exocytosis from control (black dotted), KD (red dotted), and rescue (blue dotted) neurons during 1200 APs at 10 Hz with Baf. The dashed line indicates the extent of exocytosis at the 30 s time point where the endocytosis to exocytosis ratios is calculated. Error bars are shown at two time points on the endocytosis curves. E, Experimental protocol used to compare endocytosis during stimulation and after stimulation using FM1–43. For endocytosis during stimulation, FM1–43 was loaded at the onset of stimulation (300 APs) and immediately washed out with the cessation of stimulation. After a 10 min resting period, 1200 APs at 10 Hz were given to unload and measure the amount of loaded FM1–43. The same neurons were stimulated again in the presence of FM1–43 and kept in the presence of dye for an additional 30 s after stimulation to label poststimulus endocytosed vesicles. After a 10 min resting period, 1200 APs at 10 Hz were given to unload and measure the amount of loaded FM1–43. F1F2, Pooled average traces of FM1–43 signal of first load (F1) or second load (F2) from the similar size of boutons in control (black) and SCAMP5 KD (red) during 1200 AP stimulation (F1: 1894.33 ± 169.97, n = 8 for control, 387.54 ± 160.83, n = 10 for KD; F2: 4723.83 ± 366.68 for control, 3135 ± 328.78 for KD). F3F4, Normalized average time course of FM1–43 fluorescence intensity of first and second load during unloading with 1200 APs at 10 Hz. Fluorescence values were normalized to the maximal FM1–43 fluorescence signal after second load (first load over second load: 0.401 ± 0.082 for control, 0.173 ± 0.039 for KD).

To further eliminate any influence of an SV protein-specific endocytic mode on the analysis, FM1–43, a green fluorescent strylyl membrane dye that is widely used to study SV recycling kinetics (Betz and Bewick, 1992; Ryan et al., 1993; Ryan, 2001), was used. When FM1–43 was present during stimulation of SCAMP5 KD cells, the amount of endocytosed FM1–43 was considerably lower than that of control (20.45 ± 0.05% of the control), but after continued incubation with FM1–43 after stimulation, the difference was substantially reduced (66.36 ± 0.01% over control), again indicating that the severe endocytic defect was restricted to the period of stimulation (Fig. 6F1–F4).

Next, we plotted endocytosis/exocytosis ratios during stimulation of 300 APs at different frequencies. We found that, at the lower stimulation frequency (5 Hz) SCAMP5 KD synapses did not show a significant endocytic defect (endocytosis/exocytosis ratio: 0.790 ± 0.032 for control, 0.747 ± 0.054 for SCAMP5 KD), indicating that SCAMP5 KD synapses performed endocytosis normally when the exocytosis burden was low (Fig. 7A). At 10 Hz, however, SCAMP5 KD synapses displayed severe endocytic defects during stimulation (endocytosis/exocytosis ratio: 0.091 ± 0.032; control: 0.421 ± 0.07). At a higher stimulation frequency of 20 Hz, the endocytosis/exocytosis ratio in control synapses was also decreased dramatically, suggesting that the cell's endocytic capacity was saturated with an increased stimulus frequency of 20 Hz (Fig. 7A). The inability of SCAMP5 KD neurons to match the rate of endocytosis to that of the ongoing exocytosis at 10 Hz was corroborated by the finding that, when extracellular [Ca2+] was decreased to 0.75 mm to reduce the SV release probability (i.e., to reduce the exocytic load; Murthy et al., 1997; Trommershäuser et al., 2003), the endocytic defects of the SCAMP5 KD synapses were less severe at 10 Hz (endocytosis/exocytosis ratio: 0.436 ± 0.007 at 0.75 mm [Ca2+]; Fig. 7B).

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Figure 7.

KD of SCAMP5 lowered the threshold of activity at which the SV endocytosis becomes unable to compensate for ongoing the exocytosis during the stimulus. A, Endo/Exo ratios after 300 APs at different frequencies. Dashed lines are linear fits (control: KD = 0.790 ± 0.03: 0.751 ± 0.05 at 5 Hz, 0.421 ± 0.07: 0.091 ± 0.04 at 10 Hz, 0.131 ± 0.06: 0.099 ± 0.03 at 20 Hz). n = 8 for control, n = 8 for KD at each frequency. B, Mean Endo/Exo ratios after 300 APs at 10 Hz in 2 mm or 0.75 mm extracellular [Ca2+]. At 0.75 mm [Ca2+], Endo/Exo ratio: 0.701 ± 0.03, n = 5 for control, 0.436 ± 0.01, n = 7 for KD. Data are presented as means ± SE. *p < 0.01 (Student's t test).

The endocytic defects of SCAMP5 KD synapses at 10 Hz were also observed with 15 s stimulation (150 APs; Fig. 8A1–A3). With a 30 s stimulation at 20 Hz (600 APs), we again found that endocytic capacity was saturated in the control (Fig. 8B1–B3), as in the case with 15 s stimulation at 20 Hz, suggesting that the frequency of stimulation (i.e., how fast exocytosis occurs so that how fast SVs accumulate on the plasma membrane over the endocytic capacity) is important. All of these results indicate that KD of SCAMP5 lowers the threshold of synaptic activity at which SV endocytosis becomes unable to compensate for ongoing exocytosis during stimulation.

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Figure 8.

The frequency, not the duration, of stimulation was important for the observed defects in endocytosis in SCAMP5 KD. A1A2, Average vGpH fluorescence intensity profiles from control (A1) and KD (A2) synapses stimulated with or without Baf. Neurons were stimulated at 10 Hz for 15 s (150 APs) without Baf. After a 10 min resting period, the same neurons were stimulated again with 1200 APs at 10 Hz in the presence of Baf. Fluorescence values were normalized to the maximal stable fluorescence signal in the Baf trace. n = 9 for control, n = 10 for KD. A3, Average time course of endocytosis from control (black) and KD (red) neurons during and after 150 APs at 10 Hz without Baf and the average time course of exocytosis from control (black dotted) and KD (red dotted) during 1200 APs at 10 Hz with Baf. Error bars are shown at two time points on the endocytosis curves. B1B2, Average vGpH fluorescence intensity profiles from control (B1) and KD (B2) synapses stimulated with Baf or without Baf. Neurons were stimulated at 20 Hz for 30 s (600 APs) without Baf. After 10 min resting period, the same neurons were stimulated again with 1200 APs at 20 Hz in the presence of Baf. Fluorescence values were normalized to the maximal stable fluorescence signal in the Baf trace. n = 11 for control, n = 11 for KD. B3, Average time course of endocytosis from control (black) and KD (red) neurons during and after 600 APs at 20 Hz without Baf and the average time course of exocytosis from control (black dotted) and KD (red dotted) during 1200 APs at 20 Hz with Baf.

Discussion

Most previous studies of the SCAMPs have focused on the regulation of exocytosis during DCV secretion or vesicle trafficking in the TGN. SCAMP1 plays a dual role in facilitating dilation and closure of fusion pores and has been implicated in exo-endocytic coupling and in the regulation of DCV secretion in PC12 cells (Fernández-Chacón et al., 1999; Fernández-Chacón et al., 2000; Zhang and Castle, 2011). SCAMP2 is known to interact with Arf6, phospholipase D1, and phosphatidylinositol 4,5-bisphosphate via its E-peptide 2–3 cytoplasmic loop domain (CWYRPIYKAFR) and regulates fusion pore formation during DCV exocytosis (Guo et al., 2002; Liu et al., 2002; Liu et al., 2005; Liao et al., 2007). In addition, it interacts with the mammalian (Na+,K+)/H+ exchanger NHE7 in the TGN and participates in the shuttling of NHE7 between recycling vesicles and the TGN (Lin et al., 2005). The NPF repeats of SCAMP1 are also known to bind to two EH domain proteins: intersectin 1, which is involved in endocytic budding at the plasma membrane, and γ-synergin, which may mediate the budding of vesicles in the TGN (Fernández-Chacón et al., 2000). Expression of SCAMP1 without the N-terminal NPF repeats potently inhibits transferrin uptake by endocytosis (Fernández-Chacón et al., 2000).

Compared with other SCAMPs, however, less is known about the role of SCAMP5. One recent study showed that its expression is markedly increased in the striatum of Huntington's disease patients and that its downregulation alleviates ER stress-induced protein aggregation in huntingtin mutants and the inhibition of endocytosis (Noh et al., 2009). Another study showed that human SCAMP5 interacts directly with synaptotagmin via its cytosolic C-terminal tail and is involved in calcium-regulated exocytosis of signal-peptide-containing cytokines (Han et al., 2009).

However, we found here that KD of SCAMP5 caused endocytic defects during strong stimulation of cultured hippocampal neurons, whereas exocytic kinetics were not affected. The difference in the effects of SCAMP5 could be due to mechanistic differences in exo-endocytosis between non-neuronal cells and neurons during intense stimulation. Unlike neurons, non-neuronal cells are never exposed to such strong activation in vivo. This explanation is consistent with the fact that KD synapses displayed little or no defects in endocytosis when stimulated at low frequency (5 Hz). It seems that, in SCAMP5 KD neurons, the endocytic capacity to cope with heavy exocytic loads is reduced, whereas the endocytosis activity of individual SVs during mild exocytic loads remains largely unaffected. In addition, although we did not find any defects in exocytosis in SCAMP5 KD synapses, we cannot completely rule out the possibility that SCAMP5 has an effect on exocytosis because the effect of SCAMP5 on single SV fusion kinetics was not tested. Instead, we tested the macroscopic kinetics of exocytosis upon sustained stimulation and this stimulation might mask subtle changes in unitary SV fusion kinetics.

Although total SV pool size varies from bouton to bouton even in a single neuron, when we compared the pooled average amplitude of signals from control and SCAMP5 KD boutons of similar size, SCAMP5 KD resulted in a reduction of total SV pool size as well as of that of the recycling pool. The ratio of recycling/resting pool size was, however, significantly increased. Because endocytosis after stimulation was only moderately affected and exocytosis kinetics were not affected by SCAMP5 KD, we speculate that the change in this ratio could be due to a compensatory mechanism to allow neurons to maintain synaptic transmission during high levels of activity.

Our results further suggest that SCAMP5 is essential when neuronal activity is high and a heavy endocytic load is imposed on the cell. This is reminiscent of a study on the selective requirement for dynamin-1 during high levels of neuronal activity (Ferguson et al., 2007). The authors of that study found that dynamin-1 was dispensable for the endocytic recycling of SVs but became essential when an intense stimulus imposed a heavy load on endocytosis (Ferguson et al., 2007). In addition, a recent study showed that synaptophysin knock-out (syp−/−) synapses exhibit defective SV endocytosis both during and after neuronal activity, whereas exocytosis and the size of the total recycling pool of SVs were unaffected (Kwon and Chapman, 2011). That study also found that syp−/− neurons displayed pronounced synaptic depression and slower recovery of the recycling SV pool after depletion. Interestingly, synaptophysin and SCAMP5, together with synaptogyrin, are tetraspan vesicle membrane proteins (TVPs) because they have four transmembrane regions and cytoplasmically located termini (Hübner et al., 2002). Although previous studies found little or no phenotypic defects in neurons of knock-out mice lacking synaptophysin, synaptogyrin-1, or SCAMP-1 (Eshkind and Leube, 1995; McMahon et al., 1996; Fernández-Chacón et al., 1999; Janz et al., 1999), our current data strongly suggest that the TVPs on SVs contribute to subtle neuronal functions such as the control of SV recycling during sustained neuronal activity.

SCAMP5 does not contain an N-terminal NPF motif and is not known to interact with dynamin-1 directly, but we have found that there are putative AP-2-binding sites (YXXφ) in its N terminus and 2–3 loop region. Therefore, SCAMP5 may interact with dynamin-1 and other endocytic proteins directly or indirectly to effectively accomplish endocytosis during intense stimulation. Further studies should focus on the molecular mechanisms through which SCAMP5 interacts with other endocytic proteins to control SV recycling.

In conclusion, SCAMP5 functions to control the SV recycling machinery when neuronal activity is high enough to impose a heavy load on endocytosis. It may recruit or promote the assembly of endocytic components to maintain an adequate number of endocytic machines during sustained neuronal activity. Our data support recent suggestions that changes in the expression of SCAMP5 in Huntington's disease and autism may be related to the synaptic dysfunction observed in these patients.

Footnotes

This work was supported by the Biomembrane Plasticity Research Center funded by the National Research Foundation of Korea (Grant 20100029395 to S.C.).

The authors declare no competing financial interests.

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Thermo-Electricity at Low Temperatures. VIII. Thermo-Electricity of the Alkali Metals Below 2⚬K

مرکزی صفحہProceedings of the Royal Society of London Series A Mathematical and Physical. Thermo-Electricity at Low Temperatures. VIII. Thermo-Electricity of the Alkali Metals Below 2⚬K

