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1. Paired recordings from monosynaptically connected CA3 interneurons and pyramidal cells of rat hippocampal slice cultures were used to compare the modulation of GABA release at synapses from distinct interneurons. 2. The group II metabotropic glutamate receptor (mGluR) agonist (2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl) glycine (DCG-IV, 5 muM) reduced the amplitude of IPSPs originating from stratum radiatum but not stratum oriens interneurons. In contrast, the GABAB receptor agonist (-)baclofen (10 muM) reduced the amplitude of unitary IPSPs elicited by all interneurons. 3. IPSPs mediated by stratum oriens interneurons were unaffected by the N-type calcium channel blocker omega-conotoxin MVIIA (1 muM) but were suppressed by the P/Q-type blocker omega-agatoxin IVA (200 nM). In contrast, IPSPs mediated by stratum radiatum interneurons were abolished by omega-conotoxin MVIIA. 4. Transmission dynamics were different at synapses from the two groups of interneurons. IPSPs mediated by stratum oriens interneurons showed marked paired-pulse depression (PPD) at intervals of 50 400 ms. IPSPs mediated by stratum radiatum interneurons showed paired-pulse facilitation (PPF) at 50 ms and PPD at longer intervals. 5. The amplitude of unitary IPSPs from all interneurons was unaffected by the GABAB receptor antagonist CGP52432 (2 muM) as was PPD at both 50 and 400 ms intervals. However, CGP52432 did reduce PPD of extracellularly evoked IPSPs. 6. Our results show that two groups of inhibitory synapses impinging onto CA3 pyramidal cells can be distinguished according to their dynamic and modulatory properties.

A series of recent studies has provided long-awaited direct evidence that enduring changes in synaptic strength, presumably underlying the formation of persistent memories, may be encoded in a lasting form as a change in synaptic structure.

Long-term potentiation (LTP) of unitary EPSPs, generated by pairs of monosynaptically connected CA3 and CA1 pyramidal cells, was compared with LTP of extracellularly evoked, multi-unitary EPSPs in rat hippocampal slice cultures. LTP was induced by repeated, synchronous pairing of low-frequency presynaptic and postsynaptic activity. Three differences were observed. First, LTP of multi-unitary EPSPs displayed two phases: transient (<5 min) and sustained. Potentiation of unitary EPSPs displayed both phases in 42% of experiments; the remainder showed sustained potentiation only. Unitary EPSPs displaying transient-sustained and only sustained potentiation could be recorded from single postsynaptic cells, indicating that excitatory synapses on a given cell are heterogeneous with respect to short-term plasticity. Second, whereas LTP of multi-unitary EPSPs never resulted in greater than twofold increases in amplitude (mean potentiation of 175% of control), maximal LTP of unitary EPSPs was as great as 13-fold (mean potentiation of 250%). Third, LTP could not be induced in 24% of unitary EPSPs. We provide here the first evidence for the coexistence of potentiatable and nonpotentiatable synapses on individual postsynaptic neurons. Thirty-seven percent of connections not displaying LTP exhibited long-term depression (LTD), suggesting that the connections were already maximally potentiated. In the remaining 63% of these pairs, neither LTP nor LTD could be induced, despite the existence of a pharmacologically identified, NMDA receptor-mediated EPSP component. In conclusion, there is considerable heterogeneity in the amplitude and time course of LTP expression at different synaptic connections. A substantial proportion of apparently nonplastic synapses probably accounts for the weaker potentiation displayed by compound EPSPs.

We investigated the influence of synaptically released glutamate on postsynaptic structure by comparing the effects of deafferentation, receptor antagonists and blockers of glutamate release in hippocampal slice cultures. CA1 pyramidal cell spine density and length decreased after transection of Schaffer collaterals and after application of AMPA receptor antagonists or botulinum toxin to unlesioned cultures. Loss of spines induced by lesion or by botulinum toxin was prevented by simultaneous AMPA application. Tetrodotoxin did not affect spine density. Synaptically released glutamate thus exerts a trophic effect on spines by acting at AMPA receptors. We conclude that AMPA receptor activation by spontaneous vesicular glutamate release is sufficient to maintain dendritic spines.

