Brain Research, 562 (1991) 291-300 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993191l$03.50 ADONIS 000689939117089H
291
BRES 17089
Enhanced synaptic transmission at identified synaptic connections in the cerebral ganglion of Aplysia Steven M. Fredman Department of Physiology, MehartT Medical College, Nashville, TN 37208 U.S.A.) (Accepted 4 June 1991)
Key words: Aplysia; Cerebral ganglion; Synaptic transmission; Plasticity
The identified A-B neuron synaptic connections in the cerebral ganglion of Aplysia exhibited a novel form of enhanced synaptic transmission. A brief high-frequency train of action potentials (2 s, 10-30 Hz) in the presynaptic A neurons produced a long-lasting increase in the amplitude of excitatory postsynaptic potentials (EPSPs) in B neurons. The increase in synaptic efficacy was termed slow developing potentiation (SDP) since the EPSP amplitude increased slowly with the peak occurring 5 rain after the tetanizing train. Pcak EPSP amplitudes increased relative to the initial EPSP by an average of >250%. SDP decayed as a single exponential with a time constant of r = 24 rain. The enhanced transmission was neuron specific. Only the connections made by the tetanized A neuron were potentiated. However, potentiation apparently occurred at all the synapses made by the tctanized A neuron. Tetanizing the postsynaptic B neurons neither induced, nor when paired with A neuron tetanization, increased SDP. SDP appears to be primarily due to increased transmitter release by the presynaptic neuron. INTRODUCTION
Synaptic plasticity is thought to be the substrate for altered nervous system function. The several forms of synaptic plasticity can be arbitrarily divided into two broad groups, heterosynaptic and homosynaptic. Heterosynaptic (presynaptic) facilitation (HSF) and inhibition (HSI) require the pairing of synaptic inputs and are thought to respectively increase or decrease transmitter release by the presynaptic neuron. In Aplysia, HSF at synapses made by sensory neurons can be mimicked by serotonin and is mediated by cyclic A M P 6"25'45'48'49. Recent evidence indicates that in Aplysia sensory neurons, HSI can be mediated by the neuropeptide FMRFamide and metabolites of arachidonic acid 4°'41. The known forms of homosynaptic plasticity, on the other hand, require only the activation of a single synapse or set of synapses. Repetitive low frequency stimulation of a single synaptic connection frequently results in synaptic depression. Depression can be the result of reduced transmitter release from the presynaptic neuron 5 or desensitization of the postsynaptic receptors. On the other hand, high frequency stimulation of a presynaptic neuron can induce several forms of homosynaptic facilitation and/or potentiation. These forms include post-tetanic potentiation (PTP), short-term facilitation (STF)
and augmentation s'28'31'32,ss'. Enhanced synaptic transmission has been demonstrated both at neuromuscular and central synapses in many animals. PTP and STF appear to be due to transiently increased calcium concentrations in the presynaptic terminal 7'9. There are longterm forms of homosynaptic plasticity as well. Crustacean neuromuscular junctions exhibit long-term facilitation (LTF). This requires many minutes of high frequency stimulation, but can last for many hours 3. Unlike STF, the long-lasting phase of LTF may not solely be due to increased presynaptic Ca 2+ (refs. 9, 11, 55). Synapses in the mammalian hippocampus undergo long-term potentiation (LTP). In contrast to other forms of enhanced synaptic transmission, LTP requires the strong firing of the presynaptic neurons sufficient to produce postsynaptic depolarization 2'4"34'44. Although, it now seems likely that there is also increased transmitter release by the presynaptic terminall'3s's6, the primary locus of LTP appears to be the postsynaptic neuron. LTP in the dentate gyrus and CA1 region appears to require the activation of N-methyl-D-aspartate-sensitive glutamate receptors 19'36 and is due at least in part to increases in postsynaptic Ca 2+ (refs. 30, 33). Like other forms of homosynaptic plasticity, LTP is synapse specific. Only the strongly stimulated synaptie input is potentiated. The cellular and molecular basis for synaptic plastic-
Correspondence: S.M. Fredman, Department of Physiology, Meharry Medical College, IIX}5 D.B. Todd Blvd., Nashville, TN 37208, U.S.A.
292 ity is presently being investigated in both v e r t e b r a t e s and invertebrates. The stimulation of second messenger systems (cyclic A M P , arachidonic acid and its metabolites, phosphatidylinositol, Ca 2+) is a c o m m o n theme. Second messengers have b e e n shown to alter ionic channels such as several K + channels or act on o t h e r m e m b r a n e , cytoskeleton and synaptic vesicle proteins 2°'25"37. The cerebral ganglion of Aplysia contains two symmetrical groups of readily identified neurons. The neurons in both cerebral A clusters m a k e excitatory monosynaptic connections with neurons in both B clusters TM 16,18,21. The transmitter is unknown, however, indirect evidence suggests that it m a y be a peptide 39. Like many synaptic connections, repetitive stimulation of an A neuron results in depression of its EPSPs in B neurons. In the results r e p o r t e d below, it is d e m o n s t r a t e d that the synapse also exhibits a novel form of enhanced transmission. Brief high frequency stimulation of an A n e u r o n results in a slowly developing, long-lasting e n h a n c e m e n t of transmission. Unlike o t h e r well known forms of longlasting plasticity in Aplysia, the e n h a n c e m e n t is homosynaptic and does not involve either m e c h a n o s e n s o r y neurons o r serotonin. A brief account of some of these results has been r e p o r t e d 12. MATERIALS AND METHODS Adult Aplysia californica, weighing 100-200 g were used. The animals were obtained from commercial suppliers (Sea Life Supply, Sand City, CA and Alacrity Marine Biological Services, Redondo Beach, CA), maintained in artificial seawater (SW) aquaria at 13 °C and fed a diet of Romaine lettuce. In most experiments, the animals were injected with 30-50 ml of isotonic MgCI 2 prior to dissection in normal SW. The entire central nervous system (CNS) was then transferred to calcium free (0 Ca 2+) SW, pinned to the bottom of a dish and the sheath over the cerebral ganglion removed surgically. The 0 Ca 2+ SW was then replaced with SW containing elevated divalent cations, usually 5x Ca 2+ (55 mM) and 3x Mg 2÷ (150 mM). Unless otherwise noted, all the experiments discussed b~low used 5 x Ca 2÷, 3 x Mg2+ SW. The CNS was maintained at 13-14 °C with a cold-plate. In a typical experiment, 2 cerebral A neurons and 2 cerebral B neurons 14-16 were impaled with micropipettes containing 2.5 M potassium citrate. To improve their current passing abilities, the electrodes were beveled and had resistances of -10 M~. Shortly after penetration, the synaptic connection between each A neuron and each B neuron was tested. A neurons were depolarized with single 35 ms duration pulses, the intensity of which were adjusted so as to trigger a single action potential per pulse. The amplitude of the initial EPSP in each B neuron was noted. Although all of the A neurons were used in the course of the experiments, the A neurons at the medial and posterior margins of the cluster were preferred as they tended to evoke the largest EPSPs in the B neurons. Following this initial testing, one A neuron, designated the 'test A neuron', was stimulated at predetermined intervals via a timing circuit. Each neuron could also be stimulated independently without altering the timing of the next scheduled test pulse. Both the presynaptic action potentials and the evoked EPSPs were digitized at 6-12 kHz/channel using a data interface (Modular Instruments, Inc.), automatically measured using Spike (Hilal Associates) data acquisition software and monitored on a storage oscilloscope. Long-term changes in membrane potential and neuronal activity were followed on a chart recorder.