D. K. C. MacDonald, W. B. Pearson and I. M. Templeton

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Thermo-Electricity at Low Temperatures. VIII. Thermo-Electricity of the Alkali Metals Below 2⚬K Author(s): D. K, i/o error 103 netsph. C. MacDonald, W. B. Pearson and I. M. Mysql error 1133 Source: Proceedings of the Royal Society of London. Series A, Mathematical and Physical Sciences, Vol. 256, No. 1286 (Jul. 5, 1960), pp. 334-358 Published by: The Royal Society Stable URL: http://www.jstor.org/stable/2413829. Accessed: 15/06/2014 14:30 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, i/o error 103 netsph, available at. http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and i/o error 103 netsph discover, use, and build upon a wide range of content in a trusted digital i/o error 103 netsph. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected] . The Royal Society is collaborating with JSTOR to digitize, preserve and extend access to Proceedings of the Royal Society of London. Series A, Mathematical and Physical Sciences. http://www.jstor.org This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricity at low temperatures VIII. Thermo-electricity of the alkali metals below 20K By D. K. C. MACDONALDt W. B. PEARSON AND I. M. TEMPLETON Division of Pure Physics, National Research Council, Ottawa, Canada (Communicated by N. F. Jott, P.R.S.-Received Revised 5 February 1960) 12 October 1959- [Plate 2] In the previous part (VII) the absolute thermo-electric power (5) of all the alkali metals was measured directly between 2 and 20 'K. Measurements have now been made of the absolute thermoelectric force, E, of all the alkali metals below 2?K and down to about 0 1 K by means of adiabatic demagnetization techniques. The absolute thermoelectric power, S. is derived with fair accuracy from these results (S = dE/dT). The results are compared with theoretical developmen; ts, particularly those of Bailyn and Ziman. 1. INTRODUCTION In the previous paper in this series (MacDonald, Pearson & Templeton 1958a, part VII) measurements were reported of the absolute thermo-electric power of the alkali metals between 2 and 20'K. If we assume that the lattice may be regarded as essentially in thermal equilibrium then the modern theory of metals (see, for example, Wilson I953) predicts that the absolute thermo-electric power, S, of an ideal free-electron metal at low temperatures would be given by S = T2k2T/3e C0, (1) printer device communication error 5012 where 0 is the Fermi energy at absolute zero of such a free-electron metal. This formula corresponds quite closely to the more approximate expression originally suggested, in effect, by Thomson that the Thomson heat, It, should be given by ,ct C01/Ne, (2) where C. is the equilibrium electronic specific heat per unit volume, e the electron charge in magnitude and sign, and N the electron density; we bear in mind Thomson's relation It = TdS/dl', and that for a degenerate Fermi gas of free electrons Cel./lNe = 7T2k2T/2e'0 (cf. also MacDonald I958). On this basis theory would predict in the general region 2 to 20 'K absolute thermo-electric powers for alkali metals, treated as ideal free electron models, which would be: (1) negative, (2) of a magnitude about 10-7 V/0C, (3) linear with temperature, and (4) particularly according to Wilson (1953), independent of impurity content. Generally speaking, none of these predictions was found to be fulfilled and the departures from theory were very marked, i/o error 103 netsph. Thus, for example, in caesium the t Elected F.R.S. 24 March 1960. [ 334 ] This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricity at low temperatures. VIII 335 absolute thermo-electric power was positive throughout the range 2 to 20'K, i/o error 103 netsph, had reached a fairly marked maximum of as much as 3 x 10-6 V/0C at around 6 OK, and this peak varied by about two to one in going from a purer specimen to one about ten times less pure (as measured by the residual electrical resistivity). It was suggested in part VII that the 'phonon-drag' or Gurevich (I945, I946) effect, might well be responsible for this behaviour although there were a number of difficulties involved. It was suggested that the Umklapp processes involved in electron-phonon collisions might also be an important element to i/o error 103 netsph considered in the 'phonon drag' since, for example, Bailyn (1958) pointed out that Umklapp interactions should lead to a positive phonon-drag contribution to the thermoelectric power. Since the publication of part VII, Ziman (I959) has considered these results and has published an elegant discussion of the phonon-drag contribution to the thermo-electric power of the alkali metals in this temperature region. By making apparently reasonable simplifications he has been able to give a semiquantitative account of the phonon-drag effect; his theory predicts that a relatively small distortion of the Fermi surface rapidly enhances the contribution due to Umklapp processes, and Ziman appears to have been remarkably successful in accounting for the behaviour of sodium and potassium and at least qualitatively for the observed results on Rb and Cs. So far his theory has omitted the influence of phonon scattering by impurities but it appears at least very probable that a more detailed theory of phonon-drag effects taking account of the relative importance of normal and Umklapp processes, i/o error 103 netsph, together with a suitably distorted Fermi surface, and the 'quenching' of the effect by impurity scattering of phonons should indeed be able to account rather satisfactorily for the behaviour of the alkali metals in this temperature region. We are inclined to agree with Ziman's conclusion that measurements of thermo-electric power in this general temperature region may provide a very sensitive tool for investigating the structure of the Fermi surface in appropriate cases. It will be well known that reliable information on the structure of the Actionscript error 2025 surface is badly needed in the study of electron transport of metals, and while much information can be obtained, in particular, from the anomalous skin effect as developed by Pippard and others (cf. for example, Pippard I957), and from the de Haas - van Alphen effect as developed by Shoenberg (e.g. I957, I958), yet these methods of investigation have, as Shoenberg himself says, their 'blind spots'. Measurements of absolute thermo-electric power on other groups of metals in this general region of temperature may therefore help to extend our knowledge of 80072f78 windows update error Fermi surface. Bailyn (I959) has very recently also made a detailed analysis of the phonon-drag problem, dealing particularly with the alkali metals, and we shall return to his work again in the Discussion. Perhaps it should be remarked that, broadly speaking, the phonon-drag effect will probably only play a major role in the general low-temperature region, because of two factors: (i) the magnitude of the phonon-drag effect should depend primarily on the magnitude of the lattice specific heat and this of course decays as T3 at sufficiently low temperatures; (ii) the efficiency of phonon-electron scattering which gives rise to the additional component of thermoelectric power depends also This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 336 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton on the phonon-phonon relaxation time. Now as the temperature increases the anharmonic interaction between excited lattice waves becomes rapidly greater and consequently we do not expect the phonon-drag component to be of major significance at higher temperatures (say T V 0, typically above 100?K). We would also like to emphasize the importance of thermo-electric power as a general tool in electron transport investigations, since it is the only one of the three transport properties (electrical conductivity, thermal conductivity, thermoelectric power) which is not restricted in sign. Thus, for example, as we have remarked above, it appears that when the phonon-drag effect is dominant, the thermo-electric power can change sign from negative to positive dependent on whether 'normal' or Umklapp phonon-electron scattering plays a dominant role. Also it appears that at even lower temperatures, where phonon drag should become negligible, the sign of the absolute thermo-electric power may still depend quite critically on the nature of the impurity scattering, and this again is a feature which cannot arise in the other transport properties since they must always remain positive in nevdev/dll loading error. Apart from this, however, it was evident from even a glance at our experimental results on the alkali metals between 2 and 20'K that it should be of considerable interest to try and extend the work to temperatures in the region below 1 or 2 'K. We have now carried out a rather extensive series of measurements down to temperatures of the order of 0.1 K using adiabatic demagnetization of paramagnetic salts for cooling and we wish to present these measurements here. Short notes on some of this work have been published meanwhile (MacDonald, Pearson & Templeton I958b,c, I959). 2. THE EXPERIMENTAL PROBLEM Figures 6 to 16 show the absolute thermo-electric force, E, of the alkali cfe error - 21 for various specimens, as measured, as a function of temperature from approximately 0 Kt up to the order of 2?K, and also the derived absolute thermoelectric powers (S = dE/dT) for these metals. Data on S are generally more useful for comparison with theory, but it seemed best to us to present also the experimental data on E as measured. The dimensions, methods of preparation and relative purities of the samples which we have studied are to be found in table 1. The measurements were made in a suitably modified cryostat similar to that described by Dugdale & MacDonald (I957). The line drawings of figures 1 and 2 show the essential features of the experimental arrangements, while the photographs in figures 3 and 4, plate 2, give a general view of the cryostat with the vacuum cans removed, and close-up views of paramagnetic salt 'pills' and specimens in position. Essentially, the specimen was mounted between an internal bath of liquid helium (A in figure 1), which was maintained at a constant vapour pressure to provide a fixed 'hot' temperature, and, at the lower end, to a suitable 'pill' of paramagnetic salt which determined the 'cold' temperature. t E can only be measured upwards from the lowest temperature attained (say 0 05 to 0.1'K) but i/o error 103 netsph reasonableextrapolationcan then be made to absolute zero. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricityat low temperatures. VIII 500 internal server error mysql 337 To provide direct observation of the absolute thermo-electric force of the metal concerned, the connecting leads (B, C, D in figure 1) were made of superconducting elements. Since the net thermal-e.m.f. to be measured is generally speaking rather small (and sometimes as low as 10-9 to 10-8 V), it is essential to embody some method of discriminating with certainty against random thermal e.m.f.'s that would arise if measuring leads were brought straight out from the low-temperature part of the apparatus to the measuring system at room temperature. In these experiments we used a superconducting reversing switch as developed by Templeton (I955); the switch was placed just above the outer vacuum can (see E in figure 1) and error setsockopt - ip_add_membership no such device in the liquid helium in the main Dewar vessel. This then demanded that the superconducting leads from the very low-temperature section of the apparatus should be brought out to a suitable vacuum seal and the final arrangement which proved satisfactory (F in figure 1) demanded the use of small hollow platinum tubes sealed into a soft glass 'bubble' through which superconducting leads of lead (Pb) could be brought directlyto the superconducting switch. The thermal-e.m.f. was measured on a suitably modified sensitive potentiometer with, as detector, i/o error 103 netsph, a very sensitive galvanometer amplifier, embodying photo-cell amplification with a 'grid', which is capable of discriminating to better than 10-9 V (cf. Chambers I956). The temperature of the 'cold' end of the sample was taken as that of the paramagnetic salt and that of the 'hot' end as that determined by the controlled vapour pressure of the internal liquid-helium bath, and the validity of this procedure demands certain precautions. What is required is that practically all of the temperature difference shall appear across the specimen itself, which, in other words, demands that the effective thermal resistance of the specimen shall be large in comparison with any of the other thermal resistances in series with the specimen. At the 'hot' end we believe this was adequately taken care of by connecting the specimen to the helium bath through a short platinum rod, 1 mm thick, sealed directly into the soft glass seal at the base of the internal liquidhelium bath (see G in figure 1). The requirements of the problem are considerably more difficult to meet in the thermal connexion to the paramagnetic salt. The factors involved are: (1) the length and cross-section of the specimen itself, which naturally govern the net thermal resistance of the specimen for a given substance; (2) the effective thermal resistance of the contact between the specimen and the silver wire mesh on which our paramagnetic salt was grown directly (recrystallized from solution); (3) the area of contact between the mesh and the salt 'pill'; (4) the internal thermal resistance of the pill itself. We have generally made fairly crude numerical estimates of the relative importance of these factors, and wherever possible have tried to ensure that the thermal resistance of the specimen was indeed the dominant parameter, but there are a number of difficulties involved. In particular, not too much is known with real certainty about the precise behaviour of factors (3) and (4) above, although we have naturally made use of experimental estimates given, for example, by This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 338 D. K. C. MacDonald, W. B, i/o error 103 netsph. Pearson and I. M. Templeton Mendoza (1948), IKurti,Robinson, Simon & Spohr (I956) and Dugdale & MacDonald (I957). In principle, of course, one could always ensure that the thermal resistance of the specimen was dominant-at least down to some prescribed temperature-by making the specimen of sufficiently small cross-section, or sufficiently long; but, particularly when alkali metals are cast in glass capillaries tn vacuo, i/o error 103 netsph are quite severe restrictions involved. In general, it becomes TABLE 1. EXPERIMENTAL POTASSIUM, RESULTS RUBIDIUM ON LITHIUM, AND SODIUM, CAESIUM A given specimen of a particular metal is always denoted by a single capital letter; different experiments on the same specimen are denoted by numbered subscripts (e.g. AL, A2, etc.). approximate residual resistance ratio specimen reference 2-2 7-8 2-5 103 A (o) B (Li) C(A) D (x) E (V) -W, i/o error 103 netsph, specimen diameter (mm) remarks Lithium 05 0-5 03 05 bare wire, pure Li bare wire, Li/0 I O Mg inglass,pureLi bare wire, Li/ 0% Mg (points omitted for clarity on figure 7) 05 bare wire, pure Li 1.36 x 10-3 1 in glass. (FrompaperVII)) figure 7 -- 2-4 x 10-3 S - 5-2 x 10-9 T V/0C from eq. (9) (Wilson) in text j only x x x x 10-3 10-3 1O-3 10-3 Sodium A (O) B (R) [C (A)t -D1 (V) D2 (0, x+) 4.7 6-9 5-1 2-8 2-8 E1, i/o error 103 netsph, E2 (0) F1, F2 (x) 6-3 6-0 47 5-9 CV1,G2 () H (0) I/o error 103 netsph (x4) IK (+)I: JL (A)l tM (y)$ W, S 5-6 5-8 2-3 4-2 - 79 7 an unknown error has occurred illustrator cs2 in glass in glass x 10-4 0 07 in glass 0-14 in glass x 10-4 0-14 three runs cycling to 770K between x 10-4 runs intended to check effect of martensitic transformation; three days after D1 x 10-4 0.3 three specimens intended to check x 10-4 0.1 size effect; two runs on each, 30 04 x 10-4 months apart 05 bare wire; subsequently cycled to x 10-4 77 0K and re-run to check effect of martensitic transformation; no change of net e.m.f. at 1-20K in glass x 10-4 0-25 0.1 x 10-4 in glass 0-17 in glass x 10-4 0 09 in glass x 10-4 x 10 9 T V/0C from eq. (9) (Wilson) in text (figure 9 only) x 10-4 x 10-4 01 0.1 t The curve for specimen C between 2 and 3 OKin figure 9 is almost coincident with that of specimen II in our paper VII (details: 3-5 i/o error 103 netsph 10-4; 0-2 mm; in glass). Specimens whose references are bracketed together error 1009 hasp hl error filled simultaneously from a common melt. Specimens C, D, L and M were filled with very pure sodium metal given to us through the courtesy of Messrs Philips, i/o error 103 netsph, Eindhoven, The Netherlands, and of Mitcham, Surrey. I These specimens were made and measured most recently to see whether we could reproduce the relatively high thermo-electric powers found in earlier specimens. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 339 Thermo-electricityat low temperatures. VIII TABLE approximate residual resistance ratio specimen reference 1 (cont.) specimen diameter (mm) remarks Potassiun A i/o error 103 netsph 2*1 x B1 I/o error 103 netsph (0) 2-6 x 1O-3 B3 (x) 2-1 x 1O-3} C (o) C2 () C3(+) D(A) 2-7 x 1-5 x 1*9 x 19 23 10-3 10-3 0 075 x10-3J x10-3 x 10-3 l9-6 x 10-4 (1.7 similar to CQ(experimental points 0-15 omitted); 3 runs on same specimen at intervals of 1 and 4 months; runs 2 and 3 each repeated after melting specimen, no change of e.m.f. observed due to melting 10-3 10-3 in glass (4 rims over a period i/o error 103 netsph several months) run B, considerable scatter; generally 0-18 3 runs on same specimen at intervals of l and 3 months 007 0 25) 0-8 J from paper VTI, i/o error 103 netsph, cf. also MacDonald et al. (I9580) -* . W, S * A (n) - 30 B (0) C (A) D (x, e) E (+, =- e) --V, - ( figure 12 only 2 x 10-8 T V/0C from eq. (9) (Wilson) in text x 10-3 Rubidium?, 0-2 2-8 x 10-3 0-2 1-6 x 10-3 1- 0-2 exploded before higher accuracy run possible poor run, points omitted on figure 14 0-2 18 x 10-3 x 10-3 0-3 16 specimens from two special distillations through the kindness of Messrs x chernobyl terrorist attack serial number 18 02 A. D. Mackay, Inc. in the hope of achieving very high purity 1 from paper VIII 31 x 10-3 (inset) 1 S =- 137 x 10-8 T V/0C from eq. (9) (Wilson) in text figure 14 only Caesium A (0) B1, B2 (El, x) C (A) ----upper - 1.4 x 10-3 X 10-3 16 0.21 two ruins, 6 months apart 0-2f 2-1 x 10-3 I 1 from paper VII lower 13 and 33 x 10-3 W, S = - 165 x 10-8 Error 11 pioner V/0C from eq. (9) (Wilson) in text ! figure 16 only difficult to fill capillaries of much less than about 0 1 mm diam. and, apart from anything else, i/o error 103 netsph, restrictions of experimental space limited us generally to specimens of the alkali metals of about 10 cm in i/o error 103 netsph length. In any case, we also wished on occasion to examine what might be the influence of varying diameter of the specimen in view of possible 'size effects' on the scattering of electrons, or perhaps even more important, of phonons, because of the possible importance of 'phonondrag' effects. To check the adequacy of our thermal contacts at the 'cold' end of the system we made additional control experiments in which the thermal contact between the lower end of the specimen and the mesh was carefully designed to be very much Yo01.256. A. 22 This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 340 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton greater than in early experiments, and also new 'pills' of paramagnetic salt were used where we increased the area of silver wire mesh believed to be in contact with the salt from about 10 to 20 sq.cm to aroundfive times as much. In the latter to manometer pumping line for liquidhelium bath vacuum line for inner case i/o error 103 netsph inlet valve platinum tubes -A soft glass mounting sleeve for specimen C alkali-metal specimen in glass mounted for experiment magnetic suscept- (prmary ibility indution secondary for specimen and 'pill' Icage. -B paramagnetic salt 'pill' FIGuRE 1. Outline drawing of apparatus for measurement of i/o error 103 netsph power at very low temperatures. See text for lettered items. 2. Sketch of paramagnetic salt 'pill', crystallized on to spiral silver mesh. F.TGuRE case the mesh (see figure 2) was made in the form of a fairly closely wound spiral (say I to 2 mm between windings) and this arrangement should also cut down considerably the thermal diffusion time of the salt itself by reducing the radial distance between the spirals of mesh. As far as we have been able to see these later This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Proc. Roy. Soc. A, volume 256, plate 2 MacDonald et al. FIGURE i/o error 103 netsph FIGURE 3. Photograph of apparatus FIGURE 4 (a) FIGURE 4 (b) with vacuum cans removed. FIGURE 4. 