Enforcement of temporal fidelity in pyramidal cells by somatic feed-forward inhibition.

The temporal resolution of neuronal integration depends on the time window within which excitatory inputs summate to reach the threshold for spike generation. Here, we show that in rat hippocampal pyramidal cells this window is very narrow (less than 2 milliseconds). This narrowness results from the short delay with which disynaptic feed-forward inhibition follows monosynaptic excitation. Simultaneous somatic and dendritic recordings indicate that feed-forward inhibition is much stronger in the soma than in the dendrites, resulting in a broader integration window in the latter compartment. Thus, the subcellular partitioning of feed-forward inhibition enforces precise coincidence detection in the soma, while allowing dendrites to sum incoming activity over broader time windows.

Acute decrease in net glutamate uptake during energy deprivation.

The extracellular glutamate concentration ([glu](o)) rises during cerebral ischemia, reaching levels capable of inducing delayed neuronal death. The mechanisms underlying this glutamate accumulation remain controversial. We used N-methyl-D-aspartate receptors on CA3 pyramidal neurons as a real-time, on-site, glutamate sensor to identify the source of glutamate release in an in vitro model of ischemia. Using glutamate and L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) as substrates and DL-threo-beta-benzyloxyaspartate (TBOA) as an inhibitor of glutamate transporters, we demonstrate that energy deprivation decreases net glutamate uptake within 2-3 min and later promotes reverse glutamate transport. This process accounts for up to 50% of the glutamate accumulation during energy deprivation. Enhanced action potential-independent vesicular release also contributes to the increase in [glu](o), by approximately 50%, but only once glutamate uptake is inhibited. These results indicate that a significant rise in [glu](o) already occurs during the first minutes of energy deprivation and is the consequence of reduced uptake and increased vesicular and nonvesicular release of glutamate.

GABA spillover activates postsynaptic GABA(B) receptors to control rhythmic hippocampal activity.

In the hippocampus, interneurons provide synaptic inhibition via the transmitter GABA, which can activate GABA(A) and GABA(B) receptors (GABA(A)Rs and GABA(B)Rs). Generally, however, GABA released by a single interneuron activates only GABA(A)Rs on its targets, despite the abundance of GABA(B)RS. Here, I show that during hippocampal rhythmic activity, simultaneous release of GABA from several interneurons activates postsynaptic GABA(B)Rs and that block of GABA(B)Rs increases oscillation frequency. Furthermore, if GABA uptake is inhibited, even GABA released by a single interneuron is enough to activate GABA(B)Rs. This occurs also on cells not directly contacted by that interneuron, indicating that GABA has to overcome uptake and exit the synaptic cleft to reach GABA(B)RS. Thus, activation of extrasynaptic GABA(B)Rs by pooling of GABA is an important mechanism regulating hippocampal network activity.

G-protein-independent signaling mediated by metabotropic glutamate receptors.

Synaptically released glutamate activates ionotropic and metabotropic receptors at central synapses. Metabotropic glutamate receptors (mGluRs) are thought to modulate membrane conductances through transduction cascades involving G proteins. Here we show, in CA3 pyramidal cells from rat hippocampus, that synaptic activation of type 1 mGluRs by mossy fiber stimulation evokes an excitatory postsynaptic response independent of G-protein function, while inhibiting an afterhyperpolarization current through a G-protein-coupled mechanism. Experiments using peptide activators and specific inhibitors identified a Src-family protein tyrosine kinase as a component of the G-protein-independent transduction pathway. These results represent the first functional evidence for a dual signaling mechanism associated with a heptahelical receptor such as mGluR1, in which intracellular transduction involves activation of either G proteins or tyrosine kinases.

Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin.

Maintaining glutamate at low extracellular concentrations in the central nervous system is necessary to protect neurons from excitotoxic injury and to ensure a high signal-to-noise ratio for glutamatergic synaptic transmission. We have used DL-threo-beta-benzyloxyaspartate (TBOA), an inhibitor of glutamate uptake, to determine the role of glutamate transporters in the regulation of extracellular glutamate concentration. By using the N-methyl-D-aspartate receptors of patched CA3 hippocampal neurons as "glutamate sensors," we observed that application of TBOA onto organotypic hippocampal slices led to a rapid increase in extracellular glutamate concentration. This increase was Ca(2+)-independent and was observed in the presence of tetrodotoxin. Moreover, prevention of vesicular glutamate release with clostridial toxins did not affect the accumulation of glutamate when uptake was inhibited. Inhibition of glutamine synthase, however, increased the rate of accumulation of extracellular glutamate, indicating that glial glutamate stores can serve as a source in this process. TBOA blocked synaptically evoked transporter currents in astrocytes without inducing a current mediated by the glutamate transporter. This indicates that this inhibitor is not transportable and does not release glutamate by heteroexchange. These results show that under basal conditions, the activity of glutamate transporters compensates for the continuous, nonvesicular release of glutamate from the intracellular compartment. As a consequence, acute disruption of transporter activity immediately results in significant accumulation of extracellular glutamate.