1. Long-term potentiation (LTP) and depression (LTD) were investigated at synapses formed by pairs of monosynaptically connected CA3 pyramidal cells in rat hippocampal slice cultures. 2. An N-methyl-D-aspartate (NMDA) receptor-mediated component of the unitary EPSP, elicited at the resting membrane potential in response to single action potentials in an individual CA3 cell, could be isolated pharmacologically. 3. Associative LTP was induced when single presynaptic action potentials were repeatedly paired with 240 ms postsynaptic depolarizing pulses that evoked five to twelve action potentials or with single postsynaptic action potentials evoked near the peak of the unitary EPSP. LTP induction was prevented by an NMDA receptor antagonist. 4. Associative LTD was induced when single presynaptic action potentials were repeatedly elicited with a certain delay after either 240 ms postsynaptic depolarizing pulses or single postsynaptic action potentials. The time window within which presynaptic activity had to occur for LTD induction was dependent on the amount of postsynaptic depolarization. LTD was induced if single pre- and postsynaptic action potentials occurred synchronously. 5. Homosynaptic LTD was induced by 3 Hz tetanization of the presynaptic neuron for 3 min and was blocked by an NMDA receptor antagonist. 6. Depotentiation was produced with stimulation protocols that elicit either homosynaptic or associative LTD. 7. Recurrent excitatory synapses between CA3 cells display associative potentiation and depression. The sign of the change in synaptic strength is a function of the relative timing of pre- and postsynaptic action potentials.

Transmitter release at most central synapses depends on multiple types of calcium channels. Identification of the channels mediating GABA release in hippocampus is complicated by the heterogeneity of interneurons. Unitary IPSPs were recorded from pairs of inhibitory and pyramidal cells in hippocampal slice cultures. The N-type channel antagonist omega-conotoxin MVIIA abolished IPSPs generated by interneurons in st. radiatum, whereas the P/Q-type antagonist omega-agatoxin IVA had no effect. In contrast, omega-agatoxin IVA abolished IPSPs generated by st. lucidum and st. oriens interneurons, but omega-conotoxin MVIIA had no effect. After unitary IPSPs were blocked by toxin, transmission could not be restored by increasing presynaptic calcium entry. The axons of the two types of interneurons terminated within distinct strata of area CA3. Thus, GABA release onto pyramidal cells, unlike glutamate release, is mediated entirely by either N- or P-type calcium channels, depending on the presynaptic cell and the postsynaptic location of the synapse.

Feedback inhibitory circuits were characterized electrophysiologically in the CA3 region of organotypic rat hippocampal cultures. Pyramidal cells were impaled with sharp microelectrodes and brief depolarizing current pulses were injected intracellularly to elicit single action potentials. An inhibitory postsynaptic potential (IPSP) was observed at fixed latency after the action potential in 27% of impaled cells (n = 131). These IPSPs were fully blocked by bicuculline, indicating that they were mediated solely by gamma-aminobutyric acid type A (GABAA) receptors. They were also blocked by 6-cyano-7-nitro-quinoxaline-2, 3-dione but not D-2-amino-5-phosphonovalerate, indicating that non-N-methyl-D-aspartate receptors were necessary and sufficient for activating interposed GABAergic interneurons. Adenosine (0.1-5 microM) increased the percentage of action potentials that were not followed by IPSPs by reducing the probability of glutamatergic activation of the interneurons. In 18 of 21 experiments adenosine also decreased the mean amplitude of successfully elicited IPSPs, indicating that more than one interneuron participated in the feedback inhibition of those pyramidal cells. In three experiments the non-failure IPSP amplitude was not affected by adenosine, suggesting that only one interneuron participated. Repetitive stimulation at 2-4 Hz decreased the amplitude of non-failure feedback IPSPs and usually increased the number of failures of transmission. These effects were transient and insensitive to the GABAB antagonist CGP 35348. We conclude that both the excitation of interneurons and the release of GABA from interneurons are modulated by repetitive stimulation.

