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Depression and frequency facilitation at a synapse inAplysia californica: Evidence for regulation by availability of transmitter
Brain Research, 76 (1974) 267-280 ~ Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
267
DEPRESSION AND F R E Q U E N C Y F A C I L I T A T I O N AT A SYNAPSE IN A P L Y S I A CALIFORNICA: EVIDENCE FOR R E G U L A T I O N BY AVAILABILITY OF T R A N S M I T T E R
WERNER T. SCHLAPFER, PAUL B. J. WOODSON, JACQUES P. TREMBLAY AND SAMUEL H. BARONDES
Department t~fPsyekiatl3', Veterans Administration Hospital, San Diego, Calif. 92161 and Departments of Psychiatry and Neuroseiences, School of Medicine, University of CaliJornia, San Diego, La Jolla, Calif 92037 (U.S.A.) (Accepted March 5th, 1974)
SUMMARY
Synaptic depression and frequency facilitation were observed in the monosynaptic and unitary excitatory postsynaptic potential (EPSP) obtained in the 'parabolic burster cell' (R15) by stimulating the right visceropleural connective in Aplysia californica. During trains of stimulation at 0.5-2 c/sec the size of the EPSP was initially depressed during the first few stimuli and subsequently slowly increased to reach a potentiated steady state after a few hundred stimuli. The relative amount of depression between the first two EPSPs depended on (a) the inter-stimulus interval, and (b) the size of the first EPSP. The larger the first EPSP the larger the percentage depression whether variations in the size of the first EPSP were due to natural interanimal variability or experimental manipulation of the concentrations of Ca 2+, Mg 2~ and Co 2~. The amount of frequency facilitation (expressed as the ratio of the 100th EPSP to the first EPSP of a train) increased with increasing frequency of stimulation and was inversely related to the log of the size of the first EPSP both for natural variations in EPSP size and manipulations of the EPSP size by changing the divalent cation concentrations. Neither the level of hyperpolarization of R 15 nor partial curarization affected the relative depression and potentiation. We suggest that the initial depression is largely due to depletion of a rather small pool of transmitter available for release, while the frequency facilitation is the result of a Ca 2~ and frequency dependent increase in the net rate of supply of transmitter into the available pool.
INTRODUCTION
During and after repetitive stimulation of many synapses the size of the post-
268
w.T. SCHLAPFERet al.
synaptic potential (PSP) or end-plate potential (EPP) may decrease (synaptic depression) or increase (synaptic facilitation or potentiation). These phenomena have been observed at many synapses throughout the nervous system4, re. They have been examined in greatest detail in neuromuscular junctions z,17,19,28 and, under physiological conditions, have usually been attributed to variations in the amount of transmitter released by the presynaptic terminal rather than to changes in the postsynaptic sensitivity to the transmitter substance. There are essentially two classes of mechanisms which may account for the presynaptic variations in transmitter release. One mechanism determines the fraction of the available pool of transmitter which is released. The second mechanism regulates the size of the available pool of neurotransmitter. Supply into the available pool is generally explained in terms of increased 'mobilization' of neurotransmitter and is presumed to be due either to increased synthesis of neurotransmitter or to increased rate of transfer from a storage pool to an available pool or both 4. The depression of the EPP, most commonly observed in vertebrate neuromuscular junctionsa, 2z has been ascribed to progressive exhaustion of the pool of available transmitter2°,24; although the depression may be accentuated by a concurrent decrease in the fractional release of transmitter ~. The increase in the size of the PSP as a result of preceding stimulation may take a variety of forms. For a brief period after a stimulus, a second action potential results in increased amounts of transmitter released ~. This short-term facilitation has been attributed to an increase in the fraction of the available pool of transmitter which is released 11. Low frequency stimulation, at stimulus intervals longer than the duration of short-term facilitation, results in a progressive increase of the EPP in MgZ -poisoned vertebrate neuromuscular preparations is. This has been termed frequencT potentiation or frequency facilitation. It is believed to be due to increased mobilization of neurotransmitter produced by repetitive stimulation ~s. In crustacean neuromuscular junctions where a similar phenomenon is observed, progressive depolarization of the nerve terminals has been suggested as a potentiating mechanism 17. Another facilitating phenomenon is post-tetanic potentiation (PTP) t2 which refers to the rather long-lasting (minutes and up to hours) increase in PSP size after a period of tetani. The relationship of PTP to short-term facilitation or frequency potentiation is at present not clear TM. Conclusive evidence regarding the mechanisms underlying history-dependent changes in transmitter release remains to be provided. In particular, the evidence that frequency facilitation is due to increased mobilization of neurotransmitter rests primarily on studies of the neuromuscular junction performed under high magnesium. In the course of an investigation of an identified cholinergic synapse on the parabolic burster cell (R 15)5,22 of the abdominal ganglion of Aplysia calijornica, we observed all four of the phenomena described above while the preparation was perfused with physiological media. Since all these phenomena could be sequentially produced at this synapse, the interaction of the mechanisms which produce them might be examined. In the present report, we describe detailed studies of depression
PLASTIC SYNAPTIC TRANSMISSION IN
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and frequency facilitation at this synapse. The results strongly support the hypothesis that both these phenomena are due to presynaptic regulatory mechanisms and that depression is due to transmitter depletion whereas frequency facilitation is due to increased transmitter mobilization. The post-tetanic potentiation of this junction will be analyzed in a subsequent communication. METHODS
Aplysia californica (100-1200 g) were collected locally and kept in tanks supplied with running seawater. The abdominal ganglion was dissected out with the visceropleural connectives, cut near the pleural ganglia and pinned to the paraffin floor of a recording chamber. The preparation was perfused at a rate of about 1 ml/min with aerated artificial seawater (460 mM NaCl, 10.4 m M KCI, 11.0 m M CaCl2, 55 mM MgCI2, 10 mM Tris-(hydroxymethyl)-aminomethane adjusted to pH 7.7) fortified with nutrients (per 1000 ml of artificial seawater: 10 g dextrose, 2 ml of 50 × concentrated amino acid mixture for minimum essential medium Eagle (MEM-E), 1 ml of 100 × concentrated vitamin mixture for MEM-E, 0.2 mmoles L-glutamine, and 500 units each of penicillin and streptomycin) to prolong survival of the isolated ganglion. Experimental changes in the ionic composition were compensated by appropriate adjustments of the NaCl content of the normal solution. A thermoelectric cooling unit kept the temperature at 15 ± 0.5 °C in the recording chamber. The right visceropleural connective was stimulated with a suction electrode driven through a stimulus-isolation unit by a Grass stimulator, lntracellular potentials were recorded in R 15 with 3 M KC1 filled microelectrodes of 2-10 Mr2 resistance which were gently tapped through the ganglion sheath. From a normal resting potential of 50-60 mV, R 15 was hyperpolarized to a level of 70-80 mV, to suppress its spontaneous bursting activity, either by injecting current through the recording electrode with a bridge circuit or through a second microelectrode. The recording electrode was connected through a high-input impedance, negative-capacitance amplifier (W-P Instruments Model M 701) to the display system (Tektronix 564 storage oscilloscope and Brush 220 high-speed pen recorder). The indifferent electrode consisted of an agar-seawater bridge connecting the recording chamber with a pool of 3 M KC1, containing a large Ag-AgCl electrode. The amplitude of the EPSP was measured from the Brush recorder trace. Up to 48 h of continuous intracellular recording from the soma of R 15 was possible under these conditions. These experiments were conducted between March and October 1973. RESULTS
Changes in synaptic efficacy during repetitive stimulation Cell R 15 receives a large excitatory input from an axon in the right visceropleural connective 5. This EPSP appears to be unitary and monosynaptic based on the criteria of all-or-none responsiveness and constant latency (Fig. 1), even during stimulation at high frequencies (10 stimuli/sec) a3. Transmission across this junction is likely to be cholinergic since the EPSP can be completely blocked by 5 × 10 4 M
270
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5.,v-I" Fig. 2. Characteristic EPSPs during a train of 100 stimuli to the right connective at 2 pulses/sec in R 15, hyperpolarized to suppress spontaneous bursting. The size of the EPSP decreases during the first few pulses (initial depression) and then slowly rises to a potentiated plateau (frequency facilitation).