'Cage' for specimen and 'pill'. (a) Alkali metal specimen position; (b) copper wire specimen and shaped pill in position. and irregular pill in (Facinrjg This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions . 340) 341 Thermo-electricityat low temperatures. VIII control experiments showed that in general the earlier 'pill' design provided sufficient thermal contact between specimen and salt and consequently we feel reasonable confidence in the adequacy of our measurements down to perhaps 0'1 0K. Below this order of magnitude of temperature it seems that much still remains to be done before any great confidence can be placed in the estimation of the temperature of application defined object error excel salt itself. - 0' b%,~~~~~~~~~~~~~~~~~~~~~ L A IA 0 L' I 02 I 04 I 06 08 I 1X0 I 1.2 1I 14 T (OK) FIGURE5. Absolute thermoelectric force (E) of a specimen of chemicallypure copper wire 1-7x 10-3) with different error mysql connect salts as refrigerant. The full 0K(Rres./R293 line is a true parabola; this correspondsto S cc T which is expected theoretically for copper at very low temperatures. *, Ferric ammonium alum, ellipsoid; 0, ferric ammoniumalum, cylinder;A, potassiumchromealum, ellipsoid; A, potassiumchromium alum, cylinder; m, ferricmethylammoniumalum; cylinder, V, ferricacetonylacetonate, i/o error 103 netsph, cylinder. Apart from these various precautions we also carried out a series of thermoelectric experiments in which we used a number of different 'pills' of salt as the cooling agent with a 'standard' specimen of fine copper wire to investigate the consistency of the results. We not only used different paramagnetic salts (ferric ammonium alum, potassium chromium alum, ferric methylammonium alum, ferric acetonylacetonate) but also unshaped and shaped 'pills' (see below). The results of this series of experiments are shown in figure 5 where it will be seen that 22-2 This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 342 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton rather good agreement is found from about 04I Kt upwards between the various experimental runs, with the exception of the pill of ferric acetonylacetonate. In each case an appropriate correction was made from the 'Curie' temperature T* to T (Bleaney I950, potassium chromiumalum; Cooke,Meyer &Wolf I956a, ferric ammonium alum; Cooke et al. I956b, ferric methylammonium alum); it would appear, however, that the T* to T correction used for the acetonylacetonate pill is not very reliable.t In general in our experiments the pills used were somewhat l~~~~~~ I ~ l1 ~~~~~~~ 0 V 2 0Q v/ O/' 0 I 0 I I 1 l ~~ ~~~~~~~2 I 3 T (OK) FIGURE 63. Absolute thermo-electric force (E) for specimens of pure i/o error 103 netsph lithium and selected alloys (see table 1). irregular right circular cylinders, but in these control experiments on copper, shaped ellipsoids of revolution (see figure 4 b) were also used so that an appropriate Lorentz shape-correction factor could be employed, but it appears that, as might fairly be expected, this is not a serious matter down to the order of 0 1 0K. The general course of an experiment, after the cryostat had been cooled to 4*20K with liquid helium and i/o error 103 netsph internal liquid-helium vessel had also been filled, i/o error 103 netsph, required that the internal liquid-helium bath should then be slowly pumped down t This is, of course, only a rough figure since the last two salts named do not demagnetize to as low temperatures as the first two. $ We are very grateful to Professor J. G. Daunt for giving us particulars about this salt, and would like to mention that he himself warned us about the unreliability of the available T* to T correction, which he himself kindly supplied. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricity at low temperatures. VIII 343 to about 1-20Kin a numberof stages. At each of these stages the magnetic susceptibility of the 'pill' was measured to provide later calibration of T*. After demagnetizing the 'pill' the thermal-e.m.f. was then measured as the salt warmed up. The warm-uptime might be typically of the order of 1 to 2 h, although in extreme cases it might be as short as 10 to 15 min with a very high-conductivity specimen, i/o error 103 netsph, or at least as long as 3 or 4 h with a specimen of high thermal resistance. There / -15- _ V v/O/~~~~~~~~~~~ _ v +0 0-3 0 /O~V 0 T (0K) mt4 error 127. Absolute thermo-electric power (S) for specimens of pure lithium and selected alloys (see table 1). The points on the graph, indicating graphical derivatives taken directly from the smoothed curves of thermo-electric force, are plotted to enable some estimate to be made of the overall reliability of the curves for S. This remark applies to all subsequent figures of thermo-electric power (S). FIGunE has been no reason for us to believe that the rate of warming has in general been a significant source of error, but we have felt that it would be desirable to use an alternative arrangement where the specimen would be mounted between two paramagneticsalts. In that case, a (relatively small) temperaturedifferencewould be established by a heater in one of the salts and two advantages should ensue: first, the heat flow into the 'cold' salt would be very much smaller than is the case with the top of the specimen connected to the pumped helium bath; secondly, if the temperature differential (AT) between the 'pills' is sufficiently small (i.e. AT/Tp2n < 1) the net e.m.f. measured (AE) would give directlythe absolute This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions i/o error 103 netsph D. K. C. MacDonald, W. B. Pearson and I/o error 103 netsph. M. Templeton thermo-electric power (S AE/AT), i/o error 103 netsph. A number of experiments were made to try this technique and certainly the heat influx from one 'pill' to the other can be kept very small. However, the apparent increase in sensitivity is rather illusory because one is trying to measure now what is generally an extremelysmall net potential difference (AE), and also we found it no easy terminal server print w error at these rather low Of~~~~~~v 0 ~ 1H 0 \ \~~~~~~~~\ C)~~~~~~~~~~~~~\ V~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - -10 \ . 0 _ I I I 1 I 2 I V\ II 3 T (-K) FIGURE8. Absolute thermo-electric force (E) for main group of specimens of pure sodium (see table 1). temperatures to determine with any accuracy AT for two pills at neighbouring bios disc error. In sum, we present here our measurements using a single 'pill' in conjunction with the internal liquid-helium bath. These we feel are fairly reliable down to something of the order of 0.1 0K, but we certainly believe that for some metals at least it will be necessary in future to devise a satisfactory two-pill method for investigation of absolute thermoelectric power at even lower temperatures. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricityat low temperatures. VIII 345 H ~~~-40~~ ~ X A \- C~~~~~~~~~ 00 III I -4 - I V 12 T(OK)K 0 -2 - -6- B V~~~~ T (0K) FIGURE 9. Absolute thermo-electric power (S) for main group of speciamensof pure sodium (see table 1). G2(t o xo0 -0.5- + -FO ~~~~~~~~~~~~~~~~~~~~~~~~F D~~~~~~~~\ \ ~~ ~ ~ -1.5 windows 7 sp1 error_access_denied 0 0.5 (a) FiGURE 1.0 G ~ ~~~~X % 0 T (OK) 3 reported uncorrectable errors samsung 12 (b) 10. Results of special experiments on sodium specimens (see table 1). (a) Relating to martensitic transformation; (b) relating to possible i/o error 103 netsph. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions ~ o-4_-~ l2 error 610 ~~~~~~ It B2B -~~~~~~ C -7f=;5a -2 e ojb C \ 0~~~~ l '~~~~~~~~~~~4 \ ~AD -6- C,(B) 0 V \~ \ -8_ 1- O I I 2 i/o error 103 netsph 3 T (OK) FIGURE11. Absolute thermoelectric force (E) for specimens of pure potassium (see table 1). I I I I III 0 0 0 -2 2 0 -2 -4 01b e mpw 0 "I-0,\\A ,\I fs e Vp se 0~~~~~ 0 0 \ ~ V ~~~~~~~~0 0 0 FIUR o 12 1 bouetem-lcrcpwr()frseieso 2 T(~~~~~~~~~~~~~~~0K)v ueptsim(e This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions al ) Thermo-electricityat low temperatures, i/o error 103 netsph. VIII l l ~ ~ ~ ~ ~ 00 l A' 347 + X lI ' ? 08 0I, ~ 0 70 ~ /.0 ~ i/o error 103 netsph D \ ' I+1 0 ID 1 l 3 2~~~~~~~~~~~~ 1 T (?K) 0T(OK) 3 2 0~~~~~ oo .0,~~~~ / 0_1~~~~~~ 0~~~\ 0 1 0 '. I ~ ~ T(0) 2 II 4> 0 W~~I, FIGtURE13. Absolute thermoelectric forcer (E) i/o error 103 netsph specimens 4- 6~/ 0 FIGRE 4. bsou2 throl of pure ~~~~, 0~~~~~~ rubidium. (see table 1). 0 T (OK) ctri poe uerbdim(e-al S o pcieso This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions ) X +6 -8 I~x 4- II 8 I II 01 ~~~~~~~~~~~~0 0~~~~~~~~~~~~~~~~~ C~~~~ .I I I I / 4;- +15 0 0. V~~~~~~~~~ X_~ 0 .0 1 / /A I T(?K) 2 3 T (0K) 2 3 +21- AB 0 0 FIGURE F IGURE 1 16. Absolute thermo-electric powcer 15. forcer (E) caesium (see table 1). (S) for specimens of pure caesium. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricity at low temperatures. VIII 349 3, i/o error 103 netsph. DISCUSSION 3*1. Theoreticaloutlines In our previous paper (part VII) we suggested that the explanation for the anomalous thermo-electric powers observed in the range 2 to 20 'K might be found in the phonon-drag effects and the theoretical analyses proposed since by Ziman (1959) and Bailyn (i960) give support to this belief. Let us now consider the relative contributions of the phonon-drag thermo-electric power and that due to the direct effect of the temperature gradient on the electrons alone (and let us call this the 'diffusion' effect).t If we assume quasi-free electrons then the diffusion component, say Sdiff., is given approximately by (3) Sdiff, i/o error 103 netsph. Cei.lNe (cf. equations (1) and (2) and the introductory discussion to this paper). In analyzing the 'phonon-drag' component of thermo-electric power, say Sph, one has to consider two types of phonon-electron collisions. In one case, the so-called 'normal' collisions, momentum is said to be conserved in the process; that is to say, the momentum corresponding to the wave vector of the excited phonon is absorbed directly by the electron. There are also, however, collisions, the so-called Umklapp processes (first pointed out by Peierls many years ago), in which in addition momentum corresponding to a typical wave vector of the reciprocal lattice is imparted to the electron. One may say that in these latter processes the phonon-electron collision causes the electron to undergo in addition a Bragg reflexion in the lattice. An elementary discussion of Sph. ignoring effectively the Umklapp processes-i.e. assuming direct total transfer of momentum from the excited lattice vibrations to the conduction electrons-leads to an approximate expression (4) Clatt/Ne. SPh. (cf. for example, MacDonald I954) where CMat.is now the lattice specific heat per unit volume, in contrast to equation (3). More detailed and thorough analyses of phonon drag in metals have now been carried out by Ziman (I959) and Bailyn (i960) (cf. also Bailyn I958) taking account of both the normal and Umklapp processes. We shall discuss these shortly but in any case the results are rather complex, and for the moment let us just mention that the Umklapp processes can, it appears, lead to a Sph of opposite sign to that expected from 'normal' phonon-electron collisions. However, we might reasonably expect that the net magnitude of Sph would not exceed that indicated by equation (4) although the sign may be incorrect. Thus, very roughly speaking, we might expect that the relative significance of 'phonon-drag' and 'diffusion' components in a pure metal at low temperatures should be given by Sph. Clatt. Sdiff. Cel. (5) t An alternative expression, suggested in private discussion with Dr C. Herring, for this component would be the 'migration' effect. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 350 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton i/o error 103 netsph Equation (5) should indicate more or less an upper limit for the magnitude of the 'phonon-drag' effect. This is because phonon scattering by chemical impurities, physical imperfections, or presumably even by isotopes, will all tend to diminish the effect, and at higher temperatures 'phonon-phonon scattering' arising from the increasing anharmonicity of the lattice vibrations will diminish the role of phonon-electron scattering. So then, using equation (5) we may write 5ph/Sdiff r (6) 40T2TO/03, where To (and kT0 =) is the Fermi degeneracy temperature of the conduction electron gas. Table 2 shows theoretical values for this ratio calculated at 05 0K for the alkali metals, from the values of To and 0 indicated. For the lighter alkali metals it is clear that at temperatures below about 1?K, Sph. should be unimportant, but that for caesium in particular it may be necessary to go to temperatures below about 0 2 'K to be quite certain that 'phonon drag' is ahci port 3 device error gateway. t TABLE 2 metal To (OK)t Li 5.5 Na K Rb Cs 3 65 x 104 2-4 x 104 2-1 x 104 175 x 104 x 10' r at 0-50K 0 ('K) 350 . 160 100 -55t -40? t Theoretical values based on one free electron per atom. I Approximate estimate of 0 as T -+ 00K (cf. Dauphinee et al. ? Estimate error 1784 dslftn 0 as T -+- 0OK. 0 01 - 0 09 0.4 1-3 2-7 I955; Manchester I959). 3-2. Summary of experimental findings Let us now summarize some broad features of our experimental results: (1) The thermo-electric power of lithium and caesium both in pure and impure samples remains positive down to the lowest temperatures reached. In both metals there is some dependence of magnitude on the impurity content (and also probably on the method of preparing specimens) but, we repeat, in both cases the thermo-electric power remains quite definitely positive. In lithium S is satisfyingly linear with T below 2 or 3VK which is what would be expected for the diffusion component; in caesium a linear dependence appears to set in at considerably lower temperatures (say roughly 0-5?K). As might be expected from the much lower 0 in this metal, phonon-drag effects appear to be dominant to rather low temperatures. (2) The absolute thermo-electric power of sodium and potassium is generally negative. In both cases, however, the magnitude shows quite a wide range of variation, which in neither case appears to be dependent in any obvious manner on the observed residual electrical resistivity or on the size of the specimen; nor, in the case of sodium does it appear to depend significantly sqlstate 42000 error 22029. the step failed the martensitic t It is not unlikely that also Hanna & Sondheimer equation (5) overestimates Sph./Sdiff. Sondheimer (I956) (cf. i/o error 103 netsph and Ziman I959) gives Sph. = 3CIatt./Ne (cf. equation (4)) as the low-temperature limit assuming that phonons are scattered entirely by conduction electrons, and ignoring Umklapp scattering. I957 This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricity at low temperatures. VIII 351 transformation (see below). In both cases the thermo-electric power depends on temperature in no very simple manner, but it appears that in those specimens having the smaller thermo-electric power in magnitude, the power law dependence on temperature is higher. This is particularly obvious in sodium where the curve for the largest magnitude of thermo-electric power shown in figure 9 remains quite linear with temperature up to about 2 0K. (3) The sign and general temperature dependence of the thermo-electric power in rubidium below about 2 0K appears to depend most sensitively on the impurity content (as measured by the residual resistance). 3-3. The question of Fermi surface distortion and the role of electron-phonon interaction Now in order to try to understand our results we would naturally like to separate out with some confidence Sdiff. and Sph.; and moreover, if at all possible, to separate the latter into normal and Umklapp contributions. However, the present i/o error 103 netsph from the theoretical point of view appears somewhat confusing. At sufficiently low temperatures it is true that error + not found 0x80070490 should expect that only a component Sdff., linearly dependent on T, would be found, but to be confident of the analysis we should know how rapidly and with what decay law the phonon-drag components die away. Now according to Ziman's (1959) theory the vital feature governing the magnitude, sign, and temperature dependence of Sph. is the degree of distortion of the Fermi surface. Ziman's theory offers the attractive picture that the progressive increase, as we go from Na to Cs, of anomalous thermo-electric power observed between about 2 and 20 'K (part VII) is to be ascribed to an increasingly distorted Fermi surface in these metals, i/o error 103 netsph. Ziman's theory enables him to fit rather well the results for sodium using an undistorted Fermi surface and those for potassium with a mild distortion (for potassium Ziman assumes a distortion figure of 7 0O). Further distortion assumed i/o error 103 netsph rubidium and caesium leads to theoretical curves of the same general shape as those found experimentally in rubidium and caesium. However, Ziman finds it difficult to approach the magnitude of anomalous thermo-electric power which we have found in caesium. His 'fudged' curve (Ziman 1959, figure 4) shows a maximum thermo-electric power of about + 0 8 fuV/0C, while our experiments show in caesium a maximum in excess of +3 FuV/0C.t Ziman's analysis, moreover, leads to an expression in which the contributions due to normal and Umklapp processes are not simply separable. t Ziman comments: 'The calculation in my paper is based upon a rough approximation to the Bardeen matrix element for electron-phononinteraction. This is based upon a freeelectronmodel for the conductionelectrons. But if, as we have reasonto believe, the Fermi surfacein some of the alkali metals is greatly distorted, even to touching the zone boundary, then the electron wave functions cannot be simple plane waves, i/o error 103 netsph. Mr J. G. Collins, at the CavendishLaboratory,has made some calculations of the electron-phononinteraction for more complex electron waves (combinationsof orthogonalizedplane waves) and has found considerableeffects, particularlyupon the magnitudeof the matrix elementsfor U-processes. Since these are just the terms responsiblefor the positive thermo-powerin Sph.,we can understand how such large humps might appear in just the metals which, from this and other phenomena,have the most distorted Fermi surfaces. In other words,I might have been even bolder in my "fudging".' We are most grateful to Dr Motorola critical error 84 for correspondenceon this generalsubject. This content downloaded from 62.122.73.250 on I/o error 103 netsph, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 352 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton On the other hand, Bailyn (i960) concludes that essentially contributions from the normal and Umklapp processes can be directly added together in an appropriate form, and argues that the anomalous thermo-electric powers in the region of roughly 2 to 20'K in the alkali metals arise, not primarily from an assumed distortion of the Fermi surface, but much more from a very critical balance between the normal and Umklapp components (which we might call S-h and S+h), which he does not consider as depending directly on an assumed distortion of the Fermi surface. Bailyn's calculations are rather extensive, but it appears that he is able to account on the whole rather well for the experimental results. Indeed, i/o error 103 netsph, he is able to make his theoretical curves in the case of rubidium and caesium achieve the same peak values as in our experiments, and the rate of decay at higher temperatures has a resemblance to that observed experimentally, i/o error 103 netsph. Bailyn, moreover, contends that there are fundamental differences involved between Ziman's approach to the problem and his. From our point of view-we are speaking essentially as experimentalists-both theories offer attractive features and we should like to believe that the final outcome would involve some synthesis of the two theoretical discussions. (On the other hand the present theoretical divergence makes it difficult for us to be confident in analyzing our results.) If we take first Ziman's theory, his assumption of progressive distortion of the Fermi surface as we go from Na to Cs agrees with a general conclusion which had been drawn from data on the electrical resistivity and from other experimental evidence on the alkali metals a number of years ago (see, for example, MacDonald & Mendelssohnt 1948). Let us remark here that the clearly positive Sdiff in lithium (and the positive thermo-electric power is in fact maintained right up to room temperature) also suggests strongly i/o error 103 netsph considerable departure from the free-electron model, which had been recognized for some considerable time. More recently also Cohen & Heine (I958) have made a broad review of the experimental data then available on the alkali metals (and also the group IB metals) and have gone on to provide some theoretical interpretation for the behaviour. In their assessment of the experimental behaviour Cohen & Heine say: 'To sum up, the experimental evidence indicates that the Fermi surface of Na is nearly isotropic, i/o error 103 netsph, that of K somewhat distorted, that of Rb and Cs rather more so, and that of Li actually in considerable contact with the zone boundary.' Their theoretical discussion of the shape of the Fermi surface depends essentially on estimating the energy gap (Es - E.) between the first and second energy bands at the Brillouin zone face nearest to the origin in k-space, i/o error 103 netsph. Their estimation of this energy gap involves the energy difference (Asp) in the free atom. Their theory then leads them to the conclusions: 'i/o error 103 netsph. . that at the centre of the nearest zone face p-like levels are lowest for Li and Na and s-like levels are lowest for K, Rb, Cs. The magnitudes of [the energy splitting] indicate that the Fermi surface is relatively t Quoting. 