RESULTS The synaptic connection between cerebral A and B neurons exhibited several forms of synaptic plasticity. The form that was initially seen was synaptie depression confirming previous findings ~4'2~. Repetitive stimulation of a single A neuron resulted in a progressive decrement of the amplitude of its EPSPs in B neurons (Figs. l, 5 and 8). D e p r e s s i o n was more rapid with higher stimulus frequencies (Fig. 8). H o w e v e r , depression occurred even at the lowest stimulus frequencies used, with interstimulus intervals (ISI) of up to 15 rain. With a 180 s ISI, after 5 trials, EPSP amplitudes decreased 49 -+- 14% (n = t0 pairs of A - B neurons) relative to the initial EPSPs. Only when the ISI was increased to .... 15 min was depression largely absent. As will be discussed in more detail below, there was also some decay of depression m but this was very limited at the ISI of 180 s usually used in the experiments. Synaptic transmission between A and B neurons could be enhanced by interposing a 'high frequency" train. The recordings were usually m a d e in 5× Ca 2+, 3× Mg 2+ SW to suppress polysynaptic connections. Unless otherwise indicated all the data shown below was o b t a i n e d using this SW. H o w e v e r , essentially identical results were o b t a i n e d in normal SW as well (see below). The high divalent cation SW had the additional advantages of reducing spontaneous synaptic input in the B neurons, raising thresholds and largely eliminating spiking, all of which facilitated measuring the EPSPs evoked by the A neurons. The following stimulus paradigm was used. A n A neuron was stimulated at constant intervals (typically ISI = 100-200 s, but unless otherwise noted the ISI = 180 s) until the depression of EPSP to the test pulses had reached a stable level, typically after 3-5 pulses. Twenty s prior to the next scheduled test pulse, the A neuron was driven with a 2 s duration train at 5 - 3 0 Hz. T h e stimulus intensity was usually increased, particularly when higher frequencies were used, to insure that the A neuron faithfully followed the train. The stimulus intensity was returned to its initial level before the arrival of the next test pulse (i.e. 20 s after the train), Test pulses continued to be p r e s e n t e d and the a m p l i t u d e of the EPSPs measured. The results of a typical e x p e r i m e n t are shown in Figs. 1 and 2. The amplitude of the E P S P e v o k e d by a test pulse 20 s after the high frequency train was either essentially unchanged or slightly depressed. H o w e v e r , the amplitudes of subsequent EPSPs were significantly increased. The EPSPs were p o t e n t i a t e d relative to both the E P S P e v o k e d by the last test pulse prior to the high frequency train, and the very first (non-depressed) EPSP evoked following the i m p a l e m e n t of tile neurons. The average p e a k increase in amplitude relative to the initial
293 EPSP (EPSPN/EPSP 0 was 258% (n = 25 experiments, range 134-500% using a 20 Hz train and 180 s ISI). The peak increase in EPSP amplitude occurred 2 . 5 - 9 min after the potentiating train. As can be seen in Figs. 1, 2 and 6 the enhanced transmission was long lasting. The mean (-+ S . E . M . ) time to peak potentiation was 4.97 -+ 1.70 min (n = 45 trials, with ISI = 0.5-4.5 min). Presenting test pulses at 30 s intervals after the tetanizing train indicated that while variable from A neuron to A neuron, the onset of enhanced transmission typically did not begin until - 1 . 5 min after the presentation of the train. Due to the slow kinetics of both the increase and decay of the enhanced transmission, it has been t e r m e d slow developing potentiation (SDP), While the above experiments were run at the same t e m p e r a t u r e at which the Aplysia were housed (13-14 °C), the time to p e a k and decay of SDP were unchanged at higher temperatures, H o w e v e r , at 22-23 °C, the onset of SDP a p p e a r e d to be more rapid. The slow kinetics clearly distinguished SDP from PTP. The slow rise in enhanced EPSP amplitude was not solely the result of infrequent sampling. When test pulses were presented at 30 s and 60 s ISis following a 20 Hz tetanizing train, the time to peak EPSP amplitude was 3.5 and 4 min respectively. While the slow rise of SDP was still present with shorter ISis,
~
depression was increased. The concomitant reduction in p e a k potentiation suggests that depression and SDP were occurring simultaneously, with depression reducing SDP when short ISis were used. In most experiments, there was little, if any, change in the resting potential of either the pre- or postsynaptic neurons following the tetanizing train. Thus SDP was not the result of a baseline shift for either the presynaptic spike or postsynaptic EPSP. This makes it unlikely that SDP was due to inactivating the conventional K+A current present in A neurons 46. It should be noted that while most experiments were done in SW containing 5 x Ca 2+ and 3 x Mg e+, SDP was also observed using normal SW (11 mM Ca 2+, 50 m M Mg 2+) and SW containing 3 x Ca 2+ and 5 x Mg 2+. SDP was not d e p e n d e n t solely on unusual ionic conditions. I n d e e d , in normal SW, SDP worked 'too well', with the B neurons spiking off the potentiated EPSPs. For example, in one experiment done in normal SW, SDP resulted in 65% of the EPSPs giving rise to action potentials in the B neurons versus none prior to the tetanizing train. However, when B neurons were hyperpolarized sufficiently to prevent EPSPs from triggering action potentials, the onset, rise and decay of SDP in normal SW were the same as in high divalent cation SW. This suggests that spiking by
s
6
'1
+20s
A
LA
'~._
,
/ ~
+3m 8
~
+6m lO
+12m 12
+18m
+36m
,•20mV 2mY
2Oral
Fig. 1. Enhanced transmission at the A-B neuron synapse. A left A cluster neuron (LA) was stimulated once every 3 rain. The numbers to the left of the spike indicate the stimulus presentation number. There was a marked depression of the amplitude of the EPSP in a right B cluster neuron (RB) between the 1st and 5th test stimuli. Twenty seconds prior to the 6th test stimulus, the LA neuron was driven at 20 Hz for 2 s. Except for slight further depression, there was no significant change in the amplitude of the EPSP to the 6th test stimulus. However, the EPSP evoked by 7th test stimulus 3 min later was significantly larger than even the first (non-depressed) EPSP. The amplitude of the EPSP to the 8th test stimulus (6 rain after the tetanizing train) was greater than twice that of the first EPSP. The amplitude of the EPSP was still potentiated 30 min later and returned to approximately the initial amplitude by 36 rain.
