Analysis of short-term plasticity at the perforant path-granule cell synapse

Brain Research, 178 (1979) 41-53 © Elsevier/North-Holland Biomedical Press

41

A N A L Y S I S OF S H O R T - T E R M P L A S T I C I T Y A T T H E P E R F O R A N T P A T H - G R A N U L E CELL SYNAPSE

w. F. WHITE*, J. V. NADLER and C. W. COTMAN** Department of Psychobiology, University of California, Irvine, Cali£ 92717 (U.S.A.) (Accepted March 22nd, 1979)

Key words:

synaptic transmission - - entorhinal cortex - - dentate gyrus - - glutamate - - 2-amino-4-phosphono-butyric acid

SUMMARY Short-term plasticity was investigated at the perforant path-granule cell synapse in the hippocampal slice preparation. A successive decrement in the amplitude of the extracellular EPSP was obtained at all stimulus frequencies above 0.05 Hz. This effect of repetitive stimulation has previously been shown to fulfill the requirements for habituation processes. If each stimulus within an habituation train was followed by a second identical test stimulus the response to the test stimulus was larger than that to the paired conditioning stimulus. This short-term plasticity has been called paired pulse potentiation. The test response potentiated only with respect to the paired conditioning response and not with respect to previous test responses. Neither form of plasticity appeared to result from changes in the amplitude of the afferent fiber volley. Both habituation and paired pulse potentiation result from an interaction of at least three changes in the efficacy of transmission after a conditioning stimulus: (1) an initial depression, (2) an intermediate relative potentiation and (3) a late depression which decays slowly. Paired pulse potentiation could be demonstrated only if the interpair interval corresponded to the period of maximal late depression and the interstimulus interval to the period of relative potentiation. The amplitudes of intermediate relative potentiation and late depression (and inhibition of transmission by 2-amino-4-phosphonobutyric acid (APB)) were inversely related to the control response amplitude. This relationship likely derives from nonlinear summation of postsynaptic ionic currents at higher stimulus intensities. In contrast, the initial depression increased with response amplitude. This is consistent with a mechanism dependent on the postsynaptic membrane potential, such as refractoriness to succeeding stimuli. * Present address: Department of Neurosciences, The Children's Hospital Medical Center, 300 Longwood Ave., Boston, Mass. 02115, U.S.A. This paper is based in part on a thesis submitted by W.F.W. to the University of California, Irvine in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ** To whom all correspondence should be sent,

42 When the response amplitudes in the presence and absence of 2.5 mM APB were equalized by adjusting the stimulus intensity, no difference was found in the magnitude of either form of plasticity. Since APB probably inhibits transmission at this site through competitive antagonism at the postsynaptic receptor, this observation suggests that habituation and paired pulse potentiation are generated presynaptically. INTRODUCTION The perforant path-granule cell synapse in the dentate gyrus shows several forms of short-term plasticity. Repetitive activation of the perforant path fibers successively decreases the efficacy of transmission1, ~4. No other hippocampal pathway shows this response to repetitive activation. This synaptic depression has been interpreted as a form of monosynaptic habituation on the basis of its similarity to behavioral habituation. Another form of short-term plasticity found at this synapse has been called paired pulse potentiation6,12, ~3. That is, when a conditioning stimulus is followed shortly (20-200 msec) by a test stimulus of the same intensity, the test response is greater than the conditioning response. Other hippocampal pathways also show paired pulse potentiation. These two forms of plasticity appear contradictory, in that a stimulus can either depress or potentiate the response to a following stimulus, if they are separated by a short interval. These types of short-term plastic phenomena have been suggested as synaptic analogues of behavioral plasticity in. Thus investigation of their mechanisms would provide insights into the mechanisms underlying behavior. To determine the underlying mechanisms of these phenomena, they must be studied at synapses where the neurotransmitter is known and drugs are available for manipulating transmission. We have previously argued that glutamate is the transmitter at the perforant path-granule cell synapse, since the perforant path boutons appear to release glutamate on stimulation in a manne~ suggestive of a transmitter function4,9-~l, 2° and synaptic transmission at this site is blocked by certain putative glutamate antagonistst8, z0. In this study we have investigated habituation and paired pulse potentiation with use of the hippocampal slice preparation. To determine whether these short-term changes are mediated by pre- or postsynaptic mechanisms, we have made use of the putative acidic amino acid antagonist, 2-amino-4-phosphonobutyric acid (APB). This glutamate analogue appears to inhibit transmission at the perforant path-granule cell synapse by interacting with the postsynaptic receptod 8, 2o. Thus APB should interfere with postsynaptically mediated processes, but not with those primarily dependent on presynaptic alterations. METHODS Procedures for the preparation, maintenance and perfusion of hippocampal slices, as well as the recording techniques employed in this study, are described in detail elsewhere 17. Briefly, 700 /~m thick transverse slices of rat hippocampal formation were placed in individual chambers, and Elliott's artificial CSF 3 (122 mM