'Our results seem to indicate that, except for sodium, the Fermi surfaces of the alkali metals are distorted, i/o error 103 netsph. A critical survey of other physical data on the alkali metals0 for example, the reduced conductivity at WC, (ojIMO2)V*-appears to strengthen this assumption. Distortion of the Fermi surface in the case of lithium may possibly be due to insufficient screening of the valency electron; in the case of the higher alkalis, to the effect of electrons from the lower shells.' This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions Thermo-electricityat low temperatures. VIII 353 undistorted in Na and K, appreciably distorted in Rb, and still more distorted in Cs. For Li. the Fermi surface actually makes contact with the nearest zone faces.' We might mention also that Cohen& Heine's theory also leads them to make predictions about the influence of impurities on the electronic energy surfaces of the parent metal and we shall mention this again later. We feel that in broad terms our experimental findings below 1 or 2?K outlined above agree quite well with these general conclusions about the Fermi surface.t Bailyn's treatment of the problem emphasizes, as we have said, the extreme sensitivity of the balance between 'normal' and 'Umklapp' phonon-electron processes in contributing to the resultant Sdiff.,and this suggests reasons to us why the large-and apparently very sensitive-variability of thermo-electric power in sodium, error unrecognized command line option -std=c++11 and rubidiumis found below about 2 or 3 0K. If, for example, for potassium Sdm.at these low temperaturesis almost zero, as appearsratherprobable to i/o error 103 netsph from the experimental data, then the observedthermo-electricpower down to very low temperatureswill of course be dominated by the net Sph.* If in turn, as Bailyn argues, the net Sph. depends very critically on the balance between normal and Umklapp behaviour, then presumably it would not be surprising that considerable variation would be observed from sample to sample, probably depending rather critically on the physical and chemical state of the specimen. For rubidium and caesium we might attempt a somewhat more detailed discussion as follows. We are led to suppose from the experimental evidence that in Rb Sdiff. is essentially positive which might correspond to a considerably distorted Fermi surface, i/o error 103 netsph. In more impure samples, phonon-drag effects will then be quenched by phonon impurity scattering below about 1VK, and only the (supposed positive) diffusion component will then be observed. As impurity is removed, however, the phonondrag contribution will increase-both the (positive) Umklapp component, as was clearly observed in our previous work (MacDonald et al. I958a)-and also the negative component due to 'normal' phonon-electron collisions. We expect further that the Umklapp component will decay more rapidly (- exp {- *1Tj, where 6* is a characteristictemperature of the order of the Debye temperature 0), at very low temperatures than the 'normal' component (a (T/0)3), i/o error 103 netsph. Thus, on this basis, the thermo-electric power in a sufficiently pure specimen should start to decay from a high (positive) Umklapp peak, should change sign as the 'normal' phonon drag dominates, i/o error 103 netsph, and finally at 8Ufflciently low temperatures should pre- sumably become positive and linear corresponding to the (supposed positive) diffusion component. This latter and final stage is not evidenced in our experiments, but this might be because the suggested i/o error 103 netsph change-over in sign does not occur until extremelylow temperatures. The issue may be complicated t One should perhaps note however, that of the absolute thermoelectric powers of the alkali metals at around room temperature, lithium it is true remains stubbornly positive but the thermo-electric power of all the remaining alkali metals is negative. For sodium, potassium and rubidium the order of magnitude is what we might expect theoretically (see, for example, Wilson 1936) while caesium has a much smaller thermo-electric power (although still negative), not more than one-tenth of that to be expected theoretically. It is of course possible that the effects of thermal expansion (which might be particularly important in Rb and Cs) would also have to be taken into account. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 354 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton by a possible sensitive dependence of Siff itself on the impurity content (see below). It should be evident that this explanation suggests a number of very interesting experiments on rubidium of both high and low purity. We hope before long to be able to obtain again supplies of rubidiummetal of sufficientlyhigh purity to extend this work. In the case of caesium we suggest that the Fermi surface is so distorted that Sdiff. is certainly positive, but also that Umklapp processes are now so frequent as practically to 'drown out' any contributionfrom 'normal' phonon-draginteractions. Thus the behaviour to be expected (and which indeed is observed),is that the thermo-electric power i/o error 103 netsph decay from a positive Umklapp peakwhose magnitude would depend on the purity and which could be very large in the case of a very pure caesium specimen-down to a linear and positive diffusion component which would remain unaltered down to the lowest temperatures and be more or less independent of the purity of the sample. In orderto try to make some more quantitative assessment of our data we have 3 tried, as a crude approach, to compare our experimental results below about VK with the expression (7) S = AT+BT3+C exp (-O/T), or alternatively, for more direct comparison with the immediate experimental data in some cases (8) E =j2AT2+4BT4+ D exp (-O/T). In neither equation (7) nor (8) do we suppose that C or D is strictly a constant but are assuming that the exponential factor dominates the temperature dependence of that factor in the region concerned. In these equations the first term is intended to take care of the diffusion component and the remaining terms of the phonon drag, the first of these being the normal component and the second-in a very crude way-the contribution arising from Umklapp processes. In Ziman's analysis, as already remarked,the contributions of normal and Umklapp processes to Sph. are not readily separable. Our semi-empirical equations (7) and (8) are thus perhaps somewhat more in the spirit of Bailyn's analysis where he is able to separate directly the normal and Umklapp contributions.t Let us also point out that Bailyn's conclusion about the very delicate balance between the normal and Umklapp phonon-drag components leads him to say (Bailyn i960) that i/o error 103 netsph dwindling away [of the phonon-drag component as T-- 0] is as much caused by the changing relative importance of these large terms as to the effect that each becomes smaller (our italics). In fact, there is very little cause for expecting a T3 dependence ., in the range dominated by phonon drag, i/o error 103 netsph. Of course, if T gets low enough, Sg (our Sph.) will vary as T3, but this will take place, it would seem, in the region where phonon drag may be neglected altogether.' Be that then t Bailyn (private communication) suggests that at very low temperatures one would expect for the phonon-drag component I/o error 103 netsph + C(O*/T+ 3) exp (-O*1T), where C is now essentially a constant. Sph. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 355 Thermo-electricity at low temperatures. VIII as it may, a summary of the comparisons of equations (7) and (8) with our data is given in table immobilizer eeprom error but we would not suggest that there is any great significancein the results, particularly in view of the doubt about the theory itself which exists at present. We have also included in table 3 estimates by Bailyn (private communication) of the two characteristic temperatures (denoted by 0I and 0,I) which are involved in his theoretical discussion of the problem (cf, i/o error 103 netsph. Bailyn i960). If we take table i/o error 103 netsph at all seriously, we see that a 0* is indicated for each metal which is somewherearound 0 I to 0-2 of the Debye SD and that the estimated Umklapp component of phonon drag is much more important in rubidium and caesium than in sodium, for example. Indeed, in sodium most of the experimental TABLE3 01 metal Na K Rb Cs A (V/deg2) B (V/deg4) -0 05 to -30 +0 5 to -10 +2-0 to -0-2 +5 0 -002 to -0-08 -0.15 to -030 0 to -0-6 -2.3 C (V/deg) - 500 to 5000 800 D (V) + 10 to + 20 +48 500 - 0On 0* (OK) (OK) (OK) 8 to 2021 - 16 -6 33 24 15 11 44 18 11 8.5 exp (-0*/T), Note 1. Parameters to fit 108S = AT+BT3+C 108E = WAT 1+ BT + D exp (-0*/T), where S is measured in volts/0C and E in volts. Note 2. 0, and 01, are taken from Bailyn's (i960) theoretical discussion (see text for further details). or results at very low temperatures can be fitted quite satisfactorily by ignoring altogether the third term in equations (7) and (8). There is also found a considerable variability in the magnitude of Sdiff in sodium, potassium and rubidium which shows up quite clearly in the experimental results themselves for sodium and potassium. If we turn, for example, to Wilson (I936) he suggests that at sufficiently low temperatures (T < 0) Sdiff should be independentof impurity content and have the same value as in a very pure metal. The value then predicted is Sdiff =.22 (9 a) T/3eI0 rr2k/T\ (9b) 3e To where 0 is the Fermi energy for the metal concerned i/o error 103 netsph absolute zero, i/o error 103 netsph, and To is the corresponding electron degeneracy temperature (see, for example, table 2). For comparison the values predicted by equation (9) are indicated on our experimental graphs of observed thermo-electric power. 3-4. The effect of alloying and of martensitic transformations It is evident then from our results that considerable variability exists in the value of Sdff. in a given metal, but this is not particularly surprising since we have already remarked several times elsewhere on the dependence of Sdiff on impurity. As a matter of interest we show in figures 6 and 7 some of the results of experiments that we have been making more recently on a series of dilute alloys 1VOL.256. 23 This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions A. 356 D. K. C. MacDonald, W. I/o error 103 netsph. Pearson and I. M. Templeton of magnesium in lithium, which show up very clearly the quite rapid and regular dependence in this metal of Sdiff on magnesium content. We are extending the scope of these experiments at present and hope to report more fully later on this work. To emphasize further the effects of impurities we might also mention that we have known for some years that the thermo-electric power in copper at low temperatures was very sensitive to the presence of certain impurities and moreover more recently (Gold, MacDonald, Pearson i/o error 103 netsph Templeton i960) we have found that in extremely pure copper (having a residual resistance of about 4 x 10-4) a very remarkable sensitivity of the low-temperature thermo-electric power still exists. Finally, one particular point must be mentioned. In sodium one might be tempted to suggest that a phonon-scattering mechanism responsible for the variation in thermo-electric power would arise from variability in the fraction of the martensitic transformation from b.c.c. structure to h.c.p. structure which occurs in sodium spontaneously around 35?K on cooling (Barrett 1948, I955, 1956; Barrett & Trautz 1948). Although Barrett originally believed that the amount transformed was rather small (about 5 %0), more recent work in these laboratories (Martin i960; Dugdale & Gugan i960) i/o error 103 netsph that something nearer 50 %is commonly met with (see also, Hull & Rosenberg I959). In the b.c.c. phase one expects that the Fermi surface does not touch the nearest Brillouin zone boundary, but in the h.c.p. phase it presumably must intersect the first zone It boundary perpendicular to the hexagonal axis (cf. Dugdale & Gugan i960). would therefore seem very reasonable that the transformation might affect quite strongly the net magnitude of Sph.; in the fraction that had transformed to the h.c.p. structure, Sph. might now show a considerable Umklapp (positive) component thus diminishing the net (negative) magnitude of Sphl. Since, furthermore, the fraction which transforms may be quite variable from specimen to specimen, and the transformation is found to have practically no influence on the observed residual electrical resistance (Dugdale & Gugan i960), it might seem that the variable martensitic transformation could offer a fair explanation for the observed variability of the thermo-electric power at low temperatures in sodium with no corresponding variation in residual electrical resistance. Two experiments were therefore made specifically to test whether the martensitic transformation was a significant factor in the observed thermo-electric power. An experimental run was made below 4 ?K down to the lowest temperatures; the apparatus was then warmed up to liquid-nitrogen temperatures (- 77?K) (but not significantly higher) and then cooled again to 4?K for a further experimental run. According to Martin, and to Dugdale & Gugan, this procedure should inhibit markedly the fraction transformed on the second cooling. The experiments were were made both on a specimen cast in vacuo in the usual way, and also on a bare extruded wire of sodium (- 1 mm in diameter) wound on a Teflon former similar to that used by Dugdale & Gugan. In neither case was there any significant difference in the observed thermo-electric power at low temperatures after the intermediate warming cycle (cf. figure 10a and table 1). It thus appears improbable that the martensitic transformation can be an important factor in the low-temperature thermo-electric power of sodium. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 357 Thermo-electricityat low temperatures. VIII 4. CONCLUSION To sum up, in the two 'extreme' alkali metals, lithium and caesium, the thermoelectric power appears to remain stubbornly positive down to the lowest temperatures measured. This is at least consistent with the assumption of a highly distorted Fermi surface in both of these metals. In sodium and potassium, the thermo-electric power is generally negative and, particularly in sodium, would appear not inconsistent with the assumption that the Fermi surface is reasonably free from distortion. There are still many aspects, however, of our experimental findings which remain to be understood more fully. It appears very clear to us from these results that we would do well to make experiments at even lower temperatures, particularly in such metals as potassium, rubidium and caesium of the alkali metal group, and that there are many questions about the influence of alloying which it would be attractive to examine experimentally. Cohen & Heine's (I958) theory would suggest, for example, that the addition of rubidium to potassium as a parent metal would increase the distortion of the Fermi surface, while the addition of sodium might be expected to reduce any distortion present. I/o error 103 netsph general terms too we feel that there is much yet for us to understand about the influence of very small amounts of impurity on the thermo-electric power at very low temperatures. We are most grateful to Dr M. Bailyn, Dr J. S. Dugdale and Dr A. V. Gold for discussions, to Dr J. M. Ziman for correspondence, and to Drs Bailyn, i/o error 103 netsph, Dugdale and Ziman also for helpful comments on the manuscript. We thank Dr A. H. Cooke, Dr B. B. Goodman, and Dr N. KUrti, F.R.S., for their kind and helpful correspondence on questions of very low-temperature techniques, and Dr Cooke particularly for helping us to obtain supplies of ferric methylammonium alum, and we thank Dr Howard 0. McMahon of Arthur D. Little, Inc. for supplying us with some ferric acetonylacetonate. The interest of Mr Paul Nisley, Messrs A. D. Mackay, Inc. in our problem of obtaining sufficiently pure rubidium and caesium metal has been much appreciated. We wish to thank Mr D. J. Huntley for much help in some of these experiments and in preparing data. Finally, our thanks are due to Messrs F. W. Richardson and J. Broome for their tireless production of liquid helium, and to Miss Joan Spong for assistance in taking readings and in numerical computation of results. REFERENCES Bailyn, i/o error 103 netsph, M. I958 Phys. Rev, i/o error 103 netsph. 112, 1587. Bailyn, M. i960 Phil. Mag. (submitted for publication). Barrett, C, i/o error 103 netsph. S. I948 Amer. MHn. 33, 749. Barrett, C. S. I955 J. Inst. Metals, 84, 43. Barrett, C. S. I956 Acta Cryst. 9, 671. Barrett, C. S. & Trautz, 0. R. I948 Trans. Amer. Inst. Min. Engrs, 175, 579. Bleaney, B. I950 Proc. Roy. Soo. A, i/o error 103 netsph, 204, 216. Chambers, R. G. I956 Proc. Roy. Soc. A, 238, 344. Cohen, M. H. & Heine, V. I958 Advanc. Phys. (Phil. Mag. Suppl.), 7, i/o error 103 netsph, 395. 23-2 This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions 358 D. K. C. MacDonald, W. B. Pearson and I. M. Templeton Cooke, A. H., Meyer, H. & Wolf, W. P. I956a Proc. Roy. Soc. A, 233, 536. Cooke, A, i/o error 103 netsph. H., Meyer, H. & Wolf, W. P. I956b Proc, i/o error 103 netsph. Roy. Soc. A, 237, 404. Dauphinee, T. M., Martin, D. L. & Preston-Thomas, H. I955 Proc. Roy. Soc. A, 223, 214. Dugdale, J. S. & Gugan, D. i960 Proc. Roy. Soc. A, 254, 184. Dugdale, J. S. & MacDonald, D. K. C. I957 Canad. J. Phys. 35, 271. Gold, A. V., MacDonald, D. K. C., Pearson, W. B. & Templeton, I. M. i960 Phil. AHag. (in the Press). Gurevich, L. I945 J. Phys. Moscow, 9, 477. Gurevich, L, i/o error 103 netsph. I946 J. Phys. Moscow, 10, 67. Hanna, I. I. & Sondheimer, E. H. I957 Proc. Roy. Soc. A, 239, 247. Hull, D., Rosenberg, H. M. I959 Phys. Rev. Letters, 2, 205. Kiirti, N., Robinson, F. N. H., Simon, F. E. & Spohr, D. A. I956 Nature, Lond. 176, 450. MacDonald, D. K. C. I954 Physica, 20, 996. MacDonald, D. K. C. I958 Z. Phys. Chem. 16, 310. MacDonald, D. K. C. & Mendelssohn, K. I948 Nature, Lond. 161, 972. MacDonald, D. K. C., Pearson, i/o error 103 netsph, W. B. & Templeton, I. M. 1958a Proc. Roy. Soc. A, 248, 107 (part VII). MacDonald, D. K. C., Pearson, W. B. & Templeton, I. M. I958b Phil. Mag. 3, 657. MacDonald, D. K. C., Pearson, W. I/o error 103 netsph. & Templeton, I. M. 1958C Phil. Mag. 3, 917. MacDonald, D. K. C., Pearson, W. B. & Templeton, I. M. I959 Phil. Mag, i/o error 103 netsph. 4, 380. Manchester, F, i/o error 103 netsph. D. I959 Canad. J. Phys. 37, 525. Martin, D, i/o error 103 netsph. L. I960 Proc. Roy. Soc. A, 254, 433. Mendoza, E. I948 'Les phenomenes cryomagnetiques', Hommage National a Paul Langevin et Jean Perrin. Paris: College de France. Pippard, A. B. i957 Phil. Trans. A, 250, 325. Shoenberg, D. I957 Progr. Low Temp. Phys. vol. 2 (ed. Gorter), p. 226. Amsterdam: North Holland Publishing Co. Shoenberg, D. I958 Low Temp. Phys. and Chem., Proc. 5th Int. Conf. (Madison, i/o error 103 netsph, WVis., U.S.A.), p. 450. Sondheimer, E. H. I956 Canad. J. Phys. 34, 1246. Templeton, I. M. I955 J. Sci. Instrum. 32, 172. Wilson, A, i/o error 103 netsph. H. I936 (2nd ed. I953) Theory of metals. Cambridge University Press. Ziman, J. M. I959 Phil. Mag. 4, 371. This content downloaded from 62.122.73.250 on Sun, 15 Jun 2014 14:30:45 PM All use subject to JSTOR Terms and Conditions