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Stimuli/ sec Fig. 3. A: facilitation or depression of the second of two EPSPs expressed as the ratio of the second to the first EPSP (EPSP~/EPSP1) depending on the interstimulus interval. Data from 9 animals were pooled. B: frequency facilitation (ratio of average size of the last few EPSPs to the first EPSP) (EPSP100/EPSPI) during a train of 100 stimuli at various frequencies. Average from 5 animals with several trains each separated by 30 min. during a train of stimuli indicating that no other EPSPs are recruited during repetitive stimulation. D o u b l e s t i m u l a t i o n . The ratio of the amplitudes of the second EPSP to the first EPSP (EPSP~/EPSP1) depends on the stimulus interval. At short intervals (I ~ 250 msec) the second EPSP is usually larger than the first one (EPSP2/EPSP1 2: 1.0) (short-term facilitation) while at larger intervals (I > 500 msec) the second EPSP is depressed (EPSP2/EPSP1 < 1.0) (Fig. 3A). There is considerable variability in the ratio EPSP2/EPSP1 from animal to animal. At a stimulus interval of 500 msec, EPSP2/EPSP1 was greater than 1.0 in 16~o, equal to 1.0 in 2 2 ~ and smaller than 1.0 in 62 ~ of the 45 animals tested in this fashion. After a rest period of about 20-30 rain from the beginning of an experiment, the amplitude of the first EPSP ranged from 0.2 m V to 36 m V in different animals at similar levels o f hyperpolarization (70-80
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mV). The variability in E P S P t correlates well with the interanimal variation in the ratio EPSP2/EPSP1; the larger the first E P S P in any particular animal the smaller the ratio EPSP~/EPSP1, i.e., the larger the depression of the second with respect to the first EPSP (Fig. 4A).
Trains of stimulation. The size of the EPSP first decreases and usually reaches a m i n i m u m after about 4-8 pulses, then slowly rises during repetitive stimulation to a steady-state amplitude. At a frequency o f 2 stimuli/see the amplitude o f the E P S P reaches a plateau after about 400 stimuli, a level which can be maintained in m o s t preparations for 3000-4000 pulses, after which it slowly falls. Because o f the p r o l o n g e d period of post-tetanic potentiation which follows a train of 400 stimuli, we have
PLASTIC S Y N A P T I C TRANSMISSION IN
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usually limited the train length to 100 stimuli so that experiments could be repeated at 20-30-rain intervals with little change in the response properties. By 100 pulses, the amplitude of the EPSP was usually about 80 9o of the maximum. The size of the EPSP reached during a train of 100 stimuli, expressed as the ratio of the average size of the last 3 EPSPs to the size of the first EPSP of the train (EPSP100/EPSP~), depends on the frequency of stimulation (Fig. 3B). Even at frequencies that correspond to intervals at which EPSP2/EPSP1 is less than 1.0 (0.05-2 c/sec), EPSP100/EPSP1 is equal to or larger than 1.0. This implies that linear summation of the curve of Fig. 3A cannot account for the potentiation observed upon multiple stimulation. As in the case of EPSP2/EPSP1, there was a large variability among animals in the magnitude of EPSPI00/EPSP1. In addition, the ratio EPSPa00/EPSP1 showed a strong inverse correlation with the size of the initial EPSP of a preparation (Fig. 4B).
Postsynaptic modifications of the EPSP size If the size of the EPSP is altered postsynaptically by changing the level of hyper-
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Fig. 5. Effect of D-tubocurarine chloride on (A) the size of EPSP1 and (B) on the ratios EPSP2/EPSP1 and EPSPlo0/EPSP1 during trains of stimulation of 100 pulses at 2 stimuli/sec repeated at 20-30 min intervals. The ganglion was bathed at each D-tubocurarine concentration until the EPSP size reached a constant level. The values obtained from several runs at constant curare concentration were averaged. Data pooled from 4 animals.
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TABLE I*
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4~ EFFECTS OF D I V A L E N T C A T I O N S ON T H E SIZE Or" T H E
EPSPs, THE 1NITfAL D E P R E S S I O N , A N D F R E Q U E N ( Y
Control
Treatment
EPSPI (mV)
Depression**
Frequency facilitation***
11.9 3.75 12.7 17.4 34.7 14.7
--0.15 --0.11 -0.08 --0.12 0.17 0.17
1.16 1.40 1.40 1.06 0.86 0.93
10.2 20.2 16.4 16.5 13.8 44.6 44.6
--0.14 0.19 -0.21 -0.38 0.07 0.21 -0.21
25.5 14.4
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2 3 3 3 3 1/5
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22.0 16.7 45,6 39.7 51.0 20.4
185 445 359 228 146 139
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1t3 182 175 150 118 135
0,89 0,70 0.76 0.81 0,72 0.86
78 50 54 76 84 92
1.08 0.93 0.99 0.74 1.25 0.66 0.66
2 < Mg ~3 x Mg -~ 1/5 >: Ca 2-~~ 2 m M C o ~= 4 m M C o e20 m M C o e 30 m M Co ~~
3.1 5.0 2.3 11.0 9.9 11.0 5.8
30 25 14 67 72 25 13
~-0.07 0.13 --0.03 -0.24 0.02 -0.15 ....0.01
50 68 14 63 29 71 5
1.64 1.60 2.39 0.97 1.46 1.00 1.32
152 172 241 131 117 151 200
0.93 1.15
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24.3 13.6
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114 93
* Except for the 20 m M and 30 m M and Co 2- treatments which were done consecutively on the same preparation, each line in this table represents a different animal. All values were averages of 3-5 trains of 100 stimuli each at 2 stimuli/sec interspersed with 30-min rest periods. Sufficient time was allowed for stabilization of the values after inf\lsion of the experimental solutions (usually, 30-60 min). In the experimental solutions the NaCI concentration was adjusted to preserve isotonicity. Similar changes in the NaCI concentration only (adjusting the isotonicity with sucrose) did not result in any changes in EPSPj, depression and freqtiency facilitation, ** Depression is defined as the fractional decrease o f the size of Ihe second E PSP (EPSP:O in a trai~ compared ,to the first E PSP (EPSP~) a t a 500 msec interval, hence depression - (EPSP2-EPSPD/EPSP~. *** Frequency facilitation is defined as the ratio of the size of the 100th EPSP (f!PSPto,,i to the first EPSP of a train at 2 stimuli/see hence frequency facilitation .... EPSPI001EPSPb
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Fig. 6. Effect on EPSPa00/EPSP1 of(A) increasing Mg~ from 55 mM to 165 mM (decreasing EPSPi from 18 mV to 3 mV) and (B) increasing Ca2~ from 11 mMto 33 mM (increasing EPSPz from 8 mV to 40 mV). Stimulationat 2/sec for 100 pulses every 20 rain. Each point corresponds to one stimulus train during slow infusion and washing out of the experimental medium. Solid line is linear regression analysis of experimental points. polarization of the postsynaptic cell or by partial curarization of the preparation, the relative depressions and potentiations are unchanged. Although a 60 mV increase in the level of hyperpolarization (from 40 to 100 mV) more than doubled the size of the EPSP (from 11 to 26 mV), the relative values of both the initial depression (EPSPe/ EPSP1) and the frequency facilitation (EPSP100/EPSP1) were unaltered. Partial blocking of the postsynaptic receptor by D-tubocurarine chloride lowered the EPSPs but did not affect the ratios EPSP2/EPSP1 or EPSPI00/EPSP1 (Fig. 5). A similar result was obtained when EPSP1 was reduced to 25 ~o of normal by adding 10 -4 M atropine to the bathing medium.