297 a potential mechanism for SDP, it indicates that, if present, it is pharmacologically distinct from the HSF seen in Aplysia sensory neurons. While SDP was neuron specific, it also appeared to occur at 'all' the followers of the tetanized neuron. As has been previously demonstrated ~4, each A neuron synapses on several B neurons. In most experiments, two B neurons were recorded simultaneously. When an A neuron was tetanized, the connections it made to both B neurons exhibited SDP. This is shown in Fig. 6. Tetanization caused essentially parallel changes in EPSP amplitude in both neurons. Since one of the B neurons was contralateral to the test A neuron, it indicated that SDP did not depend on strictly 'local' (e,g. within a few hundred /~m) changes. Similar results have also been obtained with more distant A neuron followers. The left pleural giant cell receives monosynaptic EPSPs from A neurons 16. This connection also exhibited SDP which paralleled SDP in B neurons (not shown). Although these results do not prove that the efficacy of transmission at all of the A neuron's connections was enhanced, they suggest that many of the connections with B neurons and more distant neurons were potentiated. This too, is consistent with a presynaptic locus for SDP. The results also indicate that generating action potentials in the A neurons was an absolute requirement for SDP. This is supported by the observation that when an A neuron failed to follow the tetanizing train (depolarizations but no spikes), SDP was greatly reduced or absent.
Changes in the presynaptic action potential during SDP Heterosynaptic facilitation of transmitter release by
Aplysia sensory neurons has been shown to be accompanied by increased action potential durations 26. In sensory neurons, there is a marked increase in the duration of the falling phase of the spike due to the inactivation of a K + current 49. Fig. 7 shows superimposed A neuron action potentials before and during SDP. Rather than there being an increase in the falling phase of the spike, there was a slight but noticeable broadening of the rising phase and a small decrease in the repolarizing phase of the spike. SPD was frequently accompanied by slightly reduced thresholds. Occasionally, the stimulus intensity had to be reduced to avoid triggering two spikes. Spike broadening due to prolongation of the falling phase of the A neuron action potential was never seen. This suggests that SDP in A neurons is due to a different cellular mechanism than HSF in Aplysia sensory neurons. This is reinforced by the observation that when a similar stimulus paradigm was tried with cerebral ganglion sensory neurons 42, enhanced transmission was accompanied by a marked prolongation of the falling phase of the ac-
tion potential. Plasticity in these neurons has not yet been examined in detail.
Decay of depression One potential mechanism for SDP is decay of depression m. According to this hypothesis, the tetanizing train might remove the accumulated depression resulting from repetitive stimulation. The slow kinetics of SDP would be due to decay of depression such as that seen at the neuromuscular junction m. To test this, an A neuron was stimulated with a 30 s ISI to induce depression. The stimulus frequency was then decreased. Since the decay of depression is proportional to amount of depression induced, one would expect a rise in EPSP amplitude similar to that seen during SDP. This was not the case. As seen in Fig. 8, reducing the stimulus frequency resulted in only a very minor recovery of EPSP amplitude. However, when a tetanizing train was presented, there was a large increase in EPSP amplitude which, as in all the other experiments, significantly exceeded the initial (non-depressed) EPSP amplitude. Once induced, depression did partially recover if followed by a long period (i.e. 20-30 min) during which the synapse was not stimulated. While this recovery was not systematically quantified, the EPSPs rarely returned to initial amplitudes and never exceeded them. DISCUSSION Although slow developing potentiation was produced by a stimulus paradigm that is very similar to that which evokes PTP, it appears to be a distinct phenomenon. The most obvious distinguishing characteristic is its time course. PTP is usually characterized by a rapid onset and rapid decay. For neurons in Aplysia the onset of PTP typically occurs within 5-10 s of terminating the tetanizing train 7~3s'43'51'52. The decay of PTP is typically also on a time scale of seconds. Ohmori 3s measured the time constant for the decay of PTP at the RC1-R15 synapse with r = 144 _+ 42 s. A slower decay, lasting several minutes has been observed using long (e.g. minutes) tetanizing trains 31. At the L10-RB synapse in the abdominal ganglion, the time constant was much shorter with r = 49 - 5 s 38. The onset of SDP, on the other hand, was only rarely observable 20 s after the tetanizing train and usually did not begin until after 1 min. The decay of SDP was also long, with r = 24 min. Even when SDP is compared to the long-lasting forms of PTP, the peak potentiation on the average occurred when PTP would have been largely or totally decayed (i.e. 5 rain). The experiments performed at 22 °C also suggest that SDP and PTP are distinct. Elevated temperatures did not significantly alter the time to peak or decay of SDP. One might
298 expect that higher temperatures would increase either the pumping or sequestration of Ca 2÷ and thus increase the rate at which PTP decays 7"9. On the other hand the more rapid onset of SDP at 22 °C is consistent with an enzymatic step being involved 13. While the poorly understood phenomenon of decay of depression ~ may make a minor contribution to the slow rise of SDP, it cannot account for most of it. The decay of depression is proportional to the train used to produce the depression 31. This was not observed with SDP. Frequent stimulation of the A neurons (e.g. ISI = 60 s), which increased synaptic depression, did not significantly delay the time to peak. The 'increase' in EPSP amplitude resulting from the decay of depression was far smaller than that seen with SDP (Fig. 8). With the decay of depression, EPSP amplitudes failed to return to initial levels. With SDP, EPSP amplitudes always exceeded the initial amplitude. The results suggest that SDP and depression are simultaneous competing processes. SDP does not appear to turn off synaptic depression; it appears to override depression. Depression at the A-B neuron synapse can be very prominent and long-lasting and appears to be due to biochemical mechanisms quite distinct from SDP (unpublished observations). Another distinguishing characteristic of SDP is the amount of tetanization needed to induce enhanced transmission. SDP could be elicited with as few as 20 spikes (10 Hz for 2 s). This also distinguishes it from PTP and other