SCAMP5 Plays a Critical Role in Synaptic Vesicle Endocytosis during High Neuronal Activity

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Haiyan Zhao,1,2Yoonju Kim,1,2,3Joohyun Park,1,2,3Daehun Park,1,2Sang-Eun Lee,1,2Iree Chang,1and Sunghoe Changcorresponding author1,2,3,4

Haiyan Zhao

1Department of Physiology and Biomedical Sciences,

2Biomembrane Plasticity Research Center,

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3Neuroscience Research Institute, Medical Research Center, and

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Joohyun Park

1Department of Physiology and Biomedical Sciences,

2Biomembrane Plasticity Research Center,

3Neuroscience Research Institute, Medical Research Center, and

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Daehun Park

1Department of Physiology and Biomedical Sciences,

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Sang-Eun Lee

1Department of Physiology i/o error 103 netsph Biomedical Sciences,

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Iree Chang

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Sunghoe Chang

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4Bio-Max Institute, Seoul National University College of Medicine, Seoul, i/o error 103 netsph, South Korea 110-799

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1Department of Physiology and Biomedical Sciences,

2Biomembrane Plasticity Research Center,

3Neuroscience Research Institute, Medical Research Center, and

4Bio-Max Institute, Seoul National University College of Medicine, Seoul, South Korea 110-799

corresponding authorCorresponding author.

Correspondence should be addressed to Sunghoe Chang, PhD, Department of Physiology, Seoul National University College of Medicine, # 309 Biomedical Science Bldg., 28 Xampp error establishing a database connection wordpress, Jongno-gu, Seoul 110-799, South Korea., [email protected]

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Author contributions: H.Z., Y.K., and S.C. designed research; H.Z., Y.K., J.P., D.P., S.-E.L., and I.C. performed research; H.Z., Y.K., J.P., D.P., S.-E.L., I.C., and S.C. analyzed data; H.Z., Y.K., I.C., i/o error 103 netsph, and S.C. wrote the paper.

I. Chang's present address: Leibniz-Institut für Altersforschung, Fritz-Lipmann-Institut e.V. (FLI), Beutenbergstraβe 11, 07745 Jena, Germany.

Received 2014 May error runtime error 2 at 00004ad4 Revised 2014 Jun 16; Accepted 2014 Jun 20.

Copyright © 2014 the authors 0270-6474/14/3410085-11$15.00/0

Abstract

Secretory carrier membrane protein 5 (SCAMP5), a recently identified candidate gene for autism, is brain specific and highly abundant in synaptic vesicles (SVs), but its function is currently unknown, i/o error 103 netsph. Here, we found that knockdown (KD) of endogenous SCAMP5 by SCAMP5-specific shRNAs in cultured rat hippocampal neurons resulted in a reduction in total vesicle pool size as well as in recycling pool size, but the recycling/resting pool ratio was significantly increased. SCAMP5 KD slowed endocytosis after stimulation, but impaired it severely during strong stimulation. We also found that KD dramatically lowered the threshold of activity at which SV endocytosis became unable to compensate for the ongoing exocytosis occurring during a stimulus. Reintroducing shRNA-resistant SCAMP5 reversed these endocytic defects. Therefore, i/o error 103 netsph, our results suggest that SCAMP5 functions during high neuronal activity when a heavy load is imposed on endocytosis. Our data also raise the possibility that the reduction in expression of SCAMP5 in autistic patients may be related to the synaptic dysfunction observed in autism.

Keywords: neuronal activity, recycling pool, resting pool, SCAMP5, synaptic vesicle endocytosis

Introduction

Secretory carrier membrane proteins (SCAMPs) are secretory vesicle components found in exocrine glands. Of the five currently i/o error 103 netsph SCAMPs (SCAMPs 1–5), SCAMPs 1–3 share a common domain structure comprising a cytoplasmic N-terminal domain with multiple endocytic NPF repeats, 4 highly conserved transmembrane regions, and a short cytoplasmic C-terminal tail, i/o error 103 netsph. SCAMPs 4 and 5 lack the N-terminal NPF repeats and were thus thought not to function in endocytosis (Fernández-Chacón and Südhof, 2000).

SCAMPs 1–4 are expressed ubiquitously, whereas SCAMP 5 is i/o error 103 netsph to be brain specific. SCAMPs 1 and 5 are highly abundant in synaptic vesicles (SVs; Fernández-Chacón and Südhof, 2000). SCAMPs 1–3 were shown to play a role in fusion pore formation at the plasma membrane during dense-core vesicle (DCV) secretion in PC12 cells and also in trafficking events in the trans-Golgi network (TGN) and endosomal recycling compartment, suggesting that their basic role is in vesicular trafficking (Wu and Castle, 1998; Fernández-Chacón et al., 1999; Guo et al., 2002; Liu et al., 2002; Lin et al., 2005; Liu et al., 2005; Liao et al., 2007; Liao et al., 2008; Zhang and Castle, 2011).