Modifications of transmitter release If the amount of transmitter release is altered by varying the concentration of calcium or magnesium ions in the medium 1°, both the initial depression and the frequency facilitation are changed (Table 1). These divalent cations affect the size of the first EPSP of a train to a larger degree than the subsequent EPSPs. Depression [(EPSP2-EPSP1)/EPSPa] is increased in comparison with the control by raising the Ca '~ concentration and is decreased by raising the Mg '~+ concentration (Table I). Conversely, frequency facilitation (EPSP~00/EPSP1) is increased by comparison with the control by raising the Mg 2~ concentration or lowering the Ca 2+ concentration (Table I). Frequency facilitation varies inversely with the log of EPSP1 (Fig. 6), when EPSP1 is varied with Mg 2~ or Ca 2=. Appropriate increases in both Ca ~+ and Mg 2~ (e.~., 1.5 > Ca e~ and 2.5 × Mg ~ ) balance out so that EPSP1, EPSP2 and EPSPn)o are identical with the control (Table !). Since cobaltous ions have been shown to interfere with spike-dependent calcium
276
w. 1. SCHLAPFERel at.
influx in Aplysia neurons and other systems v and thereby reduce transmitter release, we tested addition of cobaltous ions to the medium. Like Mg 2+, cobaltous irons reduce the size of all EPSPs, but decrease the relative depression and increase the relative amplitude of frequency facilitation (Table 1). DISCUSSION
The large monosynaptic, unitary, and presumably cholinergic EPSP observed in R 15 by stimulation of the right visceropleural connective particularly lends itself to the study of factors which modify the efficacy of a synapse in the central nervous system. Not only can one return to the same junction in animal after animal, but the large size of the postsynaptic cell allows prolonged recordings so that many phenomena can be studied in the same specimen. In the present study, depression and frequency facilitation have been identified and studied at this synapse. The evidence suggests that both are based on presynaptic changes. This interpretation rests heavily on previous studies with neuromuscular junctions in which the quantal nature of cholinergic end-plate potentials has been studied in detail 3. Although quantal studies have not yet been done on this EPSP the phenomena lend themselves well to explanations based on changes in the amount of transmitter released during repetitive stimulation. Depression is apparently due to transient transmitter depletion, whereas frequency facilitation is apparently due to increased net supply of available transmitter. Changes in the amplitude of postsynaptic potentials could be due to changes in either the amount of transmitter released or in the receptor sensitivity. Receptor desensitization and sensitization (decreased or increased responsiveness to a constant amount of transmitter) has been suggested as the cause of decrementing or potentiating responses to presynaptic stimulation in some dual-action synapses in Aplysia 6,2~. The depression observed in the present study does not appear to be due to receptor desensitization because of the effects of curare and atropine. Were desensitization responsible for the depression which we observed, curare, which competes with acetylcholine for the receptor, should reduce the amount of transmitter reaching the receptor and interfere with desensitization or sensitization. Therefore if the depression is due to receptor desensitization, curare would be expected to change the ratio of EPSP2 to EPSP1. Similarly, if the frequency potentiation were due to receptor sensitization a change in the ratio of EPSP100 to EPSP1 would be predicted with curare. However, as shown in Fig. 5, these ratios are uninfluenced by the addition of D-tubocurarine chloride to the medium, in clear distinction to the change in these ratios which is observed when the amount of transmitter reaching the receptor is varied presynaptically with Ca z+ and Mg 2+. The differential action of curare, on one hand, and Ca z+ and Mg ~+ on the other, is analogous to the action of these agents in vertebrate neuromuscular preparations, where it has been shown by more direct means 3,~1 that depression and facilitation have a presynaptic origin. This suggests that in the system under investigation short-term facilitation, frequency facilitation and depression are due to presynaptic regulatory processes. In the absence of changes in the receptor sensitivity of the postsynaptic cell, the size of an EPSP can be taken as an index of the amount of transmitter released ~,
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although a 'ceiling' effect is possible when large amounts of transmitter are released 14. The amount of transmitter release (R) may be considered a function of two parameters: the size of the available transmitter pool (A) and the fraction (F) of the available transmitter released by a presynaptic action potential; hence R F.A. Buring repetitive stimulation either or both of these parameters can change to produce alterations in the amplitudes of the EPSPs. The present experiments suggest that depression is due to a depletion of A and that frequency facilitation is due to an increased rate of supply into A.
Depression and transmitter depletion The argument that depression is due to a decrease in available transmitter is based largely on the change in intensity of depression when transmitter release was modulated by calcium and magnesium. Since increasing the magnesium concentration (which reduces transmitter release) 1° decreases the depression, while increasing the calcium concentration (which augments transmitter release)lO, 14 increases the depression it is very likely that the depression we observe is mainly due to a stimulus dependent depletion of a rather small pool of either transmitter or some other material needed for release. A similar conclusion is reached if the transmitter release is decreased by adding cobalt chloride which decreases spike dependent Ca ~+ influx into Aplysia neuronsL Such treatment greatly reduces the size of the first EPSP, but as with magnesium, EPSP2 is less affected, so that the depression decreases or sometimes even reverses to a facilitation (Table 1). This agrees well with similar observations in neuromuscular junctions where the depressions have usually been interpreted to be at least partially due to transmitter depletion z3. In addition to these experimental manipulations of the size of the EPSPs, the natural interanimal variability in the size of the first EPSP correlated with the depression between the first and second EPSP in that the larger the first EPSP (the more transmitter released), the larger the depression of the second EPSP (Fig. 4A). This also suggests that depletion of a pool is responsible for the initial depression.
FrequencyJacilitation and transmitter mobilization The argument that frequency facilitation is based on increased availability of transmitter rests largely on the inference that availability of transmitter may limit the efficacy of this junction as shown by its initial depression. Since frequency facilitation can be maintained during prolonged periods of stimulation, the available transmitter pool either has to be very large or transmitter has to be mobilized from a storage pool 2 or synthesized at a rate equal to the rate of release. The initial depressed phase has indicated that the available pool of material needed for release is rather small in this system, as in others4, 9. Therefore, an increased rate of supply of material into this pool is a necessary and sufficient condition for frequency potentiation at this junction. An increase in the fractional release during repetitive stimulation, although possible, cannot by itself account for the frequency potentiation. In the absence of a net increase in the supply process, the small available pool would be rapidly depleted and an increased fractional release would only exaggerate the depletion and therefore de-
278
w.T. SCHLAPI~ERet at.