Target cell-specific modulation of transmitter release at terminals from a single axon.

In the hippocampus, a CA3 pyramidal cell forms excitatory synapses with thousands of other pyramidal cells and inhibitory interneurons. By using sequential paired recordings from three connected cells, we show that the presynaptic properties of CA3 pyramidal cell terminals, belonging to the same axon, differ according to the type of target cell. Activation of presynaptic group III metabotropic glutamate receptors decreases transmitter release only at terminals contacting CA1 interneurons but not CA1 pyramidal cells. Furthermore, terminals contacting distinct target cells show different frequency facilitation. On the basis of these results, we conclude that the pharmacological and physiological properties of presynaptic terminals are determined, at least in part, by the target cells.

Dual effects of anandamide on NMDA receptor-mediated responses and neurotransmission.

Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as delta9-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (deltaCa2+NMDA) in rat brain slices. The presence of anandamide reduced deltaCa2+NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting deltaCa2+NMDA, they also revealed another, underlying mechanism by which anandamide influences deltaCa2+NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated deltaCa2+NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.

Use-dependent increases in glutamate concentration activate presynaptic metabotropic glutamate receptors.

The classical view of fast chemical synaptic transmission is that released neurotransmitter acts locally on postsynaptic receptors and is cleared from the synaptic cleft within a few milliseconds by diffusion and by specific reuptake mechanisms. This rapid clearance restricts the spread of neurotransmitter and, combined with the low affinities of many ionotropic receptors, ensures that synaptic transmission occurs in a point-to-point fashion. We now show, however, that when transmitter release is enhanced at hippocampal mossy fibre synapses, the concentration of glutamate increases and its clearance is delayed; this allows it to spread away from the synapse and to activate presynaptic inhibitory metabotropic glutamate receptors (mGluRs). At normal levels of glutamate release during low-frequency activity, these presynaptic receptors are not activated. When glutamate concentration is increased by higher-frequency activity or by blocking glutamate uptake, however, these receptors become activated, leading to a rapid inhibition of transmitter release. This effect may be related to the long-term depression of mossy fibre synaptic responses that has recently been shown after prolonged activation of presynaptic mGluRs (refs 2, 3). The use-dependent activation of presynaptic mGluRs that we describe here thus represents a negative feedback mechanism for controlling the strength of synaptic transmission.

Distinct short-term plasticity at two excitatory synapses in the hippocampus.

A single mossy fiber input contains several release sites and is located on the proximal portion of the apical dendrite of CA3 neurons. It is, therefore, well suited to exert a strong influence on pyramidal cell excitability. Accordingly, the mossy fiber synapse has been referred to as a detonator or teacher synapse in autoassociative network models of the hippocampus. The very low firing rates of granule cells [Jung, M. W. & McNaughton, B. L. (1993) Hippocampus 3, 165-182], which give rise to the mossy fibers, raise the question of how the mossy fiber synapse temporally integrates synaptic activity. We have therefore addressed the frequency dependence of mossy fiber transmission and compared it to associational/commissural synapses in the CA3 region of the hippocampus. Paired pulse facilitation had a similar time course, but was 2-fold greater for mossy fiber synapses. Frequency facilitation, during which repetitive stimulation causes a reversible growth in synaptic transmission, was markedly different at the two synapses. At associational/ commissural synapses facilitation occurred only at frequencies greater than once every 10 s and reached a magnitude of about 125% of control. At mossy fiber synapses, facilitation occurred at frequencies as low as once every 40 s and reached a magnitude of 6-fold. Frequency facilitation was dependent on a rise in intraterminal Ca2+ and activation of Ca2+/calmodulin-dependent kinase II, and was greatly reduced at synapses expressing mossy fiber long-term potentiation. These results indicate that the mossy fiber synapse is able to integrate granule cell spiking activity over a broad range of frequencies, and this dynamic range is substantially reduced by long-term potentiation.

Functional characterization and modulation of feedback inhibitory circuits in area CA3 of rat hippocampal slice cultures.

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.

Role of intercellular interactions in heterosynaptic long-term depression.