1. Excitatory synaptic transmission between pairs of monosynaptically coupled pyramidal cells was examined in rat hippocampal slice cultures. Action potentials were elicited in single CA3 pyramidal cells impaled with microelectrodes and unitary excitatory postsynaptic currents (EPSCs) were recorded in whole-cell voltage-clamped CA1 or CA3 cells. 2. The amplitude of successive unitary EPSCs in response to single action potentials varied. The amplitude of EPSCs was altered by adenosine or changes in the [Mg2+]/[CA2+] ratio. We conclude that single action potentials triggered the release of multiple quanta of glutamate. 3. When two action potentials were elicited in the presynaptic cell, the amplitude of the second EPSC was inversely related to the amplitude of the first. Paired-pulse facilitation (PPF) was observed when the first EPSC was small, i.e. the second EPSC was larger than the first, whereas paired-pulse depression (PPD) was observed when the first EPSC was large. 4. The number of trials displaying PPD was greater when release probability was increased, and smaller when release probability was decreased. 5. PPD was not postsynaptically mediated because it was unaffected by decreasing ionic flux with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or receptor desensitization with aniracetam. 6. PPF was maximal at an interstimulus interval of 70 ms and recovered within 500 ms. Recovery from PPD occurred within 5 s. 7. We propose that multiple release sites are formed by the axon of a CA3 pyramidal cell and a single postsynaptic CA1 or CA3 cell. PPF is observed if the first action potential fails to release transmitter at most release sites. PPD is observed if the first action potential successfully triggers release at most release sites. 8. Our observations of PPF are consistent with the residual calcium hypothesis. We conclude that PPD results from a decrease in quantal content, perhaps due to short-term depletion of readily releasable vesicles.

The effects of the benzodiazepine agonist midazolam on GABAA receptor-mediated inhibition were investigated in area CA3 of hippocampal slice cultures. Midazolam (100 nM) increased the decay time constant (tau OFF) of miniature inhibitory postsynaptic currents (mIPSCs) recorded from pyramidal cells by approximately 40%, but did not significantly affect their activation rate or amplitude, consistent with saturation of postsynaptic GABAA receptors by a quantum of GABA. Non-stationary variance analysis of mIPSCs revealed that the unitary conductance of synaptic GABAA channels (approximately 31 pS) was unaffected by midazolam. Midazolam increased not only the tau OFF (51%), but also the amplitude (23%) of unitary IPSPs, recorded from pairs of monosynaptically connected inhibitory and pyramidal cells. Simulation of unitary IPSPs indicated that the increased amplitude was primarily due to the slow time constant of pyramidal cells. Finally, the mean amplitude, tau OFF, and single-channel conductance of mIPSCs recorded in cultures chronically exposed to midazolam (0.1-10 microM) for 2 weeks were not different from control mIPSCs, nor was their response to midazolam. We conclude that benzodiazepines increase synaptic GABAA channel open time, as described previously, and that this results in an increase in both the amplitude and duration of IPSPs in pyramidal cells.

We report here two forms of activity-dependent plasticity of the transfer of neuronal information between pairs of monosynaptically coupled pyramidal cells. In a first part, we discuss the induction of long-term bidirectional changes in excitatory synaptic transmission following defined regimes of neuronal activity. In a second part, we provide evidence that the conditions in which the presynaptic action potential is elicited determine whether it will successfully propagate along the presynaptic axon.

