Elsevier

Brain Research

Volume 962, Issues 1–2, 7 February 2003, Pages 78-91
Brain Research

Research report
A paired-pulse facilitation analysis of long-term synaptic depression at excitatory synapses in rat hippocampal CA1 and CA3 regions

https://doi.org/10.1016/S0006-8993(02)03846-5Get rights and content

Abstract

Paired-pulse facilitation (PPF) is a form of short-term, activity-dependent synaptic plasticity common to most chemically transmitting synapses, manifested as an enhancement in the amplitude of the second of two rapidly evoked excitatory postsynaptic potentials (EPSPs). The generally accepted explanation of PPF posits that residual intraterminal free [Ca2+] from the first action potential facilitates the probability of transmitter release evoked by the second stimulus. A common extension of this hypothesis postulates that any plastic change which alters the probability of transmitter release, should also alter the magnitude of PPF. In the present study, we examined the relationship between PPF and both stimulus- and chemically-evoked long-term depression of synaptic strength (LTD) at Schaffer collateral-CA1, commissural/associational-CA3 and mossy fiber-CA3 synapses in rat hippocampal slices. We observed no significant change in mean PPF associated with either electrically- or chemically-induced LTD at any of these synapses. However, a correlation analysis revealed a complex pattern of PPF changes with LTD, such that low initial PPF was correlated with increases in PPF, while high initial PPF was associated with decreases. Combined with previous findings supporting a presynaptic site for chemical and stimulus-evoked LTD, our current data suggests a complex set of neurosecretory modifications downstream of presynaptic Ca2+ influx, may, at least in part, underlie the expression of LTD.

Introduction

Paired-pulse facilitation (PPF) is a relatively short-term, use-dependent form of synaptic plasticity, which occurs at most chemically-transmitting synapses (including hippocampal synapses; [2], [46]). When a synapse is activated twice in rapid succession (typically at a 20–200 ms interval), the magnitude of the second response is typically larger because of a facilitation of transmitter release, thought to be due to residual free [Ca2+] in the terminal left unbuffered from the first ‘conditioning’ pulse [17]. In the simplest formulation, residual Ca2+ summates with Ca2+ influx from the second stimulus, to enhance the probability of release. This is the phenomenon of PPF, and, based on the preceding view, it is considered an example of purely presynaptic plasticity, though postsynaptic effects on paired-pulse responses can be superimposed. The PPF ratio is a way of quantifying this effect as an enhancement of the second response relative to the first, or: EPSP2/EPSP1.

It has been predicted that this property can be used as a tool to assess the locus of changes underlying other longer-term forms of synaptic plasticity, including long-term potentiation (LTP; [1], [14], [19], [20], [25], [33], [34], [38], [45]) and long-term depression (LTD; [11], [13], [24]). The reasoning behind this idea is as follows: The magnitude of an excitatory postsynaptic potential (EPSP) is proposed to be a function of the product of 3 parameters: (1) the number of neurotransmitter release sites (n), (2) the probability of neurotransmitter release (pr), and (3) the magnitude of the postsynaptic unit response at each release site (q; quantal size), related by the following equation:EPSP=n·pr·q

Since PPF is believed to involve a transient increase in the probability of transmitter release, pr, during the second response, it should follow that PPF is an inverse function of the initial probability of release associated with the first pulse, (pri), or:PPF1/pri

In other words, if initial probability of release is high (close to 1), meaning most terminals will release neurotransmitter in response to the first pulse, the effect of any residual calcium to enhance pr in response to a second stimulus will be minimal, and PPF ratio will be small (in the limit where pri=1, PPF=1). Conversely, when pri is very low, few terminals release in response to the first stimulus, so the effect of increased residual [Ca2+] will be that many more terminals will release in response to a second stimulus, and PPF ratio is high.

We can extend this reasoning to other forms of synaptic plasticity, provided two assumptions are satisfied: (1) the absolute number of release sites, and the amount of transmitter released per quantum must be fixed, and (2) any manipulation which changes pri must also affect the magnitude of PPF ratio. If LTP causes an increase in pri, there should be a parallel decrease in PPF. Similarly, if LTD involves a decrease in neurotransmitter release, a parallel increase in PPF should be observed. If this simple hypothesis is valid and the above assumptions hold, this line of investigation could be powerful and appealing, since it is based upon minimal assumptions, and PPF is a robust and easily-measured property of chemical synapses.