forms of plasticity such as long-term facilitation (LTF) which require much longer tetanizing trains 58. Typically PTP requires 100-600 action potentials 43. With PTP, tetanizing frequencies can be as low as 2 Hz. Wojtowicz and Atw o o d 53"54 used 20 Hz tetanization for 10 min to induce LTE SDP on the other hand required frequencies >5 Hz, but with correspondingly much shorter train durations. Low frequency stimulation for longer durations (i.e. 5 Hz for up to 10 s) was not effective. Thus SDP appears to be a distinct phenomenon in terms of its onset, time-to-peak, decay time constant and the stimulus parameters which induce it. The data presented above suggest that while SDP was primarily due to a presynaptic mechanism it also appeared to be homosynaptic. Most experiments were done under conditions of high divalent cations, which should have largely suppressed polysynaptic connections. This in itself tended to eliminate heterosynaptic facilitation as a mechanism. The persistence of SDP in the presence of elevated divalent cations however, is by no means conclusive that SDP was homosynaptic. Nor is the fact that the A-B neuron connection is monosynaptic. In intact ganglia it is virtually impossible to prove that SDP is, in fact, homosynaptic, since it is mediated by synapses within the CNS. Like PTP, but unlike HSF, SDP was
neuron specific. Tetanizing a second A neuron did not potentiate the EPSPs of the test A neuron. This also argues that SDP was homosynaptic. If it was heterosynaptic, then there must be one recurrently excited interneuron for each A neuron. In addition, the interneurons must have had a much lower threshold than the B neurons, because the latter rarely spiked (due to elevated divalent cations) during the tetanizing train. If, like LTP, SDP required depolarization and spiking in the postsynaptic neuron then tetanizing either A neuron should have been effective. This was not the case. Stimulating only the postsynaptic neuron also failed to produce SDP and hyperpolarizing the B neurons to prevent spiking, which was infrequent in any case, never blocked it. This was also true in normal SW, where while hyperpolarizing the B neurons was needed to prevent spiking, the failure of the B neurons to generate spikes at the cell body in no way inhibited SDP. Simultaneously stimulating both the pre- and postsynaptic neurons did not increase SDP over that produced by A neuron stimulation alone. Thus, unlike LTP which is synapse specific, SDP appears to have a primarily presynaptic locus. However, it is very difficult to exclude the possibility that SDP is due in part to a postsynaptic mechanism, with different A neuron synapses being sufficiently far apart that changes at one site would not affect the other. One must also postulate that unlike UI'P, increased postsynaptic firing (paired or unpaired) is insufficient to duplicate the effects of synaptic activation. The results are most consistent with SDP being homosynaptic and due to increased transmission by the presynaptic neuron. However, the possibility of either a heterosynaptic or a postsynaptic component to SDP has not been completely eliminated. One possible explanation fi~r SDP is that it might be due to a pump mechanism. The tetanizing train might have increased intracellular N a ' concentrations and activated a Na+/Ca 2+ exchange pump. The net result would be potentiation of transmitter release. There are several difficulties with this hypothesis. First, a pump should be sensitive to the number of action potentials, not their frequency. As noted above, the opposite was true. Second, it is difficult to imagine how a 2 s train could increase Na + sufficiently as to produce potentiation which in several experiments took 9 rain to reach its peak and lasted longer than 30 rain. The kinetics of a pump mechanism appear to be inconsistent with SDP. While SDP had a r = 24 min, Levy and Tillotson 29 report a time constant for the Aplysia Na+/Ca 2~ pump of r = 3.14 s. An additional argument against a pump mechanism is that the Na+/Ca 2+ pump usually pumps Ca 2+ out of the cell not in. The cerebral A neurons have been shown to possess an effective electrogenic Na+/K + pump 2a- If a
299 p u m p m e c h a n i s m is r e s p o n s i b l e for SDP, it is not clear
rent. R e c e n t results suggest that S D P m a y in part be
why a N a + / C a 2+ p u m p r u n n i n g in r e v e r s e , and not the
m e d i a t e d by p r o t e i n kinase C 13 activating C a 2+ channels.
e l e c t r o g e n i c p u m p w o u l d be activated. T h e i n v o l v e m e n t
N e e d l e s s to say, m u c h m o r e w o r k is n e e d e d b e f o r e de-
of the latter is d u b i o u s since the m e m b r a n e p o t e n t i a l of
finitive s t a t e m e n t s can be m a d e a b o u t the u n d e r l y i n g
the A n e u r o n s did not c h a n g e during SDP. While a
m e c h a n i s m s m e d i a t i n g SDP.
p u m p m e c h a n i s m 5~ m a y c o n t r i b u t e to SDP, it d o e s not a p p e a r to be the p r i m a r y m e c h a n i s m .
R e g a r d l e s s of the m e c h a n i s m s , S D P is likely to be of physiological significance. It p r o v i d e s a m e a n s for re-
A l t h o u g h the m e c h a n i s m s r e s p o n s i b l e for S D P are un-
m o v i n g d e p r e s s i o n at the A - B n e u r o n synapse and sug-
clear, o n e possibility is that S D P might w o r k by a m e c h -
gests that t h e r e is b o t h a c t i v i t y - d e p e n d e n t up and d o w n
anism similar to that s e e n in Aplysia sensory n e u r o n s 26'
r e g u l a t i o n of synaptic efficacy. In n o r m a l SW, p o t e n t i -
49. T h e t e t a n i z i n g train may be inactivating K + channels,
a t e d E P S P s f r e q u e n t l y t r i g g e r e d spikes in B n e u r o n s .
allowing m o r e Ca 2+ to e n t e r during the action potential.
Since the B n e u r o n s are m o t o r n e u r o n s m e d i a t i n g de-
15,23,50,
H o w e v e r , unlike sensory n e u r o n s t h e r e was little c h a n g e
fensive b e h a v i o r s such as t e n t a c l e w i t h d r a w a l
in the falling phase of the action p o t e n t i a l . M o s t of the
can potentially m o d i f y the d e f e n s i v e b e h a v i o r of A p l y -
spike b r o a d e n i n g a p p e a r e d to be on the rising phase. It
sia.
SDP
is not clear if the m o d e s t changes in the w a v e - f o r m of the action p o t e n t i a l can a c c o u n t for the substantial increases in synaptic efficacy o b s e r v e d . This still leaves o p e n the possibility that the p r o l o n g a t i o n of the spike might h a v e b e e n d u e to inactivation of an early K + cur-
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Acknowledgements. This work was supported by N1NDS Grant NS28199 to S.M.E, NIGMS MBRS Grant GM (18037, RCMI Grant RR03032 and NSF/MRCE Grant 8714805 to Meharry Medical College.