Bidirectional control of synaptic strength is thought to be important for the development of neuronal circuits and information storage. The demonstration of homosynaptic long-term depression greatly enhances the usefulness of the synapse as a mnemonic device, but theoreticians have also seen the need for heterosynaptic decreases in synaptic efficacy, both in neuronal development and information storage. Indeed, induction of long-term potentiation in one population of synapses can be associated with a modest depression at neighbouring inactive synapses in the same population of cells. Here we report that in the CA1 region of the hippocampus this heterosynaptic long-term depression has the property that its sites of induction and expression occur in different populations of cells and thus requires the spread of a signal between neurons. Such a mechanism ensures a widespread distribution of this form of plasticity.

Heterosynaptic long-term depression in the hippocampus.

Heterosynaptic long-term depression (hetLTD) at one input can be induced by applying a conditioning stimulus to an adjacent set of synapses. In hippocampal CA1 pyramidal cells, our results suggest that hetLTD is triggered by an extracellular diffusible factor that is released following tetanic activation of NMDA receptors. This hetLTD occludes with homosynaptic LTD suggesting common underlying mechanisms.

Presynaptic inhibition of excitatory synaptic transmission by muscarinic and metabotropic glutamate receptor activation in the hippocampus: are Ca2+ channels involved?

Activation of either muscarinic cholinergic or metabotropic glutamatergic presynaptic receptors inhibits evoked excitatory synaptic responses in the hippocampus. We have investigated two possible mechanisms underlying these actions using whole-cell recording from CA3 pyramidal cells in hippocampal slice cultures. Application of either methacholine (MCh, 10 microM) or trans-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 10 microM) was found to reduce the frequency of miniature excitatory postsynaptic currents (mEPSCs) by roughly 50%, without changing their mean amplitude. The voltage-dependent Ca2+ channel blocker Cd2+ (100 microM), in contrast, had no effect on the mEPSC frequency. When the extracellular [K+] was increased from 2.7 to 16 mM, the mEPSC frequency increased from 1.7 to 4.9 Hz. This increase could be completely reversed by applying Cd2+, indicating that it was triggered by voltage-dependent Ca2+ influx. MCh and t-ACPD each decreased the mEPSC frequency by roughly 50% under these conditions. Because the agonists were equally effective in inhibiting spontaneous release whether voltage-dependent channels were activated or not, we conclude that presynaptic cholinergic and glutamatergic inhibition is not mediated by inhibition of presynaptic Ca2+ channels, but rather by a direct interference in the neurotransmitter release process at some point subsequent to Ca2+ influx.

Role of excitatory amino acid and GABAB receptors in the generation of epileptiform activity in disinhibited hippocampal slice cultures.

Selective excitatory amino acid- and GABAB-receptor antagonists were used to examine the role these receptors play in epileptiform burst discharge elicited by blocking GABAA receptor-mediated inhibition in hippocampal slice cultures of the rat. Application of bicuculline caused a single ictal burst followed by interictal bursting. The N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonovalerate, reduced the depolarizing envelope underlying interictal discharge, and accentuated the appearance of concomitant slow oscillatory potentials, which occurred synchronously in all CA3 cells. The non-N-methyl-D-aspartate receptor antagonists, 6-nitro-7sulphamoyl-benzo(F) quinoxaline and 6-cyano-7-nitro-quinoxaline-2,3-dione, blocked interictal bursting at high concentrations, and low concentrations of 6-cyano-7-nitro-quinoxaline-2,3-dione selectively eliminated the slow oscillations in an all-or-none manner, leaving the depolarizing envelope. No effects of either metabotropic glutamate receptor antagonists or of dihydropyridine Ca2+ channel agonists or antagonists on evoked interictal discharge were observed. 6-Cyano-7-nitro-quinoxaline-2,3-dione-resistant interictal-like discharge could be obtained in the presence of bicuculline when the external Mg2+ concentration was reduced from 1.5-0.5 mM. The GABAB receptor antagonist CGP 35348 prolonged individual evoked interictal bursts, and caused the appearance of spontaneous ictal-like discharges. The implications of these results are discussed with regard to the mechanisms of epileptogenesis and to potential therapeutic intervention.

Presynaptic inhibition of excitatory synaptic transmission mediated by alpha adrenergic receptors in area CA3 of the rat hippocampus in vitro.

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 inhibition 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.

Presynaptic inhibition of miniature excitatory synaptic currents by baclofen and adenosine in the hippocampus.

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.

Paroxysmal inhibitory potentials mediated by GABAB receptors in partially disinhibited rat hippocampal slice cultures.

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)