Therefore, we used PPF to probe the mechanisms underlying multiple forms of LTD in the hippocampus. In a previous report [32], we described a purely chemical means for the induction of LTD (CLTD) at Schaffer collateral-CA1 synapses, produced by simultaneous elevation of intracellular [cGMP] and inhibition of cyclic AMP-dependent protein kinase (PKA). Despite different modes of induction, chemically-induced LTD (CLTD) is occluded by LTD elicited by prolonged low frequency stimulation (SLTD), suggesting convergence of expression mechanisms for the two phenomena. Data from intracellular experiments, where pharmacologic agents were applied selectively to a postsynaptic CA1 pyramidal neuron, suggested that the primary locus of induction of chemically-induced LTD was presynaptic [32]. However, CLTD did not appear to be paralleled by an increase in PPF ratio. In the present study, we extended PPF analyses to include both stimulus-induced LTD, and an examination of chemical LTD, at multiple hippocampal synapses in CA1 and CA3 regions of the hippocampus.

There are two anatomically distinct excitatory pathways which terminate on separate portions of apical dendrites of CA3 pyramidal neurons; a mixed commissural/associational input, and a mossy fiber input (for review see [7], [40]). Mossy fibers are the axons of dentate granule cells which synapse on the proximal portion of apical dendrites of CA3 pyramidal cells in stratum lucidum. The mixed commissural/associational input, on the other hand, includes a recurrent network of fibers, where axons from CA3 neurons (Schaffer collaterals) feedback onto their neighbors, synapsing more distally on apical dendrites in stratum radiatum. These two inputs appear to exhibit mechanistically distinct forms of both LTP [28], [44], and LTD [13], [24]. Most evidence indicates that mossy fiber-CA3 synapses exhibit a purely presynaptic form of LTP and LTD (but see [44]), whereas commissural/associational-CA3 synapses require Ca2+ influx into postsynaptic pyramidal cells for the induction of either LTP or LTD. This makes CA3 an ideal area in which to independently test the hypothesis that chemical LTD, and a portion of SLTD, are expressed presynaptically. In area CA3, we compared, at both mossy fiber and commissural/associational synapses on a single population of pyramidal cells, PPF changes associated with CLTD and SLTD.

Section snippets

Hippocampal slice preparation

Experiments were performed on hippocampal slices from 14–21 day old Sprague Dawley rats of both genders. Animals were decapitated under deep ether anesthesia and the brain rapidly removed. Both hippocampi plus overlying entorhinal cortex were dissected free of surrounding tissue at room temperature, and transverse slices (400 μm thick) prepared using a spring-loaded ‘egg slicer’ grid chopper. With this method, a parallel grid of 20 μm diameter tungsten wires, spaced 400 μm apart, is forced

Effects of SLTD on Schaffer collateral-CA1 PPF

In an earlier study [32], we found no significant change in PPF ratio following the induction of chemical LTD (CLTD) at Schaffer-collateral-CA1 synapses, a form of LTD induced by simultaneous elevation of intracellular [cGMP] and inhibition of PKA. Since stimulus-induced LTD is believed to share some common expression mechanisms with CLTD, we followed the time course of PPF before and after the induction of electrically-evoked LTD (SLTD). Fig. 2A (n=10) shows the induction of SLTD following

Discussion

In this study, we found no net change in average paired-pulse facilitation (PPF) ratio following the induction of either chemical- or stimulus-evoked LTD at a number of synapses in the hippocampus. This does not necessarily undermine the hypothesis that a component of LTD is expressed by presynaptic alterations, a hypothesis supported by multiple studies on chemically and stimulus-induced LTD. Data utilizing intracellular sharp microelectrodes to selectively infuse drugs into single

Acknowledgments

We thank C. Bailey, A. Kyrozis and S. Nawy for helpful discussions. Supported by Whitehall Foundation grant #A-32 to PKS.

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