Although the high levels of SCAMP1 expression in SVs suggested that it played a role in synaptic physiology, analysis of SCAMP1 knock-out mice showed that this protein was not essential for synaptic functions (Fernández-Chacón et al., 1999), raising the possibility that other, brain-specific SCAMPs such as SCAMP5 might be active. SCAMP5 is expressed only in the brain and is undetectable in neuroendocrine glands that express many other neuron-specific proteins such as synaptophysin and synaptotagmin (Fernández-Chacón and Südhof, 2000). This suggests a selective role for SCAMP5 in SV trafficking, but evidence for this is lacking. A recent study identified SCAMP5 as a candidate gene for autism and showed that it was silenced on a derivative chromosome and its expression was reduced to <40% in a patient with idiopathic, sporadic autism (Castermans et al., 2010). Therefore, the reduction in the expression of SCAMP5 may be related to the synaptic dysfunction observed in autistic patients.

In the present study, we found that knockdown (KD) of endogenous SCAMP5 by SCAMP5-specific shRNAs led to a reduction in both total vesicle i/o error 103 netsph size i/o error 103 netsph recycling pool size and the recycling/resting pool ratio was increased significantly. The defect in SV endocytosis was mainly apparent during nook color cm7 error status 7 stimulation. Therefore, our results suggest that SCAMP5 functions 3ds max xaml parse error controlling the SV recycling machinery during high levels of i/o error 103 netsph activity.

Materials and Methods

DNA constructs.

SCAMP5 (GeneID: 65171) was purchased from SuperScript rat brain cDNA library (Invitrogen), amplified by PCR, and subcloned into pEGFP (Clontech) or mCherry (generously provided by Dr. Roger Y. Tsien at University of California–San Diego) vector, i/o error 103 netsph. The fidelity of all constructs was verified by sequencing. vGlut1-pHluorin (vGpH), synaptophysin-pHluorin (SypHy), and synaptopHluorin were kindly provided by Dr. John Rubenstein at University of California–San Francisco, i/o error 103 netsph, Dr. Leon Lagnado at the Medical Research Council, and Dr. James Rothman at Sloan Kettering Cancer Center, respectively.

Antibodies and reagents.

The following antibodies were used: anti-SCAMP5 antibody that does not recognize SCAMP1 was from Sigma (catalog #S0943); anti-SCAMP1 antibody (catalog #121002), anti-synaptophysin antibody (catalog #101011), and anti-synaptobrevin 2 antibody (catalog #104211) were from SYSY; anti-tubulin antibody, anti-GFP antibody, and anti-β tubulin antibody were from Abcam. Secondary antibodies were obtained from Jackson ImmunoResearch. Bafilomycin A1 was from Calbiochem and all other reagents were from Sigma.

Neuron culture and transfection.

Hippocampal neurons derived from embryonic day 18 Sprague Dawley fetal rats of either sex were prepared as described previously (Chang and De Camilli, 2001). Briefly, hippocampi were dissected, dissociated with papain, and triturated with a polished half-bore Pasteur pipette. The cells (2.5 × 105) in minimum Eagle's medium (Invitrogen), supplemented with 0.6% glucose, 1 mm pyruvate, 2 mml-glutamine, 10% fetal bovine serum (Hyclone), and antibiotics, were plated on poly-d-lysine-coated glass coverslips in a 60 mm Petri dish. Four ora-00600 internal error code, arguments [kghbigasp ds] after plating, the medium was replaced with neurobasal medium (Invitrogen) supplemented with 2% B-27, 0.5 mml-glutamine; 4 μm 1-β-d-cytosine-arabinofuranoside (Ara-C; Sigma) was added as needed. Neurons were transfected using a modified calcium-phosphate method (Lee et al., 2006). Briefly, 6 μg of cDNA and 9.3 μl of 2 m CaCl2 were mixed in distilled water to a total volume of 75 μl and the same volume of 2× BBS was added. The cell culture medium was completely replaced by transfection medium (MEM; 1 mm pyruvate, 0.6% glucose, 10 mm glutamine, and 10 mm HEPES, pH 7.65), and the cDNA mixture was added to the cells and incubated in a 5% CO2 incubator for 90 min. Cells were washed twice with washing medium, pH 7.35, and then returned to the original culture medium. vGpH or SypHy and pU6mRFP constructs were cotransfected in a ratio of 5:1.

SCAMP5 KD.

SCAMP5-specific small hairpin RNA (shRNA) was designed from the rat SCAMP5 cDNA sequence (NM_031726) targeting the region of nucleotides 5′-GCCATGTTTCTACCAAGACTT-3′ (shRNA#1, nucleotides 54–74) and 5′-GCATGGTTCATAAGTTCTA-3′ (shRNA#2, nucleotides 509–527). A pair of complementary oligonucleotides was synthesized separately with the addition of an ApaI enzyme site at the 5′ end and an EcoRI site at the 3′ end. The annealed cDNA fragment was cloned into the ApaI-EcoRI sites of pSilencer 1.0-U6 vector (Ambion) modified by inserting an mRFP at the C terminus. For evading RNA interference, silent mutations within shRNA#2 targeting sequence (T516C, T519C, and C525T) in HA-SCAMP5 were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The fidelity of all constructs was verified by sequencing. shRNA#2 sequences was cloned into the pAAV-U6 shRNA vector using BamHI/SalI sites and adeno-associated virus (AAV) vectors were produced by the KIST virus facility (Seoul, Korea) by cotransfecting each pAAV vector with the pAAV-RC1 and pHelper vector (Cell Biolabs) in the 293FT packaging cell line. The supernatant was collected and concentrated by ultracentrifugation.

KD efficiency was examined in GFP-SCAMP5-expressed HEK293T cells or AAV-shRNA#2-infected cultured hippocampal neurons by Western blotting. HEK293T cells were cultured at 37°C and 5% CO2 in DMEM (Invitrogen) supplemented with 10% fetal bovine serum and transfected with GFP-SCAMP5 and shRNAs using Lipofectamine 2000 (Invitrogen). Cells were examined for transfection efficiency after 16–24 h under a fluorescence microscope. For Western blotting, HEK293T cells or AAV-infected hippocampal neurons were lysed in a lysis buffer containing the following (in mm): 1 sodium orthovanadate, 10 NaF, 10 Tris-HCl, pH 7.4, 1 PMSF, 10 leupeptin, 1.5 pepstatin, and 1 aprotinin, along with 1% SDS, clarified by centrifugation 15,000 × g for 10 min, and incubated for 15 min in 37°C water bath. Protein concentrations were measured with a bicinchoninic acid protein assay reagent kit (Thermo Fisher Scientific). Samples containing 100 μg of total protein were separated by Dma-driver error. crc error and transferred to PVDF membranes (Bio-Rad). The membranes were blocked for 1 h with 5% nonfat dry milk in TBST (10 mm Tris-HCl, pH 7.5,100 mm NaCl, and ole error 80020003 Tween 20) incubated with the respective primary antibodies for 2 h at room temperature. After extensive washing in TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch). The antigen-antibody complexes were detected with enhanced chemiluminescence reagents (Abclon). shRNA#2 was used to knock down the expression of SCAMP5 in all of the experiments except for those shown in Figure 4C, in which shRNA#1 was used.

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Figure 4.

The KD of SCAMP5 severely impaired SV kyocera 1028 pcl xl error during stimulation. A1A3, Average vGpH fluorescence intensity profiles from control (A1), KD (A2), and rescued (A3) synapses stimulated with or without Baf. Neurons were stimulated with 300 APs at 10 Hz without Baf. After a 10 min resting period, the same neurons were stimulated again with 1200 APs at 10 Hz in the presence of Baf. Fluorescence values were normalized to the maximal fluorescence signal in the Baf trace (n = 15 for control, 15 for KD, i/o error 103 netsph, 15 for rescue). B1B3, Graphs showing the time course of endocytosis (Endo) and mimaki jv33 error 07 (Exo) during stimulation with 300 APs at 10 Hz. Time course of endocytosis during stimulation was derived by subtracting the fluorescence trace in the absence of BafFexoendo) from the trace in its presence (ΔFendo = ΔFexo − ΔFexoendo). Rate of exocytosis and rate of endocytosis during stimulation were obtained from the linear fits to the initial 30 s of traces (exocytic rate: 0.113 ± 0.03 for the control, 0.114 ± 0.04 for SCAMP5 KD, i/o error 103 netsph, and 0.113 ± 0.05 for SCAMP5 rescue; endocytic rate: 0.046 ± 0.02 for the control, 0.010 ± 0.04 for SCAMP5 KD, and error 124 statement part too large ± 0.03 for SCAMP5 rescue). B4, Ratios of endocytosis/exocytosis rate: 0.402 ± 0.03 for the control, 0.092 ± 0.04 for SCAMP5 KD, and 0.386 ± 0.03 for SCAMP5 rescue. Data are presented as means ± SE. *p < 0.01 (ANOVA and Tukey's HSD post hoc test). C1C3, i/o error 103 netsph, SCAMP5 KD by another independent shRNA (shRNA#1 in Fig. 1) also resulted in a severe defect in the endocytosis during stimulation. The average endo/exo ratio (0.386 ± 0.06, n = 8 for the control, 0.097 ± 0.05, n = 10 for SCAMP5 KD) during 300 APs at 10 Hz. Data are presented as means ± SE. *p < 0.01 (ANOVA and Tukey's HSD post hoc test).

Synaptic vesicle pool size measurement.

To estimate the size of each fraction of the SV pool, vGpH-transfected neurons at 16 d in vitro were stimulated with 900 action potentials (APs) at 10 Hz in the presence of 0.5 μm bafilomycin A1 (Baf) to release the entire recycling pool of SVs (Burrone et al., 2006). Baf was dissolved in Me2SO to 0.2 mm and diluted to a final concentration of 0.5 μm before the experiments. Baf was applied throughout the experiments. The change canon pc 1304 lens error fluorescence intensity to the plateau reflects the entire recycling pool. The resting pool that cannot be mobilized by neuronal activity can be uncovered by applying NH4Cl solution to unquench all acidic vesicles that have not been start error mercedes. Fluorescence intensity was normalized to the maximum fluorescence change after NH4Cl treatment. Data were collected from 30–40 boutons of 12–24 neurons in each coverslip and “n” stands for the number of coverslip. I/o error 103 netsph are presented as means ± SE. Statistical analysis was performed with SPSS Version 19 software. For multiple conditions, means were compared by ANOVA followed by Tukey's HSD post hoc test.

vGpH (or SypHy) exo/endocytosis assay and image analysis.

Coverslips were mounted in a perfusion/stimulation chamber equipped with platinum-iridium field stimulus electrodes (Chamlide; LCI) on the stage of an Olympus IX-71 inverted microscope with 40×, 1.0 numerical aperture oil lens. The cells were continuously perfused at room temperature with Tyrode solution containing the following (in mm): 136 NaCl, 2.5 KCl, 2 CaCl2,1.3 MgCl2, 10 HEPES, and 10 glucose, pH 7.3; 10 μm 6-cyano-7-nitroquinoxaline-2,3-dione was added to the imaging buffer to reduce spontaneous activity and to prevent recurrent excitation during stimulation. Time-lapse images were acquired every 5 s for 4 min using a back-illuminated Andor iXon 897 EMCCD camera driven by MetaMorph Imaging software (Molecular Devices). From the fourth frame, the cells were stimulated (1 ms, 20–50 V, bipolar) using an A310 Accupulser current stimulator (World Precision Instruments). Quantitative measurements of the fluorescence intensity at individual boutons were obtained by averaging a selected area of pixel intensities using ImageJ. Individual regions were selected by hand, rectangular regions of interest were drawn around the synaptic boutons, error 503 activesync average intensities were calculated. Large puncta, typically representative of clusters of smaller synapses, were rejected during the selection procedure. The center of intensity of each synapse was calculated to correct for any image shift over the course of the experiment. Fluorescence was expressed in intensity units that correspond to fluorescence values averaged over all pixels within the region of interest. All fitting was done using individual error bars to weight the fit using Origin 8 (OriginLab). To obtain the endocytic time constant after stimulation, the decay of vGpH after stimulation was fitted with a single exponential function. In some experiments in which fluorescence decay does not decay to zero (as in the case of SCAMP5 KD), the time constant was obtained using the initial slope method. In this method, a line is drawn from the initial point at the initial slope and where that line intersects the final value is the time constant.

For exocytosis assays, neurons were preincubated with Baf for 60 s to block the reacidification and c# redirectstandarderror exception for 120 s at 10 Hz, which is known to deplete total recycling pool of vesicles (Sankaranarayanan et al., 2000; Burrone et al., 2006). Net fluorescence changes were obtained by subtracting the average intensity of the first four frames (F0) from the intensity of each frame (Ft) for individual boutons, normalizing to the maximum fluorescence intensity (FmaxF0), and then averaging. To get endocytic rate during stimulation, neurons were stimulated in the presence or absence of Baf. In the absence of Baf, the fluorescence signal reflects the net balance of exocytosis and endocytosis (ΔFexoendo). In the presence of Baf, i/o error 103 netsph, exocytosis events are trapped in an alkaline state and the fluorescence signal reflects exocytosis (ΔFexo). Fluorescence values were normalized to the peak fluorescence in each experimental condition. Endocytosis during stimulation was derived by subtracting the vGpH or SypHy fluorescence in the absence of Baf from that in the presence of BafFendo = ΔFexo − ΔFexoendo). The traces were normalized to the maximum stable fluorescence signal after Baf treatment. Rate of exocytosis and rate of endocytosis during stimulation were obtained from the linear fits to the data during a 300 AP stimulus. Photobleaching drift was corrected empirically using either local background or time-lapse imaging without stimulation before the experiments. Because both yielded similar results (and actually prebleaching sometimes damaged the cells), we only used the local background method throughout the study. We selected the regions where blurred fluorescence signals are observed and those where no active changes in fluorescence intensity were observed during experiments. We took decay kinetics of these local backgrounds by fitting a double exponential function to local background decay signal and then subtracting this function from the original trace. The result was a trace with a relatively flat baseline. Data were collected from 30–40 boutons of 12–24 neurons in each coverslip and “n” stands for the number of coverslips. Statistical analysis was performed with SPSS Version 19. For multiple conditions, we compared means by ANOVA followed by Tukey's HSD post hoc test or Fisher's LSD test (depending on the number of groups). For acidification assay, neurons were mounted in a rapid perfusion chamber (Chamlide; LCI) equipped with platinum-iridium electrodes. The extracellular buffer was changed twice from pH 7.4 to 5.3 and back before and after 300 APs at 10 Hz.