crease the amplitude of the EPSP. At the potentiated plateau there is an equilibrium between the amount of transmitter released and supplied. Therefore the size of the EPSPs at the plateau is an indication of the amount of transmitter supplied into the available pool between any two stimuli. A process of transmitter mobilization responsible for potentiating phenomena in neuromuscular junctions has been proposed is, but disputed more recently ~J. However, frequency facilitation in vertebrate neuromuscular systems is only seen when release of transmitter has been reduced to a very low leveP s. In crayfish neuromuscular junctions, frequency facilitation has been shown to be due to an elevation of p, the statistical parameter of q uantal probability of release "7,2s. However, p is the compound of two probabilities, pl, the probability of occupying a release site, and p2, the probability that a site will release transmitter, the two parts of p being indistinguishable by the statistical methods used ~,~. In the analysis presented here, changes in p~ would be reflected as changes in the rate of supply into the available pool. The increased rate of supply into the available pool could be realized either by an increased rate of synthesis or by a process of mobilization from a larger storage pool. We cannot, at present, distinguish between these alternatives. The supply process has to be frequency dependent, since a larger steady-state EPSP amplitude is obtained at higher frequencies of stimulation (Fig. 3B). This process is also influenced by the external calcium and magnesium concentrations, since the absolute size of the steady-state EPSP during repetitive stimulation is increased at higher Ca ~4 concentrations and decreased at higher Mg 2~ concentration. The changes in the amount of frequency facilitation brought about by variations in the divalent cation concentration in the bath (Table I and Fig. 6) are due to the relatively greater effect that these agents have on the size of the first EPSP of a train with the subsequent EPSPs affected progressively less. The size of the first EPSP of a train is primarily a function of the fraction of available transmitter which is released, whereas the size of the steady-state EPSP is determined by the rate of transmitter supply into the available pool. Between different but similarly treated ani reals, considerable variability was found in the size of the control EPSPs (the first EPSP after the start of an experiment), their susceptibility to depression and their ability to show frequency l'acilitation. The larger the amplitude of the first EPSP in a particular animal, the tess frequency facilitation (EPSPt00/EPSP1) it exhibited. The interanimal variability qualitatively resembled the experimentally induced changes in EPSP2/EPSPI (Table 1) and EPSP100/EPSP1 (Fig. 6 ) w i t h Mg 2~, Ca 2~ and Co ~ . This suggests that different animals have different probabilities of transmitter release. This may be due to variations in the size of the synaptic release areas aS,''l, different sizes of terminal action potentials I v or the past history of the synapse in different animals. From this series of arguments, we conclude that depression at the synapse under investigation is due to transmitter depletion and that frequency facilitation is due to an increased net rate of transmitter supply into a pool available for release. Since preparation of this report, we have learned that Halstead and Jacklet ~ have studied frequency facilitation at this synapse. The preparation which they studied was not hyperpolarized so that the spontaneous activity of the parabolic bursle~" cell was
PLASTIC SYNAPTIC TRANSMISSION IN
Aplysia
279
s u p e r i m p o s e d on their records. Depression was n o t observed in their study possibly because the r h y t h m i c v a r i a t i o n in the m e m b r a n e potential p a r t i a l l y obscured the depression. Because o f this a p p a r e n t discrepancy, we have sought and repeatedly observed depression under conditions like those used by H a l s t e a d and Jacklet, especially in cells where the interval between bursts was long. Effects o f calcium and magnesium on frequency facilitation which they observed are similar to those reported here. These a u t h o r s also concluded t h a t the facilitation o f the EPSP in this system has a presynaptic origin since no change in p o s t s y n a p t i c m e m b r a n e resistance was seen during facilitation. ACKNOWLEDGEMENTS This w o r k was s p o n s o r e d by the Veterans A d m i n i s t r a t i o n Hospital, San Diego, under M R I S Nos. 7734(01) and 0818(01) a n d by N 1 M H G r a n t No. 18282. P.B.J.W. was s u p p o r t e d by U S P H S P r e d o c t o r a l Traineeship in Neurosciences, U S P H S 5-T01NS-05628-05, a n d J.P.T. by the C a n a d i a n Medical Research Council. W e are very grateful to Dr. E. R. K a n d e l , who p o i n t e d out to us the usefulness o f this j u n c t i o n for the study o f facilitation. We t h a n k Mr. G a r y Smith for his excellent technical support.
REFERENCES 1 BETZ, W. J., Depression of transmitter release at the neuromuscular junction of the frog, J.
Physiol. (Lond.), 206 (1970) 629-644. 2 BmKS, R., AND MACINTOSH,F. C., Acetylcholine metabolism of a sympathetic ganglion, Canad. J. Biochem., 39 (1961) 787-827. 3 DEL CASTmLO,J., AND KATZ, B., Statistical factors involved in neuromuscular facilitation and depression, J. Physiol. (Lond.), 124 (1954) 574-585. 4 ECCLES,J. C., The Physiology of Synapses, Springer, Berlin, 1964, pp. 82 93. 5 FRAZ1ER, W. T., KANDEL, E. R., KUPFERMANN, L, WAZIRI, R., AND COGGESHALL, R. E., Morphological and functional properties of identified neurons in the abdominal ganglion of Aplysia caliJbrnica, J. Neurophysiol., 30 (1967) 1288-1351. 6 GARDNER, D., AND KANDEL, E. R., Diphasic postsynaptic potential: a chemical synapse capable of mediating conjoint excitation and inhibition, Science, 176 0972) 675 678. 7 GEDULDIG, D., AND JUNGE, D., Sodium and calcium components of action potentials in the Aplysia giant neurone, J. Physiol. (Lond.), 199 (1968) 347-365. 8 HALSTEAD,D. C., AND JACKLET, J, W., Effects of calcium and magnesium on facilitation of a unitary synaptic potential in neuron R 15 of Aplysia, Comp. Biochem. Physiol., 47A (1974) 991-1003. 9 HUBBARD, J. I., Repetitive stimulation at the mammalian neuromuscular junction, and the mobilization of transmitter, J. Physiol. (Lond.), 169 (1963) 641-662. 10 HUBBARD,J. I., JONES, S. F., AND LANDAU,E. M., On the mechanism by which calcium and magnesium affect the release of transmitter by nerve impulse, J. Physiol. (Lond.), 196 0968) 75-86. ] 1 HUBBARD,J. L, JONES, S. F., AND LANDAU, E. M., The effect of temperature change upon transmitter release, facilitation and post-tetanic potentiation, J. Physiol. (Lond.), 216 (1971) 591-609. 12 HUGHES,J. R., Post-tetanic potentiation, Physiol. Rev., 38 (1958)91-113. 13 KANDEL, E. R., AND TAUC, L., Mechanism of heterosynaptic facilitation in the giant cell of the abdominal ganglion of Aplysia depilans, J. Physiol. (Lond.), 181 (1965) 28 47. 14 KATZ, B., AND MILEDI, R., Further study of the role of calcium in synaptic transmission, J. Physiol. (Lond.), 207 (1970) 789-801.