1. Paired intracellular recordings were made in rat hippocampal slice cultures, with the use of either sharp microelectrodes or the whole cell configuration of the patch-clamp technique. Unitary synaptic connections were studied between pyramidal and nonpyramidal cells within and between areas CA1 and CA3. 2. Monosynaptic excitatory synaptic responses between CA3 pyramidal neurons were found in 56% of cell pairs (n = 91, 28 postsynaptic cells). Monosynaptic connections from a CA3 cell to a CA1 cell were observed in 76% of cell pairs (n = 125, 26 postsynaptic cells), but from CA1 to CA3 neurons in only 8% of cell pairs (n = 13, 13 postsynaptic cells). Monosynaptic excitatory connections were found in only 16% of CA1/CA1 cell pairs (n = 25, 10 postsynaptic cells). 3. Disynaptic inhibition was commonly observed between CA3 cell pairs (43%), but rarely found between CA3-CA1 pyramidal cell pairs (2%). In 50% of CA3 pyramidal cell pairs, synchronous inhibitory postsynaptic potentials (IPSPs) in both cells could be triggered by an action potential in one pyramidal cell. Reciprocal monosynaptic connections were found between 75% of interneuron and pyramidal cell pairs within area CA3. 4. The latency of monosynaptic CA3- to CA1-cell responses was significantly longer than for responses between two CA3 cells. Within area CA3 the latencies for inhibitory synaptic responses between interneurons and pyramidal cells were significantly shorter than those for excitatory responses between pyramidal cells. Monosynaptic excitatory postsynaptic potentials (EPSPs) in interneurons had a significantly shorter time-to-peak than those recorded in pyramidal neurons. 5. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX)- and D-2-amino-5-phosphonovalerate (AP5)-sensitive components were identified in unitary monosynaptic EPSPs in CA3-CA3 and CA3-CA1 pyramidal cell pairs. The CNQX-sensitive component had a mean time-to-peak and duration of 6.2 +/- 0.3 (SE) ms and 61.2 +/- 2.0 ms, respectively, and an amplitude of approximately 1 mV (n = 93). The AP5-sensitive component of EPSPs was only detected when the cell was depolarized with respect to the resting potential, had a mean time-to-peak of 41 +/- 5 ms and duration of 121 +/- 11 ms (n = 6), and increased in amplitude with postsynaptic depolarization. 6. Unitary monosynaptic IPSPs between an interneuron and a pyramidal cell had a mean amplitude of approximately 1 mV and were fully blocked by gamma-aminobutyric acid-A (GABAA) receptor antagonists (n = 3). 7. Unitary inhibitory responses were found only within, but not between, areas CA3 or CA1.(ABSTRACT TRUNCATED AT 400 WORDS)

Associative long-term depression (LTD) was induced in hippocampal slice cultures with repeated low-frequency (0.3 Hz) stimulation of the Schaffer collateral pathway, only when such stimuli were preceded by intracellular injection of brief depolarizing current pulses in the postsynaptic CA1 pyramidal cell. The decrease in excitatory postsynaptic potential amplitude lasted > 30 min, could be reversed by induction of potentiation, could be induced at previously potentiated inputs, was input-specific, and did not require activation or potentiation of other inputs. The magnitude of the depression depended upon the time interval between depolarization and stimulation and upon the duration of the depolarization pulse. LTD was not observed in neurons impaled with electrodes containing a Ca2+ chelator. LTD could not be induced in the presence of an N-methyl-D-aspartate receptor antagonist, suggesting that voltage-dependent Ca2+ influx is necessary but not sufficient for LTD induction. We conclude that associative LTD results when synaptic activity follows postsynaptic depolarization within a circumscribed time window.

We have investigated the action of norepinephrine (NE) on excitatory synaptic transmission in the hippocampus by recording from CA3 pyramidal cells in organotypic slice cultures. NE (5 microM) was found to decrease the amplitude of pharmacologically isolated EPSPs elicited with stimulation of mossy fibers or recurrent axon collaterals (mean decrease in EPSP amplitude, 44%). Desensitization was observed with repetitive applications. NE did not affect the sensitivity of CA3 cells to iontophoretically applied AMPA, and did not affect the amplitude distribution of TTX-resistant, miniature excitatory synaptic currents. These data suggest that NE acts at presynaptic receptors to decrease glutamate release. This action of NE was blocked by the alpha receptor antagonist phentolamine and the specific alpha 1 receptor antagonist prazosine, but not by the beta receptor antagonist timolol or the alpha 2 receptor antagonist idazoxan. Inhibition of EPSPs by NE was prevented by pretreatment of cultures with pertussis toxin, indicating that G-proteins couple these receptors to their effectors. Stimulation of protein kinase C with phorbol ester blocked the action of NE on EPSPs. This effect, as well as the desensitization of NE responses, was reduced by application of the protein kinase inhibitor staurosporin. Presynaptic inhibition of excitatory synaptic transmission, mediated by alpha adrenergic receptors, represents a novel modulatory action of NE in the hippocampus.