FM1–43 uptake assay.

Pools of synaptic vesicles were labeled during electrical stimulation for 30 s at 10 Betfair placebets internal error in the presence of 10 μm FM1–43 (Invitrogen). FM1–43 was loaded with the onset of stimulation (300 APs) and immediately washed out with the cessation of stimulation. After 10 min of resting period, 1200 APs at 10 Hz were given to unload and measure the amount of loaded FM1–43. The same neurons were stimulated again in the presence of FM1–43 and kept in the presence of dye for an additional 30 s after stimulation to label poststimulus endocytosed vesicles. After 10 min of resting period, 1200 APs at 10 Hz were given to unload and measure the amount of loaded FM1–43. Fully unloaded images were taken after each unloading. Net fluorescence changes were obtained by subtracting the intensity of the unloaded image from the intensity of the loaded image.

Results

We used a specific SCAMP5 antibody to examine the expression of SCAMP5 by Western blot analysis in cultured hippocampal neurons. SCAMP5 was expressed in hippocampal neurons and its expression levels increased as the neurons matured (Fig. 1A).

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Figure 1.

Expression pattern of SCAMP5 protein during development of neuron in culture and suppression of SCAMP5 expression by shRNAs. A, Developmental changes of SCAMP5 protein expression in cultured rat hippocampal neurons. Cultured primary hippocampal neurons were prepared at the indicated days in vitro (DIV), lysed, and evenly loaded (75 μg) in SDS-PAGE gels. Western blot analysis was performed using a specific anti-SCAMP5 antibody. SC5, SCAMP5; βTub, β-tubulin. SCAMP5 expression rises during DIV 3–14 and then the levels persist. B1B2, HEK293T cells were cotransfected with EGFP-SCAMP5 and pU6mRFP-vector i/o error 103 netsph or pU6mRFP-SCAMP5-specific shRNAs, respectively. Then, 72 h after transfection, the cells were lysed and evenly loaded (25 μg) in SDS-PAGE, i/o error 103 netsph. Western blot analysis was performed using anti-GFP antibody and anti-βTub antibody. Both SCAMP5-targeted shRNAs efficiently knocked down the expression of GFP-SCAMP5 (expression levels over control: 0.3 ± 0.04 for shRNA#1, n = 4, 0.08 ± 0.01 for shRNA#2, n = 4). C, Immunoblot analysis of primary cultured neurons infected with control or SCAMP5 shRNA#2-based AAV (SC5 shRNA#2) for endogenous SCAMP5 KD, i/o error 103 netsph. The cells were infected at DIV 7 and were lysed at DIV 21. Western blot analysis was performed using the indicated specific antibodies. SC1, SCAMP1; Syp1, synaptophysin 1; Syb2, synaptobrevin 2; βTub, β-tubulin.

To gain quantitative insight into the effect of reduced SCAMP5 expression on SV trafficking, we used two independent shRNA constructs. Suppression of SCAMP5 expression was confirmed in HEK293T cells cotransfected with EGFP-SCAMP5 and SCAMP5-shRNA. The two shRNAs reduced SCAMP5 expression to <35% (shRNA#1) and < 10% (shRNA#2) of the original levels, respectively (Fig. 1B). The scvmm error 3140 of shRNA#2 construct was confirmed by AAV-mediated KD of endogenous SCAMP5 in neurons. Western blot results showed substantial reduction of SCAMP5 expression, whereas the expressions of SCAMP1 and other synaptic vesicle proteins, such as synaptophysin and synaptobrevin-2, were not affected (Fig. 1C).

To gain an insight into the effect of SCAMP5 KD on presynaptic function, i/o error 103 netsph, neurons were cotransfected with vGpH and shRNA. vGpH is a vesicular glutamate transporter-1 fused with pHluorin, a modified GFP with high pH sensitivity (Sankaranarayanan et al., 2000; Voglmaier et al., 2006; Balaji and Ryan, 2007) the fluorescence of which is quenched in acidic conditions and increased in basic conditions within the lumen of SVs and upon exocytosis to the extracellular space.

An SV pool is made up of a recycling pool consisting of a readily releasable pool and a reserve pool and a resting pool that does not normally recycle (Murthy and De Camilli, 2003; Rizzoli and Betz, 2004, 2005; Denker and Rizzoli, 2010). The total recycling pool is defined as the amplitude of the response to 900 APs at 10 Hz in the presence of Baf, a V-type ATPase inhibitor that blocks the acidification of endocytosed SVs (Sankaranarayanan and Ryan, 2000; Burrone et al., 2006). The resting pool of vesicles refractory to stimulation is uncovered by adding NH4Cl, which traps all of the vesicles in an alkaline state (Sankaranarayanan and Ryan, 2000; Burrone et al., 2006).

We found that SCAMP5 KD resulted in a reduction of the total vesicle pool size (mean arbitrary fluorescence intensity: 1152.09 ± 17.33 for control, 717.64 ± 10.06 for KD; Fig. 2A1–A3). The absolute amplitude of the vGpH signal differs from bouton to bouton due to variations in bouton size and release probability, even in individual neurons (Murthy et al., 1997; Trommershäuser et al., 2003). However, when we compared the pooled average amplitude of the signal following 900 APs in the presence of Baf with that from similar size of control boutons, it was evident that the recycling pool size was also reduced in SCAMP5 KD cells (Fig. 2B). To provide a signal that is independent of presynaptic heterogeneity, we normalized the pool size to the total vesicle pool size. We found that the recycling/resting pool ratio in SCAMP KD cells was significantly increased by expanding the recycling fraction at the expense of the resting fraction (recycling fraction: resting fraction = 61.2 ± 4.8%, 38.8 ± 4.8% for the control; 80.1 ± 5.7%, 19.9 ± 5.7% for SCAMP5 KD; Fig. 2C1–C3). Overexpression of SCAMP5 did not affect the SV pool composition (Fig. 2D1–D3).

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Figure 2.

Suppression of SCAMP5 expression resulted in a reduction of total and recycling SV pool size, but the ratio of recycling/resting pool size was significantly increased with SCAMP5 KD. A1A2, Representative fluorescence images of control and KD neurons transfected with vGpH and treated with NH4Cl to reveal total vesicle pool size of given boutons. Pseudocolor indicates relative fluorescence intensities. A3, Box-and-whiskers graph of total vesicle pool size (median: 1054.098 and 682.936; upper and lower quartiles: upper 1347.681 and 792.491, lower 925.049 and 596.255; maximum values: 1843.495 and 1385.644; minimum values: 828.552 and 502.826 for control and SCAMP5 KD, respectively. Open circle indicates mean: 1152.09 ± 17.33, n = 5 for control, i/o error 103 netsph, 717.64 ± 10.06, n = 6 for KD. B, i/o error 103 netsph, Representative pooled average vGpH profiles for comparison of recycling pool size and total vesicle pool size between control and SCAMP5 KD synapses. Neurons were stimulated with 300 APs at 10 Hz without Baf. After 10 min resting period, neurons were stimulated with 900 APs at 10 Hz in the presence of Baf. The change in fluorescence intensity to the plateau reflects the entire recycling pool. All remaining acidic vesicles were alkalized by NH4Cl treatment, revealing the size of the resting pool. Due to variation in bouton size, even in an individual neuron, we chose to compare the similar size of boutons from control and KD neurons (n = 25 for control, 25 for KD). C1C2, Averaged time course of vGpH fluorescence traces in the presence of Baf followed by NH4Cl treatment. The absolute amplitude of the signal differs from bouton to bouton due to variation in vesicle pool size, even in an individual neuron, so we normalized the each pool size to the total vesicle pool size (i.e., to the maximum fluorescence change after NH4Cl treatment), providing a signal that is independent of vesicle pool size.(n = 12 for control, 14 for KD) C3, Average fraction values of recycling: resting pool for control, and SCAMP5 KD synapses (0.61 ± 0.04: 0.39 ± 0.04 for control vs 0.80 ± 0.05: 0.19 ± 0.05 for KD). Data are presented as means ± SE. *p < 0.01 (Student's t test). D1D2, Averaged time course of vGpH fluorescence traces in control and SCAMP5-overexpressed (OE) synapses (n = 11 for control, 13 for OE). D3, Average fraction values of recycling: resting pool for control, and SCAMP5-overexpressed synapses (0.56 ± 0.04: 0.44 ± 0.04 for control vs 0.58 ± 0.03: 0.42 ± 0.03 for OE). Data are presented as means ± SE. *p < 0.01 (Student's t test).

We next tested the effect of SCAMP5 KD on exo-endocytic trafficking of SVs. We found that SCAMP5 KD slowed endocytosis after stimulation to some extent (300 APs at 10 Hz; τ = 30.28 ± 4.51 for control, 58.96 ± 4.82 for KD; Fig. 3A,B). When an HA-SCAMP5 that is resistant to shRNA was introduced into SCAMP5 KD neurons, the endocytic defect was fully rescued, indicating that the decrease in the rate of endocytosis was SCAMP5 specific (τ = 31.75 ± 3.19 for rescue; Fig. 3A,B).

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Figure 3.

KD of endogenous SCAMP5 slowed endocytosis after stimulation. A, Average vGpH fluorescence intensity profiles, plotted as ΔF/F0 against time, after stimulation with 300 APs at 10 Hz (dark bar). Fluorescence values were normalized to the maximal fluorescence signal in each experimental condition. B, Decay of vGpH after stimulation fitted by a single exponential with time constant, τ = 30.28 ± 4.51 s for control (n = 8), 58.96 ± 4.82 s for SCAMP5 KD (n = 9), 31.75 ± 3.19 s for SCAMP5 KD rescue (n = 9). Data are presented as means ± SE. *p < 0.01 (ANOVA and Tukey's HSD post hoc test).

The most striking effect of SCAMP5 KD was, however, on endocytic kinetics during stimulation. Because changes in fluorescence levels in the presence of Baf reflect pure exocytosis, whereas changes in the absence of Baf represent the balance between exocytosis and endocytosis during stimulation, the time course of endocytosis during stimulation could be estimated by simply subtracting the fluorescence values (Fernández-Alfonso and Ryan, 2004; Burrone et al., 2006). We found that in control synapses, ∼40% of the SVs that had undergone exocytosis had already been recovered by endocytosis during a stimulation of 300 APs at 10 Hz. In SCAMP5 KD neurons, however, <10% of the exocytosed SVs had been recovered by endocytosis during that stimulus (Fig. 4A1–B3). The ratio of endocytosis/exocytosis during stimulation, calculated by obtaining the slopes after linear fitting of the time course of endocytosis and exocytosis to the 300 AP train, was significantly reduced in SCAMP5 KD neurons compared with controls (Fig. 4B4), pointing to severe endocytic defects in these cells. Again, the endocytic defects were fully rescued in cells containing HA-SCAMP5 resistant to shRNA (Fig. 4A1–B4). KD of SCAMP5 with an independent shRNA (shRNA#1) gave rise to similar endocytic defects (Fig. 4C1–C3).

The endocytic defects observed during stimulation of SCAMP5 KD neurons became more i/o error 103 netsph when the average time courses of exocytosis and endocytosis in i/o error 103 netsph and KD neurons were compared. There was no statistically significant difference between the average time course of exocytosis from control and KD neurons, i/o error 103 netsph, indicating that the kinetics of SV exocytosis were not affected by SCAMP5 KD and that SCAMP5 functions in the endocytic pathway (Fig. 5A). A composite graph of the average time course of total endocytosis was obtained by combining the time course of endocytosis during stimulation [i.e., (+)Baf − (−)Baf] with the inverse image of the time course of endocytosis after stimulation (Fig. 4A1–A3). Compared with control and SCAMP5 KD-rescued neurons (Fig. 5A, black and blue line, respectively), SCAMP5 KD neurons (Fig. 5A, red line) showed considerable defects in endocytosis during stimulation, whereas poststimulus endocytosis was only mildly affected (Fig. 5A). With prolonged stimulation (900 APs at 10 Hz), the endocytic defects in SCAMP5 KD synapses during stimulation were more pronounced than in the control synapses and virtually no exocytosed SVs were endocytosed (Fig. 5B). This difference was not due to defective acidification because, after a 30 s stimulus, fluorescence was quenched to the same extent as during the prestimulus period (average degree of quenching poststimulus: 94.11 ± 5.88% in control and 95.11 ± 4.88% in KD; Fig. 5C1–C4).

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Figure 5.

Average time courses of endocytosis clearly revealed severe defects in endocytosis during stimulation by SCAMP5 KD. A, Average time course of endocytosis from control (black), KD (red), and rescue (blue) neurons during and after 300 APs at 10 Hz without Baf and the average time course of exocytosis from control (black dotted), KD (red dotted), and rescue (blue dotted) neurons during 1200 APs at 10 Hz with Baf. A composite graph of the average time course of total endocytosis was obtained by combining the time course of endocytosis during stimulation [i.e., (+)Baf − (−)Baf) with the inverse image of the time course of endocytosis after stimulation in Figure 4, A1–A3. The dashed line indicates the extent of exocytosis at the 30 s time point i/o error 103 netsph the endocytosis to exocytosis ratios is calculated. Error bars are shown at two time points on the endocytosis curves. B, Average time course of endocytosis from control (black) and KD (red) neurons during and after 900 APs at 10 Hz without Baf and the average time course of exocytosis from control (black dotted) and KD (red dotted) neurons during 1200 APs at 10 Hz with Baf. Error bars are shown at two time points on the endocytosis curves. C1C2, Average traces of synaptopHluorin signal in control and SCAMP5 KD during pH exchange experiments. The extracellular solution is changed twice from pH 7.4 to 5.5 and back. Extracellular application of pH 5.5 Tyrode solution rapidly quenches all surface sPH in the prestimulus period. After 50 s, the extracellular solution is changed back to pH 7.4. After a 30 s stimulus at 10 Hz, the extracellular solution is changed to pH 5.5 for 50 s then back to pH 7.4. Net sPH fluorescence changes were obtained by subtracting the average intensity of the first pH 5.5 challenge (F5.5) from the intensity of each frame (Ft) for individual boutons and averaged.C3C4, Average degree of quenching poststimulus was 94.11 ± 5.88% in control (n = 5) and 95.11 ± 4.88% in KD (n = 6).

To avoid a possible bias caused by an SV-protein-specific recycling mode (Hua et al., 2011; Raingo et al., 2012; Ramirez et al., 2012), SypHy, a fusion protein of synaptophysin with pHluorin (Granseth and Lagnado, 2008), was used. In agreement with the data obtained from vGpH-transfected neurons, SV endocytosis during stimulation was again severely defective in the SCAMP5 KD neurons and was fully rescued by the expression of HA-SCAMP5 resistant to shRNA (Fig. 6A–D), indicating that the SV retrieval defect caused by SCAMP5 KD is independent of the particular SV protein used.