280
W.T. SCHLAPFER et aL
15 KUNO, M., TURKANIS, S. A., AND WEAKLY, J. N., Correlation between nerve terminal size and transmitter release at the neuromuscular junction of the frog, J. Physiol. (Lond.), 213 (1971) 545556. 16 LANDAU, E. M., SMOLINSKY, A., ANO LASS, Y., Post-tetanic potentiation and facilitation do not share a common calcium-dependent mechanism, Nature New Biol., 244 (1973) 155-157. 17 LANG, F., AND ATWOOD, H. L., Crustacean neuromuscular mechanisms: functional morphology of nerve terminals and the mechanism of facilitation, Amer. Zool., 13 (1973) 337--355. 18 MAENO, T., AND EDWARDS, C., Neuromuscular facilitation with low-frequency stimulation and effects of some drugs, J. Neurophysiol., 32 (1969) 785-792. 19 MALLART, A., AND MARTIN, A. R., An analysis of facilitation of transmitter release at the neuromuscular junction of the frog, J. Physiol. (Lond.), 193 (t967) 679-694. 20 OTSUKA, M., ENDO, M., AND NONOMURA, Y., Presynaptic nature of neuromuscular depression, Jap. J. Physiol., 12 (1962) 573-584. 21 SHERMAN, R. G., AND ATWOOD, H. L., Correlated electrophysiological and ultrastructurat studies of a crustacean motor unit, J. gen. Physiol., 59 (1972) 58(~615. 22 STRUMWASSER,F., The demonstration and manipulation of a circadian rhythm in a single neuron. In J. ASCHOFF(Ed.), Circadian Clocks, North Holland Publ., Amsterdam, 1965, pp. 442-462. 23 TAKEUCHI, A., The long-lasting depression in neuromuscular transmission of frog, ,lap. Ji Phy~iol., 8 (1958) 102-113. 24 THIES, R. E., Neuromuscular depression and the apparent depletion of transmitter in mammalian muscle, J. Neurophysiol., 28 (1965) 427-442. 25 VERRE-JONES, D., Simple stochastic models for the release of quanta of transmitter from a nerve terminal, Aust. J. Statist., 8 (1966) 53-63. 26 WACHTEL, H., AND KANDEL, E. R., Conversion of synaptic excitation to inhibition at ~ dual chemical synapse, J. Neurophysiol., 34 (1971) 56 68. 27 WERN1G, A., Changes in statistical parameters during facilitation at the crayfish nettromuscular junction, J. Physiol. (Lond.), 226 (1972) 751-759. 28 ZUCKER, R. S., Changes in the statistics of transmitter release during facilitation. 7. Physiol. (Lond.), 229 (1973) 787-810.
267
DEPRESSION AND F R E Q U E N C Y F A C I L I T A T I O N AT A SYNAPSE IN A P L Y S I A CALIFORNICA: EVIDENCE FOR R E G U L A T I O N BY AVAILABILITY OF T R A N S M I T T E R
WERNER T. SCHLAPFER, PAUL B. J. WOODSON, JACQUES P. TREMBLAY AND SAMUEL H. BARONDES
Department t~fPsyekiatl3', Veterans Administration Hospital, San Diego, Calif. 92161 and Departments of Psychiatry and Neuroseiences, School of Medicine, University of CaliJornia, San Diego, La Jolla, Calif 92037 (U.S.A.) (Accepted March 5th, 1974)
SUMMARY
Synaptic depression and frequency facilitation were observed in the monosynaptic and unitary excitatory postsynaptic potential (EPSP) obtained in the 'parabolic burster cell' (R15) by stimulating the right visceropleural connective in Aplysia californica. During trains of stimulation at 0.5-2 c/sec the size of the EPSP was initially depressed during the first few stimuli and subsequently slowly increased to reach a potentiated steady state after a few hundred stimuli. The relative amount of depression between the first two EPSPs depended on (a) the inter-stimulus interval, and (b) the size of the first EPSP. The larger the first EPSP the larger the percentage depression whether variations in the size of the first EPSP were due to natural interanimal variability or experimental manipulation of the concentrations of Ca 2+, Mg 2~ and Co 2~. The amount of frequency facilitation (expressed as the ratio of the 100th EPSP to the first EPSP of a train) increased with increasing frequency of stimulation and was inversely related to the log of the size of the first EPSP both for natural variations in EPSP size and manipulations of the EPSP size by changing the divalent cation concentrations. Neither the level of hyperpolarization of R 15 nor partial curarization affected the relative depression and potentiation. We suggest that the initial depression is largely due to depletion of a rather small pool of transmitter available for release, while the frequency facilitation is the result of a Ca 2~ and frequency dependent increase in the net rate of supply of transmitter into the available pool.
INTRODUCTION
During and after repetitive stimulation of many synapses the size of the post-
268
w.T. SCHLAPFERet al.
synaptic potential (PSP) or end-plate potential (EPP) may decrease (synaptic depression) or increase (synaptic facilitation or potentiation). These phenomena have been observed at many synapses throughout the nervous system4, re. They have been examined in greatest detail in neuromuscular junctions z,17,19,28 and, under physiological conditions, have usually been attributed to variations in the amount of transmitter released by the presynaptic terminal rather than to changes in the postsynaptic sensitivity to the transmitter substance. There are essentially two classes of mechanisms which may account for the presynaptic variations in transmitter release. One mechanism determines the fraction of the available pool of transmitter which is released. The second mechanism regulates the size of the available pool of neurotransmitter. Supply into the available pool is generally explained in terms of increased 'mobilization' of neurotransmitter and is presumed to be due either to increased synthesis of neurotransmitter or to increased rate of transfer from a storage pool to an available pool or both 4. The depression of the EPP, most commonly observed in vertebrate neuromuscular junctionsa, 2z has been ascribed to progressive exhaustion of the pool of available transmitter2°,24; although the depression may be accentuated by a concurrent decrease in the fractional release of transmitter ~. The increase in the size of the PSP as a result of preceding stimulation may take a variety of forms. For a brief period after a stimulus, a second action potential results in increased amounts of transmitter released ~. This short-term facilitation has been attributed to an increase in the fraction of the available pool of transmitter which is released 11. Low frequency stimulation, at stimulus intervals longer than the duration of short-term facilitation, results in a progressive increase of the EPP in MgZ -poisoned vertebrate neuromuscular preparations is. This has been termed frequencT potentiation or frequency facilitation. It is believed to be due to increased mobilization of neurotransmitter produced by repetitive stimulation ~s. In crustacean neuromuscular junctions where a similar phenomenon is observed, progressive depolarization of the nerve terminals has been suggested as a potentiating mechanism 17. Another facilitating phenomenon is post-tetanic potentiation (PTP) t2 which refers to the rather long-lasting (minutes and up to hours) increase in PSP size after a period of tetani. The relationship of PTP to short-term facilitation or frequency potentiation is at present not clear TM. Conclusive evidence regarding the mechanisms underlying history-dependent changes in transmitter release remains to be provided. In particular, the evidence that frequency facilitation is due to increased mobilization of neurotransmitter rests primarily on studies of the neuromuscular junction performed under high magnesium. In the course of an investigation of an identified cholinergic synapse on the parabolic burster cell (R 15)5,22 of the abdominal ganglion of Aplysia calijornica, we observed all four of the phenomena described above while the preparation was perfused with physiological media. Since all these phenomena could be sequentially produced at this synapse, the interaction of the mechanisms which produce them might be examined. In the present report, we describe detailed studies of depression
PLASTIC SYNAPTIC TRANSMISSION IN
Aplysia
269
and frequency facilitation at this synapse. The results strongly support the hypothesis that both these phenomena are due to presynaptic regulatory mechanisms and that depression is due to transmitter depletion whereas frequency facilitation is due to increased transmitter mobilization. The post-tetanic potentiation of this junction will be analyzed in a subsequent communication. METHODS
Aplysia californica (100-1200 g) were collected locally and kept in tanks supplied with running seawater. The abdominal ganglion was dissected out with the visceropleural connectives, cut near the pleural ganglia and pinned to the paraffin floor of a recording chamber. The preparation was perfused at a rate of about 1 ml/min with aerated artificial seawater (460 mM NaCl, 10.4 m M KCI, 11.0 m M CaCl2, 55 mM MgCI2, 10 mM Tris-(hydroxymethyl)-aminomethane adjusted to pH 7.7) fortified with nutrients (per 1000 ml of artificial seawater: 10 g dextrose, 2 ml of 50 × concentrated amino acid mixture for minimum essential medium Eagle (MEM-E), 1 ml of 100 × concentrated vitamin mixture for MEM-E, 0.2 mmoles L-glutamine, and 500 units each of penicillin and streptomycin) to prolong survival of the isolated ganglion. Experimental changes in the ionic composition were compensated by appropriate adjustments of the NaCl content of the normal solution. A thermoelectric cooling unit kept the temperature at 15 ± 0.5 °C in the recording chamber. The right visceropleural connective was stimulated with a suction electrode driven through a stimulus-isolation unit by a Grass stimulator, lntracellular potentials were recorded in R 15 with 3 M KC1 filled microelectrodes of 2-10 Mr2 resistance which were gently tapped through the ganglion sheath. From a normal resting potential of 50-60 mV, R 15 was hyperpolarized to a level of 70-80 mV, to suppress its spontaneous bursting activity, either by injecting current through the recording electrode with a bridge circuit or through a second microelectrode. The recording electrode was connected through a high-input impedance, negative-capacitance amplifier (W-P Instruments Model M 701) to the display system (Tektronix 564 storage oscilloscope and Brush 220 high-speed pen recorder). The indifferent electrode consisted of an agar-seawater bridge connecting the recording chamber with a pool of 3 M KC1, containing a large Ag-AgCl electrode. The amplitude of the EPSP was measured from the Brush recorder trace. Up to 48 h of continuous intracellular recording from the soma of R 15 was possible under these conditions. These experiments were conducted between March and October 1973. RESULTS
Changes in synaptic efficacy during repetitive stimulation Cell R 15 receives a large excitatory input from an axon in the right visceropleural connective 5. This EPSP appears to be unitary and monosynaptic based on the criteria of all-or-none responsiveness and constant latency (Fig. 1), even during stimulation at high frequencies (10 stimuli/sec) a3. Transmission across this junction is likely to be cholinergic since the EPSP can be completely blocked by 5 × 10 4 M
270
w.T. SCHLAPFER el al.