Presynaptic receptors for virtually all transmitters have been identified throughout the nervous system. Recent studies in the hippocampus provide new insights into the mechanisms by which the activation of these receptors leads to presynaptic inhibition of transmitter release, and characterize the second messengers involved in coupling presynaptic receptors to their effectors. Presynaptic receptors also provide a tractable route via which the amount of transmitter release may be selectively regulated in therapeutically useful ways.

The morphological and functional consequences of epileptic activity were investigated by applying the convulsants bicuculline and/or picrotoxin to mature rat hippocampal slice cultures. After 3 days, some cells in all hippocampal subfields showed signs of degeneration, including swollen somata, vacuolation, and dendritic deformities, whereas others displayed only a massive reduction in the number of their dendritic spines. Intracellular recordings from CA3 pyramidal cells revealed a decrease in the amplitude of evoked excitatory synaptic potentials. gamma-Aminobutyric acid-releasing interneurons and inhibitory synaptic potentials were unaffected. Seven days after withdrawal of convulsants, remaining cells possessed a normal number of dendritic spines, thus demonstrating a considerable capacity for recovery. The pathological changes induced by convulsants are similar to those found in the hippocampi of human epileptics, suggesting that they are a consequence, rather than a cause, of epilepsy.

Presynaptic inhibition of neurotransmitter release is thought to be mediated by a reduction of axon terminal Ca2+ current. We have compared the actions of several known inhibitors of evoked glutamate release with the actions of the Ca2+ channel antagonist Cd2+ on action potential-independent synaptic currents recorded from CA3 neurons in hippocampal slice cultures. Baclofen and adenosine decreased the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting the distribution of their amplitudes. Cd2+ blocked evoked synaptic transmission, but had no effect on the frequency or amplitude of either mEPSCs or inhibitory postsynaptic currents (IPSCs). Inhibition of presynaptic Ca2+ current therefore appears not to be required for the inhibition of glutamate release by adenosine and baclofen. Baclofen had no effect on the frequency of miniature IPSCs, indicating that gamma-aminobutyric acid B-type receptors exert distinct presynaptic actions at excitatory and inhibitory synapses.

1. The effects of the gamma-aminobutyric acid (GABA) uptake blocker tiagabine on inhibitory synaptic potentials (IPSPs) were examined with microelectrode and whole-cell recording from CA3 pyramidal cells in rat hippocampal slice cultures. 2. Tiagabine (10-25 microM) greatly prolonged the duration of monosynaptic IPSPs elicited in the presence of excitatory amino acid antagonists but had no effect on their amplitude. Part of the prolonged time course resulted from a GABAB receptor-mediated component that was not detectable under control conditions. 3. The mean decay time constant of the underlying GABAA receptor-mediated synaptic current was increased from 16 to 250 ms. Spontaneous miniature IPSPs recorded with whole-cell clamp were unaffected by tiagabine. Pentobarbital sodium, in contrast, increased the decay time constant of both evoked and spontaneous GABAA-mediated currents. 4. Tiagabine (25 microM) inhibited spontaneous and evoked epileptiform bursting induced by increasing the extracellular potassium concentration to 8 mM. 5. We conclude that GABA uptake plays a significant role in determining the time course of evoked IPSPs and also limits the likelihood that GABAB receptors are activated.