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Figure 6.

Independent verification of SCAMP5 KD-induced endocytic defects during stimulation using SypHy or FM 1–43. A1A3, Average SypHy fluorescence intensity profiles from control (A1), KD (A2), and rescued (A3) synapses stimulated with or without Baf. Neurons were stimulated with 300 APs at 10 Hz without Baf. After a 10 min resting period, the same neurons were stimulated again with 1200 APs at seagate ss error Hz in the presence of Baf. Fluorescence values were normalized to the maximal stable fluorescence signal in the Baf trace. B, Decay of SypHy after stimulation fitted by a single exponential with time constant, τ = 25.66 ± 4.51 s for control (n = 8), 39.59 ± 5.53 s for I/o error 103 netsph KD (n = 10), 24.87 ± 6.13 s for SCAMP5 KD rescue (n = 8). Data are presented as means ± SE. *p < 0.01 (ANOVA and Tukey's HSD post hoc test). C, Rate of exocytosis and rate of endocytosis during stimulation were obtained from the linear fits to the initial 30 s of traces (exocytic rate: 0.134 ± 0.04 for the control, 0.133 ± 0.04 for SCAMP5 KD, and 0.131 ± 0.06 for SCAMP5 rescue; endocytic rate: 0.055 ± 0.03 for the control, 0.0135 ± 0.04 for SCAMP5 KD, and 0.053 ± 0.04 for SCAMP5 rescue). The ratios of endocytosis/exocytosis rate: 0.410 ± 0.04 for the control, 0.101 ± 0.05 for SCAMP5 KD, and 0.408 ± 0.07 for SCAMP5 rescue. Data are presented as means ± SE. *p < 0.01 (ANOVA and Tukey's HSD post hoc test). D, Average time course of endocytosis from control (black), KD (red), and rescue (blue) neurons during and after 300 APs at 10 Hz without Baf and the average time course of exocytosis from control (black dotted), i/o error 103 netsph, KD (red dotted), and rescue (blue dotted) neurons during 1200 APs at 10 Hz with Baf. The dashed line indicates the extent of exocytosis at the 30 s time point where the endocytosis to exocytosis ratios is calculated. Error bars are shown at two time points on the endocytosis curves. E, Experimental protocol used to compare endocytosis during stimulation and after stimulation using FM1–43. For endocytosis during stimulation, FM1–43 was loaded at the onset of stimulation (300 APs) and immediately washed out with the cessation of stimulation. After a 10 min resting period, 1200 APs at 10 Hz were given to unload and measure the amount of loaded FM1–43. The same i/o error 103 netsph were stimulated again in the presence of FM1–43 and kept in the presence of dye for an additional 30 s after stimulation to label poststimulus endocytosed vesicles. After a 10 min resting period, 1200 APs at 10 Hz were given to unload and measure the amount of loaded FM1–43. F1F2, Pooled average traces of FM1–43 signal of first load (F1) or second load (F2) from the similar size of boutons in control (black) and SCAMP5 KD (red) during 1200 AP stimulation (F1: 1894.33 ± 169.97, n = 8 for control, 387.54 ± 160.83, n = 10 for KD; F2: 4723.83 ± 366.68 for control, 3135 ± 328.78 for KD). F3F4, Normalized average time course of FM1–43 fluorescence intensity of i/o error 103 netsph and second load during unloading with 1200 APs at 10 Hz. Fluorescence values were normalized to the maximal FM1–43 fluorescence signal after second load (first load over second load: i/o error 103 netsph ± 0.082 for control, 0.173 ± 0.039 for KD).

To further eliminate any influence of an SV protein-specific endocytic mode on the analysis, FM1–43, a green fluorescent strylyl membrane dye that is widely used to study SV recycling kinetics (Betz and Bewick, 1992; Ryan et al., 1993; Ryan, 2001), was used. When FM1–43 was present during stimulation of SCAMP5 KD cells, the amount of endocytosed FM1–43 was considerably lower than that of control (20.45 ± 0.05% of the control), but after continued incubation with FM1–43 i/o error 103 netsph stimulation, the difference was substantially reduced (66.36 ± 0.01% over control), again indicating that the severe endocytic defect was restricted to the period of stimulation (Fig. 6F1–F4).

Next, we plotted endocytosis/exocytosis ratios during stimulation of 300 APs at different frequencies. We found that, at the lower stimulation frequency (5 Hz) SCAMP5 KD synapses did not show a significant endocytic defect (endocytosis/exocytosis ratio: 0.790 ± 0.032 for control, 0.747 ± 0.054 for SCAMP5 KD), indicating that SCAMP5 KD synapses performed endocytosis normally when the exocytosis burden was low (Fig, i/o error 103 netsph. 7A). At 10 Hz, however, SCAMP5 KD synapses displayed severe endocytic defects during stimulation (endocytosis/exocytosis ratio: 0.091 ± 0.032; control: 0.421 ± 0.07). At a higher stimulation frequency of 20 Hz, the endocytosis/exocytosis ratio in control synapses was also decreased dramatically, suggesting that the cell's endocytic capacity was saturated terrorism szkoly kavkaza bieslan 2004 video an increased stimulus frequency of 20 Hz (Fig. 7A). The inability of SCAMP5 KD neurons to match the rate of endocytosis to that of the ongoing exocytosis at 10 Hz was corroborated by the finding that, when extracellular [Ca2+] was decreased to 0.75 mm to reduce the SV release probability (i.e., to reduce the exocytic load; Murthy et al., 1997; Trommershäuser et al., 2003), the endocytic defects of the SCAMP5 KD synapses were less severe at 10 Hz (endocytosis/exocytosis ratio: 0.436 ± 0.007 at 0.75 mm [Ca2+]; Fig. 7B).

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Figure 7.

KD of SCAMP5 lowered the threshold of activity at which the SV endocytosis becomes unable to compensate for ongoing the exocytosis during the stimulus. A, Endo/Exo ratios after 300 APs at different frequencies. Dashed lines are linear fits (control: KD = 0.790 ± 0.03: 0.751 ± 0.05 at 5 Hz, 0.421 ± 0.07: 0.091 ± 0.04 at 10 Hz, 0.131 ± 0.06: 0.099 ± 0.03 at 20 Hz). n = 8 for control, n = 8 for KD at each frequency. B, Mean Endo/Exo ratios after 300 APs at 10 Hz in 2 mm or 0.75 mm extracellular [Ca2+]. At 0.75 mm [Ca2+], Endo/Exo ratio: 0.701 ± 0.03, n = 5 for control, 0.436 ± 0.01, n = 7 for KD. Data are presented as means ± SE. *p < 0.01 (Student's t test).

The endocytic defects of SCAMP5 KD synapses at 10 Hz were i/o error 103 netsph observed with 15 s stimulation (150 APs; Fig. 8A1–A3). With a 30 s stimulation at 20 Hz (600 APs), we again found that endocytic capacity was saturated in the control (Fig. 8B1–B3), as in the case with 15 s stimulation at 20 Hz, suggesting that the frequency of stimulation (i.e., how fast exocytosis occurs so that how fast SVs accumulate on the plasma membrane over the endocytic capacity) is important. All of these results indicate that KD of SCAMP5 lowers the threshold of synaptic activity at which SV endocytosis becomes unable to compensate for ongoing exocytosis during stimulation.

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Figure 8.

The frequency, not the duration, of stimulation was important for the observed defects in endocytosis in SCAMP5 KD. A1A2, Average vGpH fluorescence intensity profiles from control (A1) and KD (A2) synapses stimulated with or without Baf. Neurons were stimulated at 10 Hz for 15 s (150 APs) without Baf. After a 10 min resting period, qip error dns lookup failed same neurons were stimulated again with 1200 APs at 10 Hz in the presence of Baf. Fluorescence values were normalized to the maximal stable fluorescence signal in the Baf trace. n = 9 for control, n = 10 for KD. A3, Average time course of endocytosis from control (black) and KD (red) neurons during and after 150 APs at 10 Hz without Baf and the average time course i/o error 103 netsph exocytosis from control (black dotted) and KD (red dotted) during 1200 APs at 10 Hz with Baf. Error bars are shown at two time points on the endocytosis curves. B1B2, Average vGpH fluorescence intensity profiles from control (B1) and KD (B2) synapses stimulated with Baf or without Baf. Neurons were stimulated at 20 Hz for 30 s (600 APs) without Baf. After 10 min resting period, the same neurons were stimulated again with 1200 APs at 20 Hz in the presence of Baf. Fluorescence values were normalized to the maximal stable fluorescence signal in the Baf trace. n = 11 for control, n = 11 for KD. B3, Average time course of endocytosis from control (black) and KD (red) neurons during and after 600 APs at 20 Hz without Baf and the average time course of exocytosis from control (black dotted) and KD (red dotted) during 1200 APs at 20 Hz with Baf.

Discussion

Most previous studies of the SCAMPs have focused on the regulation of exocytosis during DCV secretion or vesicle trafficking in the TGN. SCAMP1 plays a dual role in facilitating dilation and closure of fusion pores and has been implicated in exo-endocytic coupling and in the regulation of DCV i/o error 103 netsph in PC12 cells (Fernández-Chacón et al., 1999; Fernández-Chacón et al., 2000; Zhang and Castle, 2011). SCAMP2 is known to interact with Arf6, phospholipase D1, and phosphatidylinositol 4,5-bisphosphate via its E-peptide 2–3 cytoplasmic loop domain (CWYRPIYKAFR) and regulates fusion pore formation during DCV exocytosis (Guo et al., 2002; Liu et al., 2002; Liu et al., 2005; Liao et al., 2007). In addition, it interacts with the mammalian (Na+,K+)/H+ exchanger NHE7 in the TGN and participates in the shuttling of NHE7 between recycling vesicles and the TGN (Lin et al., 2005). The NPF repeats of SCAMP1 are also known to bind to two EH domain proteins: intersectin 1, which is involved in endocytic budding at the plasma membrane, and γ-synergin, which may mediate the budding of vesicles in the I/o error 103 netsph (Fernández-Chacón et al., 2000). Expression of SCAMP1 without the N-terminal NPF repeats potently inhibits transferrin uptake by endocytosis (Fernández-Chacón et al., 2000).

Compared with other SCAMPs, however, less is known about the role of SCAMP5. One recent study showed that its expression is markedly increased in the striatum of Huntington's disease patients and that its downregulation alleviates ER stress-induced protein aggregation in huntingtin mutants and the inhibition of endocytosis (Noh et al., 2009). Another study showed that human SCAMP5 interacts directly with synaptotagmin via its cytosolic C-terminal tail and is involved in calcium-regulated exocytosis of signal-peptide-containing cytokines (Han et al., 2009).

However, we found here that KD of SCAMP5 caused endocytic defects during strong stimulation of cultured hippocampal neurons, whereas exocytic kinetics were not affected. The difference in the effects of SCAMP5 could be due to mechanistic differences in exo-endocytosis between non-neuronal cells and neurons during intense stimulation. Unlike neurons, non-neuronal cells are never exposed to such strong activation in vivo. This explanation is consistent with the fact that KD synapses displayed little or no defects in endocytosis when stimulated at low frequency (5 Hz). It seems that, in SCAMP5 KD neurons, the endocytic capacity to cope with heavy exocytic loads is reduced, whereas the endocytosis activity of individual SVs during mild exocytic loads remains largely unaffected. In addition, although we did not find any defects in exocytosis in SCAMP5 KD synapses, we cannot completely rule out the possibility that SCAMP5 has an effect on exocytosis because the effect of SCAMP5 on single SV fusion kinetics was not tested. Instead, we tested the macroscopic kinetics of exocytosis upon sustained stimulation and this stimulation might mask subtle changes in unitary SV fusion kinetics.

Although total SV pool size varies from bouton to bouton even in a single neuron, when we compared the pooled average amplitude of signals from control and SCAMP5 KD boutons of similar size, SCAMP5 KD resulted in a reduction of total SV pool size as well as of that of the recycling pool. The ratio of recycling/resting pool size was, however, significantly increased. Because endocytosis after stimulation was only moderately affected and exocytosis kinetics were not affected by SCAMP5 KD, we speculate that the change in this ratio could be due to a compensatory mechanism to allow neurons to maintain synaptic transmission during high levels of activity.

Our results further i/o error 103 netsph that I/o error 103 netsph is essential when neuronal activity is high and a heavy endocytic load is imposed on the cell. This is reminiscent of a study on the selective requirement for dynamin-1 during high levels of neuronal activity (Ferguson et al., 2007). The authors of that study found that dynamin-1 was dispensable for the endocytic recycling of SVs but became essential when an intense stimulus imposed a heavy load on endocytosis (Ferguson et al., 2007). In addition, a recent study showed that synaptophysin knock-out (syp−/−) synapses exhibit defective SV endocytosis both during and after neuronal activity, whereas exocytosis and the size of the total recycling pool of SVs were unaffected (Kwon and Chapman, 2011). That study also found that syp−/− neurons displayed pronounced synaptic depression and slower recovery final cut pro voice over error the recycling SV pool after depletion. Interestingly, synaptophysin and SCAMP5, together with synaptogyrin, are tetraspan vesicle membrane proteins (TVPs) because they have four transmembrane regions and cytoplasmically located termini (Hübner i/o error 103 netsph al., 2002). Although previous studies found little or no phenotypic defects in neurons of knock-out mice lacking synaptophysin, synaptogyrin-1, or SCAMP-1 (Eshkind and Leube, 1995; McMahon et al., 1996; Fernández-Chacón et al., 1999; Janz et al., i/o error 103 netsph, 1999), our current data strongly suggest that the TVPs on SVs contribute to subtle neuronal functions such as the control of SV recycling during sustained neuronal activity.

SCAMP5 does not contain an N-terminal NPF motif and is not known to interact with dynamin-1 directly, but we have found that there i/o error 103 netsph putative AP-2-binding sites (YXXφ) in its N terminus and 2–3 loop region. Therefore, SCAMP5 may interact with dynamin-1 and other endocytic proteins directly or indirectly to effectively accomplish endocytosis during intense stimulation. Further studies should focus on the molecular mechanisms through which SCAMP5 interacts with other endocytic proteins to control SV recycling.

In conclusion, SCAMP5 functions to control the SV recycling machinery when neuronal activity is high enough to impose a heavy load on endocytosis. It may recruit or promote the assembly of endocytic components to maintain an adequate number of endocytic machines during sustained neuronal activity. Our data support recent suggestions that changes in the expression of SCAMP5 in Huntington's disease and autism may be related to the synaptic dysfunction observed in these patients.

Footnotes

This work was supported by the Biomembrane Plasticity Research Center funded by the National Research Foundation of Korea (Grant 20100029395 to S.C.).

The authors declare no competing financial interests.

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