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Fig. 1. Examples of EPSPs in a hyperpolarized R 15 evoked by stimulating the right visceropleural connective with progressively higher stimulus intensities (A-E). Note the all-or-none character of the EPSP (A,B) over a wide range of stimulus intensities (B,C) and the constant latencies. D-tubocurarine, 5 X 10 -4 M hexamethonium or 5 :~: 10 .4 M atropine, and since the soma o f R 15 depolarizes in response to iontophoretically applied acetylcholine '~. During repetitive low-frequency stimulation (0.5-2 c/sec) of the right visceropleural connective, the size o f the E P S P recorded in R 15 (which had been hyperpolarized to suppress its spontaneous bursting activity, as well as its rhythmic variation in m e m b r a n e potential) usually decreases during the first few stimuli and then increases gradually to reach a plateau after a few hundred pulses (Fig. 2). This potentiated plateau can be maintained for prolonged periods of stimulation, slightly decreasing only af!er several t h o u s a n d stimuli. The shape of the E P S P is unchanged
5.,v-I" Fig. 2. Characteristic EPSPs during a train of 100 stimuli to the right connective at 2 pulses/sec in R 15, hyperpolarized to suppress spontaneous bursting. The size of the EPSP decreases during the first few pulses (initial depression) and then slowly rises to a potentiated plateau (frequency facilitation).
PLASTIC SYNAPTIC TRANSMISSION IN A p l y s i a
271
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Stimuli/ sec Fig. 3. A: facilitation or depression of the second of two EPSPs expressed as the ratio of the second to the first EPSP (EPSP~/EPSP1) depending on the interstimulus interval. Data from 9 animals were pooled. B: frequency facilitation (ratio of average size of the last few EPSPs to the first EPSP) (EPSP100/EPSPI) during a train of 100 stimuli at various frequencies. Average from 5 animals with several trains each separated by 30 min. during a train of stimuli indicating that no other EPSPs are recruited during repetitive stimulation. D o u b l e s t i m u l a t i o n . The ratio of the amplitudes of the second EPSP to the first EPSP (EPSP~/EPSP1) depends on the stimulus interval. At short intervals (I ~ 250 msec) the second EPSP is usually larger than the first one (EPSP2/EPSP1 2: 1.0) (short-term facilitation) while at larger intervals (I > 500 msec) the second EPSP is depressed (EPSP2/EPSP1 < 1.0) (Fig. 3A). There is considerable variability in the ratio EPSP2/EPSP1 from animal to animal. At a stimulus interval of 500 msec, EPSP2/EPSP1 was greater than 1.0 in 16~o, equal to 1.0 in 2 2 ~ and smaller than 1.0 in 62 ~ of the 45 animals tested in this fashion. After a rest period of about 20-30 rain from the beginning of an experiment, the amplitude of the first EPSP ranged from 0.2 m V to 36 m V in different animals at similar levels o f hyperpolarization (70-80
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Fig. 4. Animal variability of the size of the first EPSP (EPSP0 and the correlation with (A) the initial depression (EPSP2/EPSP1) and (B) the frequency facilitation (EPSP100/EPSPO. R 15 was hyperpolarized to a similar level in all animals (70-80 mV) and 30-45 rain were allowed to elapse between the set-up of the preparation and the measurements presented in this figure. Each point represents the first train of 100 pulses at 2 stimuli/sec after the rest period in different animals (45 animals). Correlation between increasing EPSP~/EPSP1 and decreasing EPSP1 is significant with P < 0~00t (Spearman rank correlation coefficient =: 0.768). Correlation of frequency facilitation (EPSP100/EPSP0 with the size of the first EPSP
has a Spearman
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mV). The variability in E P S P t correlates well with the interanimal variation in the ratio EPSP2/EPSP1; the larger the first E P S P in any particular animal the smaller the ratio EPSP~/EPSP1, i.e., the larger the depression of the second with respect to the first EPSP (Fig. 4A).
Trains of stimulation. The size of the EPSP first decreases and usually reaches a m i n i m u m after about 4-8 pulses, then slowly rises during repetitive stimulation to a steady-state amplitude. At a frequency o f 2 stimuli/see the amplitude o f the E P S P reaches a plateau after about 400 stimuli, a level which can be maintained in m o s t preparations for 3000-4000 pulses, after which it slowly falls. Because o f the p r o l o n g e d period of post-tetanic potentiation which follows a train of 400 stimuli, we have
PLASTIC S Y N A P T I C TRANSMISSION IN
Aplysia
273
usually limited the train length to 100 stimuli so that experiments could be repeated at 20-30-rain intervals with little change in the response properties. By 100 pulses, the amplitude of the EPSP was usually about 80 9o of the maximum. The size of the EPSP reached during a train of 100 stimuli, expressed as the ratio of the average size of the last 3 EPSPs to the size of the first EPSP of the train (EPSP100/EPSP~), depends on the frequency of stimulation (Fig. 3B). Even at frequencies that correspond to intervals at which EPSP2/EPSP1 is less than 1.0 (0.05-2 c/sec), EPSP100/EPSP1 is equal to or larger than 1.0. This implies that linear summation of the curve of Fig. 3A cannot account for the potentiation observed upon multiple stimulation. As in the case of EPSP2/EPSP1, there was a large variability among animals in the magnitude of EPSPI00/EPSP1. In addition, the ratio EPSPa00/EPSP1 showed a strong inverse correlation with the size of the initial EPSP of a preparation (Fig. 4B).