43 NaCI, 25 mM NaHCO3, 3.1 mM KC1, 1.3 mM CaC12, 1.2 mM MgSO4, 0.4 mM KH2PO4, 10 mM D-glucose, pH 7.4 with O2/CO2 (95 ~/5 ~)), warmed to 30-33 °C, was perfused through the chambers at 2.5 ml/min. Conventional stimulating and recording techniques were employed, and the baseline to peak amplitude of each evoked perforant path extracellular EPSP was manually determined, either directly from the oscilloscope or from continuous film records. A bipolar stimulating electrode was placed in the perforant path fibers as they course through the subiculum and a micropipette recording electrode filled with 4 M NaC1 (3-15 Mf~) in their termination field within the dentate molecular layer (Fig. 1A). Although optimal positioning of the electrodes required some stimulation of the perforant path fibers, no stimulation was applied for at least a 5 rain interval before the start of the experiment, in order to avoid any stimulus induced changes in synaptic efficacy. In addition, the stimulus intensity (usually about 1 V) was maintained below threshold for the population spike, as recorded in the dendrites. Since the extracellular EPSP was uncontaminated by population spikes, its amplitude could be measured accurately and reflected the synaptic drive imparted by the stimulus. RESULTS

Stimulation of the perforant path projection generated a negative extracellular EPSP in the molecular layer of the dentate gyrus (Fig. 1B). This potential results from the summed synaptic currents produced at the perforant path-granule cell synapses in the vicinity of the recording electrode, and its amplitude is proportional to the synaptic drive conveyed by perforant path activationL Thus in the present study the amplitude of the extracellular EPSP was employed as a measure of synaptic efficacy. Habituation was studied as a function of the stimulus frequency and paired pulse

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Fig. 1. Hippocampal slice illustrating stimulating and recording sites and the form of the perforant path extracellular EPSP. A: hippocampal slice with stimulating (S) and recording (R) sites indicated. B: evoked perforant path extracellular EPSP. The control response amplitude varied between 0.8 mV and 2 mV, positive up. PP, perforant path; MF, mossy fibers; Corn, commissural fibers to area CA1 ; SC, Schaffer collaterals.

44 B PAIRED-PULSE POTENTIATION

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Fig. 2. Habituation (A) and paired pulse potentiation (B) at the perforant path-granule cell synapse. Values are percentages of control response (amplitude of extracellular EPSP). In this and subsequent figures the stimulation paradigm is given below the plot of response amplitudes. C, conditioning stimuli or responses; T, test stimuli or responses, Sample extracellular EPSPs are shown at the times they were recorded. A: habituation of the perforant path extracellular EPSP during stimulation at 1 Hz. B: paired pulse potentiation of the perforant path extracellular EPSP. Twenty pairs of stimuli were delivered at an IP[ of 1 sec and a ISI of 40 msec. Note that, after the first pair, the test response is larger than its paired conditioning response.

A.

Fig. 3. Fiber potential amplitude to paired-pulse stimulation. A: the only case in which the fiber potential was easily measured in the absence of added Mg 2+. Paired-pulse potentiation is quite apparent in this record while the amplitude of the test fiber potential (arrow) is slightly less than the conditioning fiber potential (B, C, D). A series of fiber potentials were taken 2 rain after the introduction of 25 m M Mg z+ (B), 5 rnin after the introduction of 25 m M Mg 2+ (C) and 5 min after the addition of 2.5 m M APB to the media containing 25 m M Mg 2+ (D). Note the potentiation of the test response in B and the lack of potentiation of the fiber potential. Also note the slightly decreased amplitude of the test fiber potential compared to the conditioningfiber potential in C and D. Finally there is a lack of an effect of 2.5 m M APB on the amplitude of the fiber potentials in C and D. Calibration bars: A, 1 mV, ] 0 msec; B, 200 pV, I0 msec; C and D, 100 pV, 10 msec.