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291
BRES 17089
Enhanced synaptic transmission at identified synaptic connections in the cerebral ganglion of Aplysia Steven M. Fredman Department of Physiology, MehartT Medical College, Nashville, TN 37208 U.S.A.) (Accepted 4 June 1991)
Key words: Aplysia; Cerebral ganglion; Synaptic transmission; Plasticity
The identified A-B neuron synaptic connections in the cerebral ganglion of Aplysia exhibited a novel form of enhanced synaptic transmission. A brief high-frequency train of action potentials (2 s, 10-30 Hz) in the presynaptic A neurons produced a long-lasting increase in the amplitude of excitatory postsynaptic potentials (EPSPs) in B neurons. The increase in synaptic efficacy was termed slow developing potentiation (SDP) since the EPSP amplitude increased slowly with the peak occurring 5 rain after the tetanizing train. Pcak EPSP amplitudes increased relative to the initial EPSP by an average of >250%. SDP decayed as a single exponential with a time constant of r = 24 rain. The enhanced transmission was neuron specific. Only the connections made by the tetanized A neuron were potentiated. However, potentiation apparently occurred at all the synapses made by the tctanized A neuron. Tetanizing the postsynaptic B neurons neither induced, nor when paired with A neuron tetanization, increased SDP. SDP appears to be primarily due to increased transmitter release by the presynaptic neuron. INTRODUCTION
Synaptic plasticity is thought to be the substrate for altered nervous system function. The several forms of synaptic plasticity can be arbitrarily divided into two broad groups, heterosynaptic and homosynaptic. Heterosynaptic (presynaptic) facilitation (HSF) and inhibition (HSI) require the pairing of synaptic inputs and are thought to respectively increase or decrease transmitter release by the presynaptic neuron. In Aplysia, HSF at synapses made by sensory neurons can be mimicked by serotonin and is mediated by cyclic A M P 6"25'45'48'49. Recent evidence indicates that in Aplysia sensory neurons, HSI can be mediated by the neuropeptide FMRFamide and metabolites of arachidonic acid 4°'41. The known forms of homosynaptic plasticity, on the other hand, require only the activation of a single synapse or set of synapses. Repetitive low frequency stimulation of a single synaptic connection frequently results in synaptic depression. Depression can be the result of reduced transmitter release from the presynaptic neuron 5 or desensitization of the postsynaptic receptors. On the other hand, high frequency stimulation of a presynaptic neuron can induce several forms of homosynaptic facilitation and/or potentiation. These forms include post-tetanic potentiation (PTP), short-term facilitation (STF)
and augmentation s'28'31'32,ss'. Enhanced synaptic transmission has been demonstrated both at neuromuscular and central synapses in many animals. PTP and STF appear to be due to transiently increased calcium concentrations in the presynaptic terminal 7'9. There are longterm forms of homosynaptic plasticity as well. Crustacean neuromuscular junctions exhibit long-term facilitation (LTF). This requires many minutes of high frequency stimulation, but can last for many hours 3. Unlike STF, the long-lasting phase of LTF may not solely be due to increased presynaptic Ca 2+ (refs. 9, 11, 55). Synapses in the mammalian hippocampus undergo long-term potentiation (LTP). In contrast to other forms of enhanced synaptic transmission, LTP requires the strong firing of the presynaptic neurons sufficient to produce postsynaptic depolarization 2'4"34'44. Although, it now seems likely that there is also increased transmitter release by the presynaptic terminall'3s's6, the primary locus of LTP appears to be the postsynaptic neuron. LTP in the dentate gyrus and CA1 region appears to require the activation of N-methyl-D-aspartate-sensitive glutamate receptors 19'36 and is due at least in part to increases in postsynaptic Ca 2+ (refs. 30, 33). Like other forms of homosynaptic plasticity, LTP is synapse specific. Only the strongly stimulated synaptie input is potentiated. The cellular and molecular basis for synaptic plastic-
Correspondence: S.M. Fredman, Department of Physiology, Meharry Medical College, IIX}5 D.B. Todd Blvd., Nashville, TN 37208, U.S.A.
292 ity is presently being investigated in both v e r t e b r a t e s and invertebrates. The stimulation of second messenger systems (cyclic A M P , arachidonic acid and its metabolites, phosphatidylinositol, Ca 2+) is a c o m m o n theme. Second messengers have b e e n shown to alter ionic channels such as several K + channels or act on o t h e r m e m b r a n e , cytoskeleton and synaptic vesicle proteins 2°'25"37. The cerebral ganglion of Aplysia contains two symmetrical groups of readily identified neurons. The neurons in both cerebral A clusters m a k e excitatory monosynaptic connections with neurons in both B clusters TM 16,18,21. The transmitter is unknown, however, indirect evidence suggests that it m a y be a peptide 39. Like many synaptic connections, repetitive stimulation of an A neuron results in depression of its EPSPs in B neurons. In the results r e p o r t e d below, it is d e m o n s t r a t e d that the synapse also exhibits a novel form of enhanced transmission. Brief high frequency stimulation of an A n e u r o n results in a slowly developing, long-lasting e n h a n c e m e n t of transmission. Unlike o t h e r well known forms of longlasting plasticity in Aplysia, the e n h a n c e m e n t is homosynaptic and does not involve either m e c h a n o s e n s o r y neurons o r serotonin. A brief account of some of these results has been r e p o r t e d 12. MATERIALS AND METHODS Adult Aplysia californica, weighing 100-200 g were used. The animals were obtained from commercial suppliers (Sea Life Supply, Sand City, CA and Alacrity Marine Biological Services, Redondo Beach, CA), maintained in artificial seawater (SW) aquaria at 13 °C and fed a diet of Romaine lettuce. In most experiments, the animals were injected with 30-50 ml of isotonic MgCI 2 prior to dissection in normal SW. The entire central nervous system (CNS) was then transferred to calcium free (0 Ca 2+) SW, pinned to the bottom of a dish and the sheath over the cerebral ganglion removed surgically. The 0 Ca 2+ SW was then replaced with SW containing elevated divalent cations, usually 5x Ca 2+ (55 mM) and 3x Mg 2÷ (150 mM). Unless otherwise noted, all the experiments discussed b~low used 5 x Ca 2÷, 3 x Mg2+ SW. The CNS was maintained at 13-14 °C with a cold-plate. In a typical experiment, 2 cerebral A neurons and 2 cerebral B neurons 14-16 were impaled with micropipettes containing 2.5 M potassium citrate. To improve their current passing abilities, the electrodes were beveled and had resistances of -10 M~. Shortly after penetration, the synaptic connection between each A neuron and each B neuron was tested. A neurons were depolarized with single 35 ms duration pulses, the intensity of which were adjusted so as to trigger a single action potential per pulse. The amplitude of the initial EPSP in each B neuron was noted. Although all of the A neurons were used in the course of the experiments, the A neurons at the medial and posterior margins of the cluster were preferred as they tended to evoke the largest EPSPs in the B neurons. Following this initial testing, one A neuron, designated the 'test A neuron', was stimulated at predetermined intervals via a timing circuit. Each neuron could also be stimulated independently without altering the timing of the next scheduled test pulse. Both the presynaptic action potentials and the evoked EPSPs were digitized at 6-12 kHz/channel using a data interface (Modular Instruments, Inc.), automatically measured using Spike (Hilal Associates) data acquisition software and monitored on a storage oscilloscope. Long-term changes in membrane potential and neuronal activity were followed on a chart recorder.