Postsynaptic modifications of the EPSP size If the size of the EPSP is altered postsynaptically by changing the level of hyper-
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t,~
TABLE I*
"...1
4~ EFFECTS OF D I V A L E N T C A T I O N S ON T H E SIZE Or" T H E
EPSPs, THE 1NITfAL D E P R E S S I O N , A N D F R E Q U E N ( Y
Control
Treatment
EPSPI (mV)
Depression**
Frequency facilitation***
11.9 3.75 12.7 17.4 34.7 14.7
--0.15 --0.11 -0.08 --0.12 0.17 0.17
1.16 1.40 1.40 1.06 0.86 0.93
10.2 20.2 16.4 16.5 13.8 44.6 44.6
--0.14 0.19 -0.21 -0.38 0.07 0.21 -0.21
25.5 14.4
- 0.07 -0.20
Experimental EPSPI (mV)
2 3 3 3 3 1/5
FA(ILITAT10N
( o~ of control.)
Depres- (% c~[" sion** control.)
Frequency (%o.[" facilitation control)
"< Ca 2~ "~: Ca 2~ :, Ca 2~ , Ca ~ :': Ca "z" Mg ~
22.0 16.7 45,6 39.7 51.0 20.4
185 445 359 228 146 139
--0.17 0,20 -0,14 -0.18 -0.20 --0.23
1t3 182 175 150 118 135
0,89 0,70 0.76 0.81 0,72 0.86
78 50 54 76 84 92
1.08 0.93 0.99 0.74 1.25 0.66 0.66
2 < Mg ~3 x Mg -~ 1/5 >: Ca 2-~~ 2 m M C o ~= 4 m M C o e20 m M C o e 30 m M Co ~~
3.1 5.0 2.3 11.0 9.9 11.0 5.8
30 25 14 67 72 25 13
~-0.07 0.13 --0.03 -0.24 0.02 -0.15 ....0.01
50 68 14 63 29 71 5
1.64 1.60 2.39 0.97 1.46 1.00 1.32
152 172 241 131 117 151 200
0.93 1.15
-~- 110 m M Sucrose ~ 1.5 :., Ca 2 I ! 2.5 ;': Mg 2+
24.3 13.6
95 95
0.06 ....0.21
86 105
1.08 1.07
114 93
* Except for the 20 m M and 30 m M and Co 2- treatments which were done consecutively on the same preparation, each line in this table represents a different animal. All values were averages of 3-5 trains of 100 stimuli each at 2 stimuli/sec interspersed with 30-min rest periods. Sufficient time was allowed for stabilization of the values after inf\lsion of the experimental solutions (usually, 30-60 min). In the experimental solutions the NaCI concentration was adjusted to preserve isotonicity. Similar changes in the NaCI concentration only (adjusting the isotonicity with sucrose) did not result in any changes in EPSPj, depression and freqtiency facilitation, ** Depression is defined as the fractional decrease o f the size of Ihe second E PSP (EPSP:O in a trai~ compared ,to the first E PSP (EPSP~) a t a 500 msec interval, hence depression - (EPSP2-EPSPD/EPSP~. *** Frequency facilitation is defined as the ratio of the size of the 100th EPSP (f!PSPto,,i to the first EPSP of a train at 2 stimuli/see hence frequency facilitation .... EPSPI001EPSPb
,r ),
Aplysia
PLASTIC SYNAPTIC TRANSMISSION IN
2o01
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I 80 I ~ N ~ e
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0.70
080
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0.90
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060
I0
'0
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40
Fig. 6. Effect on EPSPa00/EPSP1 of(A) increasing Mg~ from 55 mM to 165 mM (decreasing EPSPi from 18 mV to 3 mV) and (B) increasing Ca2~ from 11 mMto 33 mM (increasing EPSPz from 8 mV to 40 mV). Stimulationat 2/sec for 100 pulses every 20 rain. Each point corresponds to one stimulus train during slow infusion and washing out of the experimental medium. Solid line is linear regression analysis of experimental points. polarization of the postsynaptic cell or by partial curarization of the preparation, the relative depressions and potentiations are unchanged. Although a 60 mV increase in the level of hyperpolarization (from 40 to 100 mV) more than doubled the size of the EPSP (from 11 to 26 mV), the relative values of both the initial depression (EPSPe/ EPSP1) and the frequency facilitation (EPSP100/EPSP1) were unaltered. Partial blocking of the postsynaptic receptor by D-tubocurarine chloride lowered the EPSPs but did not affect the ratios EPSP2/EPSP1 or EPSPI00/EPSP1 (Fig. 5). A similar result was obtained when EPSP1 was reduced to 25 ~o of normal by adding 10 -4 M atropine to the bathing medium.
Modifications of transmitter release If the amount of transmitter release is altered by varying the concentration of calcium or magnesium ions in the medium 1°, both the initial depression and the frequency facilitation are changed (Table 1). These divalent cations affect the size of the first EPSP of a train to a larger degree than the subsequent EPSPs. Depression [(EPSP2-EPSP1)/EPSPa] is increased in comparison with the control by raising the Ca '~ concentration and is decreased by raising the Mg '~+ concentration (Table I). Conversely, frequency facilitation (EPSP~00/EPSP1) is increased by comparison with the control by raising the Mg 2~ concentration or lowering the Ca 2+ concentration (Table I). Frequency facilitation varies inversely with the log of EPSP1 (Fig. 6), when EPSP1 is varied with Mg 2~ or Ca 2=. Appropriate increases in both Ca ~+ and Mg 2~ (e.~., 1.5 > Ca e~ and 2.5 × Mg ~ ) balance out so that EPSP1, EPSP2 and EPSPn)o are identical with the control (Table !). Since cobaltous ions have been shown to interfere with spike-dependent calcium
276
w. 1. SCHLAPFERel at.
influx in Aplysia neurons and other systems v and thereby reduce transmitter release, we tested addition of cobaltous ions to the medium. Like Mg 2+, cobaltous irons reduce the size of all EPSPs, but decrease the relative depression and increase the relative amplitude of frequency facilitation (Table 1). DISCUSSION
The large monosynaptic, unitary, and presumably cholinergic EPSP observed in R 15 by stimulation of the right visceropleural connective particularly lends itself to the study of factors which modify the efficacy of a synapse in the central nervous system. Not only can one return to the same junction in animal after animal, but the large size of the postsynaptic cell allows prolonged recordings so that many phenomena can be studied in the same specimen. In the present study, depression and frequency facilitation have been identified and studied at this synapse. The evidence suggests that both are based on presynaptic changes. This interpretation rests heavily on previous studies with neuromuscular junctions in which the quantal nature of cholinergic end-plate potentials has been studied in detail 3. Although quantal studies have not yet been done on this EPSP the phenomena lend themselves well to explanations based on changes in the amount of transmitter released during repetitive stimulation. Depression is apparently due to transient transmitter depletion, whereas frequency facilitation is apparently due to increased net supply of available transmitter. Changes in the amplitude of postsynaptic potentials could be due to changes in either the amount of transmitter released or in the receptor sensitivity. Receptor desensitization and sensitization (decreased or increased responsiveness to a constant amount of transmitter) has been suggested as the cause of decrementing or potentiating responses to presynaptic stimulation in some dual-action synapses in Aplysia 6,2~. The depression observed in the present study does not appear to be due to receptor desensitization because of the effects of curare and atropine. Were desensitization responsible for the depression which we observed, curare, which competes with acetylcholine for the receptor, should reduce the amount of transmitter reaching the receptor and interfere with desensitization or sensitization. Therefore if the depression is due to receptor desensitization, curare would be expected to change the ratio of EPSP2 to EPSP1. Similarly, if the frequency potentiation were due to receptor sensitization a change in the ratio of EPSP100 to EPSP1 would be predicted with curare. However, as shown in Fig. 5, these ratios are uninfluenced by the addition of D-tubocurarine chloride to the medium, in clear distinction to the change in these ratios which is observed when the amount of transmitter reaching the receptor is varied presynaptically with Ca z+ and Mg 2+. The differential action of curare, on one hand, and Ca z+ and Mg ~+ on the other, is analogous to the action of these agents in vertebrate neuromuscular preparations, where it has been shown by more direct means 3,~1 that depression and facilitation have a presynaptic origin. This suggests that in the system under investigation short-term facilitation, frequency facilitation and depression are due to presynaptic regulatory processes. In the absence of changes in the receptor sensitivity of the postsynaptic cell, the size of an EPSP can be taken as an index of the amount of transmitter released ~,
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although a 'ceiling' effect is possible when large amounts of transmitter are released 14. The amount of transmitter release (R) may be considered a function of two parameters: the size of the available transmitter pool (A) and the fraction (F) of the available transmitter released by a presynaptic action potential; hence R F.A. Buring repetitive stimulation either or both of these parameters can change to produce alterations in the amplitudes of the EPSPs. The present experiments suggest that depression is due to a depletion of A and that frequency facilitation is due to an increased rate of supply into A.