45 potentiation was studied as a function of the time between members of a pair of stimuli (interstimulus interval, ISI) and the time between pairs (interpair interval, IPI). The response to the first stimulus in a train of unpaired stimuli or to the first conditioning stimulus in a train of paired stimuli is referred to as the control or initial response. Repetitive activation of the perforant path fibers at 1 Hz successively decreased the amplitude of the evoked extracellular EPSP (Fig. 2A). When a second test stimulus followed 40 msec after each of the stimuli in the 1 Hz train, the test (second) response was greater than the conditioning (first) response in all pairs but the first (Fig. 2B). In approximately 25 ~ of the cases the first test response also showed potentiation. In all cases the amplitude of both conditioning and test responses underwent a successive depression similar to that of responses to unpaired stimuli. Repetitive activation affected only the amplitude, not the shape, latency or duration, of the extracellular EPSP. The presynaptic fiber potential was monitored in a parallel series of'experiments to determine whether these changes in synaptic efficacy result from changes in the number of fibers being activated. As the amplitude of the fiber potential is difficult to measure in the presence of the much larger extracellular EPSP these experiments were performed in the presence of 25 mM Mg z+ which totally inhibits the extracellular EPSP. The amplitude of the presynaptic fiber potential did not parallel the plasticity seen in the extracellular EPSP. In fact, under conditions which produce paired-pulse potentiation of the extracellular EPSP there was frequently a depression of the test fiber potential rather than a potentiation (Fig. 3). These observations support the notion that paired-pulse potentiation and habituation result from changes occurring at the synapse. In order to investigate the processes which underlie these phenomena, pairs of stimuli were delivered at various ISis (10 msec to 20 sec) separated by a long IPI (50 sec) to avoid interactions between successive pairs. At very short ISis the response to the test stimulus was depressed relative to that evoked by the conditioning stimulus (Fig. 4A). Lengthening the ISI up to 50 msec reversed this depression, but further increases in the ISI once again reduced the amplitude of the test response. Finally, this late depression became increasingly less evident at ISis above 0.5 sec, and no effect of the conditioning stimulus could be detected at ISis of 20 sec or longer. In all but one experiment in this series the amplitude of the test response never equaled or exceeded that of the control response. In the one exceptional case no late depression was observed and potentiation of the test response was seen at intermediate ISis (Fig. 4B). This result suggested that the relative potentiation observed at ISis around 50 msec represents a true facilitation of transmission rather than simply a reversal or delay of synaptic depression and that at least two distinct processes are involved. This study indicated at least three changes in the efficacy of transmission at the perforant path-granule cell synapse following a conditioning stimulus: (1) an initial depression, (2) an intermediate relative potentiation and finally (3) a depression that decays slowly. Each of these alterations in synaptic excitability appears to decay linearly with log time. Presumably the short-term plastic phenomena observed at this synapse result from an interaction of these processes. Thus habituation would be

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Fig. 6. Effect of response amplitude on habituation and paired pulse potentiation. A: habituation of the perforant path extracellular EPSP during stimulation at 1 Hz and at stimulus intensities which produced control responses of the amplitudes indicated. Values are percentages of the response to the first stimulus in the train (control response). Trains were separated by 5 min. Results of a typical experiment on a single slice are shown. Similar results were obtained in 5 additional experiments. B: paired pulse potentiation obtained at different control response amplitudes. Each point is the average potentiation of 10 test pulses expressed as a percentage of the paired conditioning response amplitude. ISI = 40 msec, IPI -- 1 sec. Line was drawn by the method of least squares. Results of a typical experiment on a single slice are shown. Similar results were obtained in 3 additional experiments.

intersected, so t h a t the test response was greater than the c o n d i t i o n i n g response (paired pulse potentiation). A t long I P I s (greater t h a n 2 sec) the c o n d i t i o n i n g response was always greater t h a n the test response even t h o u g h the c o n d i t i o n i n g response sometimes h a b i t u a t e d to a greater extent. A t short IPIs (200 msec or less) the two responses were a b o u t equal. Hence only at IPIs c o r r e s p o n d i n g to the p e r i o d o f m a x i m a l late depression was p a i r e d pulse p o t e n t i a t i o n obtained. Clearly, the d e m o n s t r a t i o n o f this f o r m o f plasticity does, in fact, d e p e n d on b o t h the I S I a n d I P I as suggested by Fig. 3. I n agreement with previous reports, the m a g n i t u d e o f b o t h h a b i t u a t i o n 14 a n d p a i r e d pulse p o t e n t i a t i o n 13 at this synapse was inversely related to the c o n t r o l response a m p l i t u d e (Fig. 6A, B). To investigate the effect o f response a m p l i t u d e on the three changes in synaptic efficacy which follows a c o n d i t i o n i n g stimulus, we delivered pairs o f stimuli at v a r i o u s I S i s (10 msec to 50 sec) a n d a c o n s t a n t I P I (50 sec) at two stimulus intensities t h a t p r o d u c e d responses o f quite different amplitudes. A l l phases o f altered excitability were affected b y a change in c o n t r o l response amplitude, larger responses t e n d i n g to accentuate the initial depression a n d flatten the r e m a i n d e r o f the curve (Fig. 7). A series o f experiments on b o t h h a b i t u a t i o n a n d p a i r e d pulse p o t e n t i a t i o n was c o n d u c t e d in the presence o f 2.5 m M A P B to d e t e r m i n e w h e t h e r these forms o f shortt e r m plasticity reflect p r e - o r p o s t s y n a p t i c alterations. A P B at this c o n c e n t r a t i o n has previously been shown to depress t r a n s m i s s i o n at the p e r f o r a n t p a t h - g r a n u l e cell