RESULTS The synaptic connection between cerebral A and B neurons exhibited several forms of synaptic plasticity. The form that was initially seen was synaptie depression confirming previous findings ~4'2~. Repetitive stimulation of a single A neuron resulted in a progressive decrement of the amplitude of its EPSPs in B neurons (Figs. l, 5 and 8). D e p r e s s i o n was more rapid with higher stimulus frequencies (Fig. 8). H o w e v e r , depression occurred even at the lowest stimulus frequencies used, with interstimulus intervals (ISI) of up to 15 rain. With a 180 s ISI, after 5 trials, EPSP amplitudes decreased 49 -+- 14% (n = t0 pairs of A - B neurons) relative to the initial EPSPs. Only when the ISI was increased to .... 15 min was depression largely absent. As will be discussed in more detail below, there was also some decay of depression m but this was very limited at the ISI of 180 s usually used in the experiments. Synaptic transmission between A and B neurons could be enhanced by interposing a 'high frequency" train. The recordings were usually m a d e in 5× Ca 2+, 3× Mg 2+ SW to suppress polysynaptic connections. Unless otherwise indicated all the data shown below was o b t a i n e d using this SW. H o w e v e r , essentially identical results were o b t a i n e d in normal SW as well (see below). The high divalent cation SW had the additional advantages of reducing spontaneous synaptic input in the B neurons, raising thresholds and largely eliminating spiking, all of which facilitated measuring the EPSPs evoked by the A neurons. The following stimulus paradigm was used. A n A neuron was stimulated at constant intervals (typically ISI = 100-200 s, but unless otherwise noted the ISI = 180 s) until the depression of EPSP to the test pulses had reached a stable level, typically after 3-5 pulses. Twenty s prior to the next scheduled test pulse, the A neuron was driven with a 2 s duration train at 5 - 3 0 Hz. T h e stimulus intensity was usually increased, particularly when higher frequencies were used, to insure that the A neuron faithfully followed the train. The stimulus intensity was returned to its initial level before the arrival of the next test pulse (i.e. 20 s after the train), Test pulses continued to be p r e s e n t e d and the a m p l i t u d e of the EPSPs measured. The results of a typical e x p e r i m e n t are shown in Figs. 1 and 2. The amplitude of the E P S P e v o k e d by a test pulse 20 s after the high frequency train was either essentially unchanged or slightly depressed. H o w e v e r , the amplitudes of subsequent EPSPs were significantly increased. The EPSPs were p o t e n t i a t e d relative to both the E P S P e v o k e d by the last test pulse prior to the high frequency train, and the very first (non-depressed) EPSP evoked following the i m p a l e m e n t of tile neurons. The average p e a k increase in amplitude relative to the initial
293 EPSP (EPSPN/EPSP 0 was 258% (n = 25 experiments, range 134-500% using a 20 Hz train and 180 s ISI). The peak increase in EPSP amplitude occurred 2 . 5 - 9 min after the potentiating train. As can be seen in Figs. 1, 2 and 6 the enhanced transmission was long lasting. The mean (-+ S . E . M . ) time to peak potentiation was 4.97 -+ 1.70 min (n = 45 trials, with ISI = 0.5-4.5 min). Presenting test pulses at 30 s intervals after the tetanizing train indicated that while variable from A neuron to A neuron, the onset of enhanced transmission typically did not begin until - 1 . 5 min after the presentation of the train. Due to the slow kinetics of both the increase and decay of the enhanced transmission, it has been t e r m e d slow developing potentiation (SDP), While the above experiments were run at the same t e m p e r a t u r e at which the Aplysia were housed (13-14 °C), the time to p e a k and decay of SDP were unchanged at higher temperatures, H o w e v e r , at 22-23 °C, the onset of SDP a p p e a r e d to be more rapid. The slow kinetics clearly distinguished SDP from PTP. The slow rise in enhanced EPSP amplitude was not solely the result of infrequent sampling. When test pulses were presented at 30 s and 60 s ISis following a 20 Hz tetanizing train, the time to peak EPSP amplitude was 3.5 and 4 min respectively. While the slow rise of SDP was still present with shorter ISis,
~
depression was increased. The concomitant reduction in p e a k potentiation suggests that depression and SDP were occurring simultaneously, with depression reducing SDP when short ISis were used. In most experiments, there was little, if any, change in the resting potential of either the pre- or postsynaptic neurons following the tetanizing train. Thus SDP was not the result of a baseline shift for either the presynaptic spike or postsynaptic EPSP. This makes it unlikely that SDP was due to inactivating the conventional K+A current present in A neurons 46. It should be noted that while most experiments were done in SW containing 5 x Ca 2+ and 3 x Mg e+, SDP was also observed using normal SW (11 mM Ca 2+, 50 m M Mg 2+) and SW containing 3 x Ca 2+ and 5 x Mg 2+. SDP was not d e p e n d e n t solely on unusual ionic conditions. I n d e e d , in normal SW, SDP worked 'too well', with the B neurons spiking off the potentiated EPSPs. For example, in one experiment done in normal SW, SDP resulted in 65% of the EPSPs giving rise to action potentials in the B neurons versus none prior to the tetanizing train. However, when B neurons were hyperpolarized sufficiently to prevent EPSPs from triggering action potentials, the onset, rise and decay of SDP in normal SW were the same as in high divalent cation SW. This suggests that spiking by
s
6
'1
+20s
A
LA
'~._
,
/ ~
+3m 8
~
+6m lO
+12m 12
+18m
+36m
,•20mV 2mY
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Fig. 1. Enhanced transmission at the A-B neuron synapse. A left A cluster neuron (LA) was stimulated once every 3 rain. The numbers to the left of the spike indicate the stimulus presentation number. There was a marked depression of the amplitude of the EPSP in a right B cluster neuron (RB) between the 1st and 5th test stimuli. Twenty seconds prior to the 6th test stimulus, the LA neuron was driven at 20 Hz for 2 s. Except for slight further depression, there was no significant change in the amplitude of the EPSP to the 6th test stimulus. However, the EPSP evoked by 7th test stimulus 3 min later was significantly larger than even the first (non-depressed) EPSP. The amplitude of the EPSP to the 8th test stimulus (6 rain after the tetanizing train) was greater than twice that of the first EPSP. The amplitude of the EPSP was still potentiated 30 min later and returned to approximately the initial amplitude by 36 rain.