Depression and transmitter depletion The argument that depression is due to a decrease in available transmitter is based largely on the change in intensity of depression when transmitter release was modulated by calcium and magnesium. Since increasing the magnesium concentration (which reduces transmitter release) 1° decreases the depression, while increasing the calcium concentration (which augments transmitter release)lO, 14 increases the depression it is very likely that the depression we observe is mainly due to a stimulus dependent depletion of a rather small pool of either transmitter or some other material needed for release. A similar conclusion is reached if the transmitter release is decreased by adding cobalt chloride which decreases spike dependent Ca ~+ influx into Aplysia neuronsL Such treatment greatly reduces the size of the first EPSP, but as with magnesium, EPSP2 is less affected, so that the depression decreases or sometimes even reverses to a facilitation (Table 1). This agrees well with similar observations in neuromuscular junctions where the depressions have usually been interpreted to be at least partially due to transmitter depletion z3. In addition to these experimental manipulations of the size of the EPSPs, the natural interanimal variability in the size of the first EPSP correlated with the depression between the first and second EPSP in that the larger the first EPSP (the more transmitter released), the larger the depression of the second EPSP (Fig. 4A). This also suggests that depletion of a pool is responsible for the initial depression.
FrequencyJacilitation and transmitter mobilization The argument that frequency facilitation is based on increased availability of transmitter rests largely on the inference that availability of transmitter may limit the efficacy of this junction as shown by its initial depression. Since frequency facilitation can be maintained during prolonged periods of stimulation, the available transmitter pool either has to be very large or transmitter has to be mobilized from a storage pool 2 or synthesized at a rate equal to the rate of release. The initial depressed phase has indicated that the available pool of material needed for release is rather small in this system, as in others4, 9. Therefore, an increased rate of supply of material into this pool is a necessary and sufficient condition for frequency potentiation at this junction. An increase in the fractional release during repetitive stimulation, although possible, cannot by itself account for the frequency potentiation. In the absence of a net increase in the supply process, the small available pool would be rapidly depleted and an increased fractional release would only exaggerate the depletion and therefore de-
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crease the amplitude of the EPSP. At the potentiated plateau there is an equilibrium between the amount of transmitter released and supplied. Therefore the size of the EPSPs at the plateau is an indication of the amount of transmitter supplied into the available pool between any two stimuli. A process of transmitter mobilization responsible for potentiating phenomena in neuromuscular junctions has been proposed is, but disputed more recently ~J. However, frequency facilitation in vertebrate neuromuscular systems is only seen when release of transmitter has been reduced to a very low leveP s. In crayfish neuromuscular junctions, frequency facilitation has been shown to be due to an elevation of p, the statistical parameter of q uantal probability of release "7,2s. However, p is the compound of two probabilities, pl, the probability of occupying a release site, and p2, the probability that a site will release transmitter, the two parts of p being indistinguishable by the statistical methods used ~,~. In the analysis presented here, changes in p~ would be reflected as changes in the rate of supply into the available pool. The increased rate of supply into the available pool could be realized either by an increased rate of synthesis or by a process of mobilization from a larger storage pool. We cannot, at present, distinguish between these alternatives. The supply process has to be frequency dependent, since a larger steady-state EPSP amplitude is obtained at higher frequencies of stimulation (Fig. 3B). This process is also influenced by the external calcium and magnesium concentrations, since the absolute size of the steady-state EPSP during repetitive stimulation is increased at higher Ca ~4 concentrations and decreased at higher Mg 2~ concentration. The changes in the amount of frequency facilitation brought about by variations in the divalent cation concentration in the bath (Table I and Fig. 6) are due to the relatively greater effect that these agents have on the size of the first EPSP of a train with the subsequent EPSPs affected progressively less. The size of the first EPSP of a train is primarily a function of the fraction of available transmitter which is released, whereas the size of the steady-state EPSP is determined by the rate of transmitter supply into the available pool. Between different but similarly treated ani reals, considerable variability was found in the size of the control EPSPs (the first EPSP after the start of an experiment), their susceptibility to depression and their ability to show frequency l'acilitation. The larger the amplitude of the first EPSP in a particular animal, the tess frequency facilitation (EPSPt00/EPSP1) it exhibited. The interanimal variability qualitatively resembled the experimentally induced changes in EPSP2/EPSPI (Table 1) and EPSP100/EPSP1 (Fig. 6 ) w i t h Mg 2~, Ca 2~ and Co ~ . This suggests that different animals have different probabilities of transmitter release. This may be due to variations in the size of the synaptic release areas aS,''l, different sizes of terminal action potentials I v or the past history of the synapse in different animals. From this series of arguments, we conclude that depression at the synapse under investigation is due to transmitter depletion and that frequency facilitation is due to an increased net rate of transmitter supply into a pool available for release. Since preparation of this report, we have learned that Halstead and Jacklet ~ have studied frequency facilitation at this synapse. The preparation which they studied was not hyperpolarized so that the spontaneous activity of the parabolic bursle~" cell was
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s u p e r i m p o s e d on their records. Depression was n o t observed in their study possibly because the r h y t h m i c v a r i a t i o n in the m e m b r a n e potential p a r t i a l l y obscured the depression. Because o f this a p p a r e n t discrepancy, we have sought and repeatedly observed depression under conditions like those used by H a l s t e a d and Jacklet, especially in cells where the interval between bursts was long. Effects o f calcium and magnesium on frequency facilitation which they observed are similar to those reported here. These a u t h o r s also concluded t h a t the facilitation o f the EPSP in this system has a presynaptic origin since no change in p o s t s y n a p t i c m e m b r a n e resistance was seen during facilitation. ACKNOWLEDGEMENTS This w o r k was s p o n s o r e d by the Veterans A d m i n i s t r a t i o n Hospital, San Diego, under M R I S Nos. 7734(01) and 0818(01) a n d by N 1 M H G r a n t No. 18282. P.B.J.W. was s u p p o r t e d by U S P H S P r e d o c t o r a l Traineeship in Neurosciences, U S P H S 5-T01NS-05628-05, a n d J.P.T. by the C a n a d i a n Medical Research Council. W e are very grateful to Dr. E. R. K a n d e l , who p o i n t e d out to us the usefulness o f this j u n c t i o n for the study o f facilitation. We t h a n k Mr. G a r y Smith for his excellent technical support.
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