1. Intracellular recording techniques were used to study synaptic potentials in CA3 pyramidal cells elicited with mossy fibre stimulation in partially disinhibited hippocampal slice cultures. Two experimental protocols were used: (1) high concentrations (20-40 microM) of the A-type gamma-aminobutyric acid (GABAA) receptor antagonist bicuculline plus low concentrations (2-4 microM) of the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or (2) low concentrations (1-2.5 microM) of bicuculline alone. 2. Under the first condition, stimulation of mossy fibre afferents evoked epileptic bursts alternating with a response consisting of an excitatory postsynaptic potential (EPSP) followed by an unusually large and long-lasting hyperpolarizing potential with a maximal amplitude in the range of -30 mV from the resting membrane potential. 3. This paroxysmal inhibitory potential (PIP) had a reversal potential near that of potassium. The amplitude of the PIP was not dependent on action potentials superimposed on the preceding EPSP, and was present in cells recorded with microelectrodes containing the Ca2+ chelator EGTA. These data suggest that the PIP is not a Ca(2+)-activated K+ potential. 4. The PIP was prolonged by the GABA-uptake blocker nipecotic acid, was reduced by hyperpolarizing interneurons with the opioid agonist FK 33-824, and was abolished by the GABAB-receptor antagonist CGP 35 348. These data indicate that the PIP is mediated by the activation of GABAB receptors following GABA release from interneurons. 5. The NMDA-receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) strongly reduced the amplitude of the PIP, but had no effect on the GABAB receptor-mediated inhibitory postsynaptic potential (IPSP) under control conditions. 6. Under the first condition, regular stimulation elicited a cyclical pattern of evoked responses. There was either an alternation between an epileptic burst and a PIP or, at shorter interstimulus intervals, a sequence of gradually increasing PIPs followed by an epileptic burst, which then reset the cycle. 7. Under the second condition, in low concentrations of bicuculline alone, the early GABAA-mediated IPSP was little affected, but the late GABAB-mediated IPSP was greatly enhanced. These enhanced late IPSPs were comparable in amplitude and duration to the PIPs seen under the first conditions, could exhibit cyclical behaviour, and were reduced by D-APV. 8. Application of CGP 35 348 abolished the late IPSP under control conditions, but had no effect on hippocampal excitability. In contrast, CGP 35 348 blocked the PIP elicited in low bicuculline, and consequently led to intense epileptic discharge.(ABSTRACT TRUNCATED AT 400 WORDS)

The temperature dependence of intrinsic membrane conductances and synaptic potentials in guinea pig hippocampal CA1 pyramidal neurons were examined in vitro as they were cooled from 37 degrees C to between 33 and 27 degrees C. Cooling reversibly increased resting input resistance in a voltage-independent manner (Q10 = 0.58 to 0.75). The amplitude and duration of orthodromically evoked action potentials were increased by cooling (Q10 = 0.87 and 0.52 to 0.53, respectively), whereas the maximum rates of rise and fall were reduced (Q10 = 1.27 to 1.49 and 2.19 to 2.44, respectively). The amplitude and duration of the afterhyperpolarization which follows a directly evoked train of action potentials were substantially increased at low temperatures. It is possible to attribute this increase to an augmentation of Ca2+ influx during the train and also to a slowing of Ca2+ removal from the cytoplasm. Spike frequency adaptation during prolonged depolarizing pulses was enhanced at low temperatures. In addition, there was a decrement in spike amplitude during the train of action potentials. These observations all suggest an increase in Ca2+-activated K+ conductance at low temperature. A late, slow, hyperpolarizing synaptic potential in response to orthodromic stimulation became apparent at low temperature. This potential had an apparent reversal potential more negative than the early inhibitory postsynaptic potential, suggesting that it was mediated by a K+ conductance, possibly activated by Ca2+ influx. We conclude that reductions in temperature of as little as 5 to 10 degrees C from normal can significantly alter the intrinsic and synaptic physiology of hippocampal neurons and should, therefore, be considered an important variable in in vitro brain slice experiments.