297 a potential mechanism for SDP, it indicates that, if present, it is pharmacologically distinct from the HSF seen in Aplysia sensory neurons. While SDP was neuron specific, it also appeared to occur at 'all' the followers of the tetanized neuron. As has been previously demonstrated ~4, each A neuron synapses on several B neurons. In most experiments, two B neurons were recorded simultaneously. When an A neuron was tetanized, the connections it made to both B neurons exhibited SDP. This is shown in Fig. 6. Tetanization caused essentially parallel changes in EPSP amplitude in both neurons. Since one of the B neurons was contralateral to the test A neuron, it indicated that SDP did not depend on strictly 'local' (e,g. within a few hundred /~m) changes. Similar results have also been obtained with more distant A neuron followers. The left pleural giant cell receives monosynaptic EPSPs from A neurons 16. This connection also exhibited SDP which paralleled SDP in B neurons (not shown). Although these results do not prove that the efficacy of transmission at all of the A neuron's connections was enhanced, they suggest that many of the connections with B neurons and more distant neurons were potentiated. This too, is consistent with a presynaptic locus for SDP. The results also indicate that generating action potentials in the A neurons was an absolute requirement for SDP. This is supported by the observation that when an A neuron failed to follow the tetanizing train (depolarizations but no spikes), SDP was greatly reduced or absent.
Changes in the presynaptic action potential during SDP Heterosynaptic facilitation of transmitter release by
Aplysia sensory neurons has been shown to be accompanied by increased action potential durations 26. In sensory neurons, there is a marked increase in the duration of the falling phase of the spike due to the inactivation of a K + current 49. Fig. 7 shows superimposed A neuron action potentials before and during SDP. Rather than there being an increase in the falling phase of the spike, there was a slight but noticeable broadening of the rising phase and a small decrease in the repolarizing phase of the spike. SPD was frequently accompanied by slightly reduced thresholds. Occasionally, the stimulus intensity had to be reduced to avoid triggering two spikes. Spike broadening due to prolongation of the falling phase of the A neuron action potential was never seen. This suggests that SDP in A neurons is due to a different cellular mechanism than HSF in Aplysia sensory neurons. This is reinforced by the observation that when a similar stimulus paradigm was tried with cerebral ganglion sensory neurons 42, enhanced transmission was accompanied by a marked prolongation of the falling phase of the ac-
tion potential. Plasticity in these neurons has not yet been examined in detail.
Decay of depression One potential mechanism for SDP is decay of depression m. According to this hypothesis, the tetanizing train might remove the accumulated depression resulting from repetitive stimulation. The slow kinetics of SDP would be due to decay of depression such as that seen at the neuromuscular junction m. To test this, an A neuron was stimulated with a 30 s ISI to induce depression. The stimulus frequency was then decreased. Since the decay of depression is proportional to amount of depression induced, one would expect a rise in EPSP amplitude similar to that seen during SDP. This was not the case. As seen in Fig. 8, reducing the stimulus frequency resulted in only a very minor recovery of EPSP amplitude. However, when a tetanizing train was presented, there was a large increase in EPSP amplitude which, as in all the other experiments, significantly exceeded the initial (non-depressed) EPSP amplitude. Once induced, depression did partially recover if followed by a long period (i.e. 20-30 min) during which the synapse was not stimulated. While this recovery was not systematically quantified, the EPSPs rarely returned to initial amplitudes and never exceeded them. DISCUSSION Although slow developing potentiation was produced by a stimulus paradigm that is very similar to that which evokes PTP, it appears to be a distinct phenomenon. The most obvious distinguishing characteristic is its time course. PTP is usually characterized by a rapid onset and rapid decay. For neurons in Aplysia the onset of PTP typically occurs within 5-10 s of terminating the tetanizing train 7~3s'43'51'52. The decay of PTP is typically also on a time scale of seconds. Ohmori 3s measured the time constant for the decay of PTP at the RC1-R15 synapse with r = 144 _+ 42 s. A slower decay, lasting several minutes has been observed using long (e.g. minutes) tetanizing trains 31. At the L10-RB synapse in the abdominal ganglion, the time constant was much shorter with r = 49 - 5 s 38. The onset of SDP, on the other hand, was only rarely observable 20 s after the tetanizing train and usually did not begin until after 1 min. The decay of SDP was also long, with r = 24 min. Even when SDP is compared to the long-lasting forms of PTP, the peak potentiation on the average occurred when PTP would have been largely or totally decayed (i.e. 5 rain). The experiments performed at 22 °C also suggest that SDP and PTP are distinct. Elevated temperatures did not significantly alter the time to peak or decay of SDP. One might
298 expect that higher temperatures would increase either the pumping or sequestration of Ca 2÷ and thus increase the rate at which PTP decays 7"9. On the other hand the more rapid onset of SDP at 22 °C is consistent with an enzymatic step being involved 13. While the poorly understood phenomenon of decay of depression ~ may make a minor contribution to the slow rise of SDP, it cannot account for most of it. The decay of depression is proportional to the train used to produce the depression 31. This was not observed with SDP. Frequent stimulation of the A neurons (e.g. ISI = 60 s), which increased synaptic depression, did not significantly delay the time to peak. The 'increase' in EPSP amplitude resulting from the decay of depression was far smaller than that seen with SDP (Fig. 8). With the decay of depression, EPSP amplitudes failed to return to initial levels. With SDP, EPSP amplitudes always exceeded the initial amplitude. The results suggest that SDP and depression are simultaneous competing processes. SDP does not appear to turn off synaptic depression; it appears to override depression. Depression at the A-B neuron synapse can be very prominent and long-lasting and appears to be due to biochemical mechanisms quite distinct from SDP (unpublished observations). Another distinguishing characteristic of SDP is the amount of tetanization needed to induce enhanced transmission. SDP could be elicited with as few as 20 spikes (10 Hz for 2 s). This also distinguishes it from PTP and other forms of plasticity such