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DISCUSSION

The results presented here confirm previous studies in showing the occurrence of both habituation and paired pulse potentiation at the perforant path-granule cell synapse. The extracellular EPSP habituates whenever the perforant path fibers are stimulated at a frequency above 0.05 Hz. Potentiation of the second member of a pair of stimuli can be demonstrated only if both the ISI and IPI are carefully selected. In these studies maximal paired pulse potentiation was obtained with an ISI of 40 msec and an

51 IPI of 1 sec. The test response is potentiated only with respect to the paired conditioning response and not with respect to the test response of the initial or immediately preceding pair. In previous studies on paired pulse potentiation averaged responses were measuredS, 13, a procedure which masks the overriding habituation at this synapse and brings out the potentiation. This is especially true if many stimulus pairs are averaged, since habituation becomes asymptotic after the first several stimuli, or if the first pair of stimuli is not included in the average. Both habituation and paired pulse potentiation result from an interaction of at least three changes in the efficacy of transmission which follow a single stimulus: (1) an initial depression, (2) an intermediate relative potentiation and (3) a late depression which slowly decays. At constant stimulus frequencies above 0.05 Hz depression predominates and the response therefore habituates. If a stimulus train is presented in pairs, however, the intermediate relative potentiation can overcome the late depression, if the ISI corresponds to the period of relative potentiation and the IPI coincides with the period of maximal late depression. If the intervals between stimuli and pairs are not optimized in this fashion, the late depression predominates and no paired pulse potentiation is observed. Our study confirms previous observations that the magnitude of both habituation and paired pulse potentiation is inversely related to the amplitude of the initial extracellular EPSP. This is attributable to a similar variation of both the intermediate potentiation and late depression with initial response amplitude. In addition, the degree of inhibition produced by the putative acidic amino acid antagonist, APB, is also inversely related to the amplitude of the control response. These observations can be explained by a non-linear relationship between synaptic input and the postsynaptic membrane potential at higher stimulus intensities. Thus any process which alters synaptic input affects the postsynaptic membrane potential less at higher intensities. This has been attributed to increased shunting of synaptic currents at the postsynaptic membrane 7,s. In contrast, greater response amplitudes augment the initial depression. This observation suggests a dependence on the magnitude of the postsynaptic depolarization. As more presynaptic elements are activated, the postsynaptic element may become increasingly refractory to stimuli which follow too closely after one another. Studies with the reversible antagonist, APB, were conducted to determine whether these forms of short-term plasticity require activation of the postsynaptic receptor. Present evidence suggests that APB depresses transmission at the perforant path-granule cell synapse by acting at the postsynaptic receptor; thus it should inhibit only postsynaptically mediated forms of plasticity. In accordance with this hypothesis, APB does not affect post-tetanic potentiation, which is generally regarded as a presynapticaUy mediated process 19. In these experiments we found no significant difference in the magnitude of either habituation or paired pulse potentiation, when control response amplitudes in the presence and absence of drug were equalized. On this basis we conclude that both forms of short-term plasticity result from changes that occur presynaptically, presumably involving modulation of transmitter release. These conclusions are consistent with mechanisms proposed to underlie similar processes at

52 other vertebrate synapsesZ, 15. This is in contrast to the i n h i b i t o r y effect of A P B o n long-term p o t e n t i a t i o n at the Schaffer-commissural-CA1 p y r a m i d a l cell synapse, a n action which suggests that receptor activation is required for this form of plasticity 19. ACKNOWLEDGEMENTS W e t h a n k Ms. J. Mueller for secretarial aid a n d Mr. D. Shelton, Ms. Ellen Lewis a n d Ms. Grace C h i a n g for their help with the figures. This study was supported by N S F G r a n t BNS 76-09973 a n d N I M H G r a n t M H 19691. C. W. C o t m a n is recipient of a Research Scientist A w a r d from N a t i o n a l Institute of D r u g A b u s e ( D A 00019).

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