as long-term facilitation (LTF) which require much longer tetanizing trains 58. Typically PTP requires 100-600 action potentials 43. With PTP, tetanizing frequencies can be as low as 2 Hz. Wojtowicz and Atw o o d 53"54 used 20 Hz tetanization for 10 min to induce LTE SDP on the other hand required frequencies >5 Hz, but with correspondingly much shorter train durations. Low frequency stimulation for longer durations (i.e. 5 Hz for up to 10 s) was not effective. Thus SDP appears to be a distinct phenomenon in terms of its onset, time-to-peak, decay time constant and the stimulus parameters which induce it. The data presented above suggest that while SDP was primarily due to a presynaptic mechanism it also appeared to be homosynaptic. Most experiments were done under conditions of high divalent cations, which should have largely suppressed polysynaptic connections. This in itself tended to eliminate heterosynaptic facilitation as a mechanism. The persistence of SDP in the presence of elevated divalent cations however, is by no means conclusive that SDP was homosynaptic. Nor is the fact that the A-B neuron connection is monosynaptic. In intact ganglia it is virtually impossible to prove that SDP is, in fact, homosynaptic, since it is mediated by synapses within the CNS. Like PTP, but unlike HSF, SDP was
neuron specific. Tetanizing a second A neuron did not potentiate the EPSPs of the test A neuron. This also argues that SDP was homosynaptic. If it was heterosynaptic, then there must be one recurrently excited interneuron for each A neuron. In addition, the interneurons must have had a much lower threshold than the B neurons, because the latter rarely spiked (due to elevated divalent cations) during the tetanizing train. If, like LTP, SDP required depolarization and spiking in the postsynaptic neuron then tetanizing either A neuron should have been effective. This was not the case. Stimulating only the postsynaptic neuron also failed to produce SDP and hyperpolarizing the B neurons to prevent spiking, which was infrequent in any case, never blocked it. This was also true in normal SW, where while hyperpolarizing the B neurons was needed to prevent spiking, the failure of the B neurons to generate spikes at the cell body in no way inhibited SDP. Simultaneously stimulating both the pre- and postsynaptic neurons did not increase SDP over that produced by A neuron stimulation alone. Thus, unlike LTP which is synapse specific, SDP appears to have a primarily presynaptic locus. However, it is very difficult to exclude the possibility that SDP is due in part to a postsynaptic mechanism, with different A neuron synapses being sufficiently far apart that changes at one site would not affect the other. One must also postulate that unlike UI'P, increased postsynaptic firing (paired or unpaired) is insufficient to duplicate the effects of synaptic activation. The results are most consistent with SDP being homosynaptic and due to increased transmission by the presynaptic neuron. However, the possibility of either a heterosynaptic or a postsynaptic component to SDP has not been completely eliminated. One possible explanation fi~r SDP is that it might be due to a pump mechanism. The tetanizing train might have increased intracellular N a ' concentrations and activated a Na+/Ca 2+ exchange pump. The net result would be potentiation of transmitter release. There are several difficulties with this hypothesis. First, a pump should be sensitive to the number of action potentials, not their frequency. As noted above, the opposite was true. Second, it is difficult to imagine how a 2 s train could increase Na + sufficiently as to produce potentiation which in several experiments took 9 rain to reach its peak and lasted longer than 30 rain. The kinetics of a pump mechanism appear to be inconsistent with SDP. While SDP had a r = 24 min, Levy and Tillotson 29 report a time constant for the Aplysia Na+/Ca 2~ pump of r = 3.14 s. An additional argument against a pump mechanism is that the Na+/Ca 2+ pump usually pumps Ca 2+ out of the cell not in. The cerebral A neurons have been shown to possess an effective electrogenic Na+/K + pump 2a- If a
299 p u m p m e c h a n i s m is r e s p o n s i b l e for SDP, it is not clear
rent. R e c e n t results suggest that S D P m a y in part be
why a N a + / C a 2+ p u m p r u n n i n g in r e v e r s e , and not the
m e d i a t e d by p r o t e i n kinase C 13 activating C a 2+ channels.
e l e c t r o g e n i c p u m p w o u l d be activated. T h e i n v o l v e m e n t
N e e d l e s s to say, m u c h m o r e w o r k is n e e d e d b e f o r e de-
of the latter is d u b i o u s since the m e m b r a n e p o t e n t i a l of
finitive s t a t e m e n t s can be m a d e a b o u t the u n d e r l y i n g
the A n e u r o n s did not c h a n g e during SDP. While a
m e c h a n i s m s m e d i a t i n g SDP.
p u m p m e c h a n i s m 5~ m a y c o n t r i b u t e to SDP, it d o e s not a p p e a r to be the p r i m a r y m e c h a n i s m .
R e g a r d l e s s of the m e c h a n i s m s , S D P is likely to be of physiological significance. It p r o v i d e s a m e a n s for re-
A l t h o u g h the m e c h a n i s m s r e s p o n s i b l e for S D P are un-
m o v i n g d e p r e s s i o n at the A - B n e u r o n synapse and sug-
clear, o n e possibility is that S D P might w o r k by a m e c h -
gests that t h e r e is b o t h a c t i v i t y - d e p e n d e n t up and d o w n
anism similar to that s e e n in Aplysia sensory n e u r o n s 26'
r e g u l a t i o n of synaptic efficacy. In n o r m a l SW, p o t e n t i -
49. T h e t e t a n i z i n g train may be inactivating K + channels,
a t e d E P S P s f r e q u e n t l y t r i g g e r e d spikes in B n e u r o n s .
allowing m o r e Ca 2+ to e n t e r during the action potential.
Since the B n e u r o n s are m o t o r n e u r o n s m e d i a t i n g de-
15,23,50,
H o w e v e r , unlike sensory n e u r o n s t h e r e was little c h a n g e
fensive b e h a v i o r s such as t e n t a c l e w i t h d r a w a l
in the falling phase of the action p o t e n t i a l . M o s t of the
can potentially m o d i f y the d e f e n s i v e b e h a v i o r of A p l y -
spike b r o a d e n i n g a p p e a r e d to be on the rising phase. It
sia.
SDP
is not clear if the m o d e s t changes in the w a v e - f o r m of the action p o t e n t i a l can a c c o u n t for the substantial increases in synaptic efficacy o b s e r v e d . This still leaves o p e n the possibility that the p r o l o n g a t i o n of the spike might h a v e b e e n d u e to inactivation of an early K + cur-
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Acknowledgements. This work was supported by N1NDS Grant NS28199 to S.M.E, NIGMS MBRS Grant GM (18037, RCMI Grant RR03032 and NSF/MRCE Grant 8714805 to Meharry Medical College.
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