NMDA receptor activation enhances inhibitory GABAergic transmission onto hippocampal pyramidal neurons via presynaptic and postsynaptic mechanisms

N-methyl-D-aspartate (NMDA) receptors (NMDARs) are implicated in synaptic plasticity and modulation of glutamatergic excitatory transmission. Effect of NMDAR activation on inhibitory GABAergic transmission remains largely unknown. Here, we report that a brief application of NMDA could induce two distinct actions in CA1 pyramidal neurons in mouse hippocampal slices: 1) an inward current attributed to activation of postsynaptic NMDARs; and 2) fast phasic synaptic currents, namely spontaneous inhibitory postsynaptic currents (sIPSCs), mediated by GABA(A) receptors in pyramidal neurons. The mean amplitude of sIPSCs was also increased by NMDA. This profound increase in the sIPSC frequency and amplitude was markedly suppressed by the sodium channel blocker TTX, whereas the frequency and mean amplitude of miniature IPSCs were not significantly affected by NMDA, suggesting that NMDA elicits repetitive firing in GABAergic interneurons, thereby leading to GABA release from multiple synaptic sites of single GABAergic axons. We found that the NMDAR open-channel blocker MK-801 injected into recorded pyramidal neurons suppressed the NMDA-induced increase of sIPSCs, which raises the possibility that the firing of interneurons may not be the sole factor and certain retrograde messengers may also be involved in the NMDA-mediated enhancement of GABAergic transmission. Our results from pharmacological tests suggest that the nitric oxide signaling pathway is mobilized by NMDAR activation in CA1 pyramidal neurons, which in turn retrogradely facilitates GABA release from the presynaptic terminals. Thus NMDARs at glutamatergic synapses on both CA1 pyramidal neurons and interneurons appear to exert feedback and feedforward inhibition for determining the spike timing of the hippocampal microcircuit.     

Excitatory GABA input directly drives seizure-like rhythmic synchronization in mature hippocampal CA1 pyramidal cells

GABA, which generally mediates inhibitory synaptic transmissions, occasionally acts as an excitatory transmitter through intense GABA(A) receptor activation even in adult animals. The excitatory effect results from alterations in the gradients of chloride, bicarbonate, and potassium ions, but its functional role still remains a mystery. Here we show that such GABAergic excitation participates in the expression of seizure-like rhythmic synchronization (afterdischarge) in the mature hippocampal CA1 region. Seizure-like afterdischarge was induced by high-frequency synaptic stimulation in the rat hippocampal CA1-isolated slice preparations. The hippocampal afterdischarge was completely blocked by selective antagonists of ionotropic glutamate receptors or of GABA(A) receptor, and also by gap-junction inhibitors. In the CA1 pyramidal cells, oscillatory depolarizing responses during the afterdischarge were largely dependent on chloride conductance, and their reversal potentials (average -38 mV) were very close to those of exogenously applied GABAergic responses. Moreover, intracellular loading of the GABA(A) receptor blocker fluoride abolished the oscillatory responses in the pyramidal cells. Finally, the GABAergic excitation-driven afterdischarge has not been inducible until the second postnatal week. Thus, excitatory GABAergic transmission seems to play an active functional role in the generation of adult hippocampal afterdischarge, in cooperation with glutamatergic transmissions and possible gap junctional communications. Our findings may elucidate the cellular mechanism of neuronal synchronization during seizure activity in temporal lobe epilepsy.     

The Effects of Activation of Kainate Receptors on Tonic and Phasic Gabaergic Inhibition in Interneurons in Field Ca1 of Guinea Pig Hippocampus Slices

Kainate receptor agonists are powerful convulsants and excitotoxins. Until recently, there have been several contradictory views as to the roles of these receptors in the CNS. We report here experiments showing that application of kainate led to concentration-dependent increases in evoked GABAergic inhibitory postsynaptic currents (phasic currents) in interneurons in field CA1 of guinea pig hippocampus slices. This evidently occurred as a result of a decrease in the action potential generation threshold in inhibitory axons and an increase in the number of endings responding at a given stimulus strength. Increases in phasic inhibitory postsynaptic currents were accompanied by increases in the tonic GABAergic current (the constant component of GABAergic conduction). Increases in the tonic current occurred because of increases in the discharge frequency of interneurons, leading to action-potential-dependent GABA release and, as a result, increases in the extracellular concentration of endogenous agonist. The high level of extracellular GABA after addition of kainate led to desensitization of synaptic GABAergic receptors, while the tonic conductivity led to shunting of synaptic currents. Thus, while 1 microM kainate increased inhibitory postsynaptic currents, this was preceded by a transient depression. The different dynamics of the effects of kainate on phasic and tonic inhibitory GABAergic currents in hippocampal interneurons and the decrease in inhibition of glutamatergic pyramidal cells which may result from these changes may explain the epileptogenic properties of kainate.     

Comparable GABAergic mechanisms of hippocampal seizurelike activity in posttetanic and low-Mg2+ conditions

It is known that GABA is a major inhibitory neurotransmitter in mature mammalian brains, but the effect of this substance is sometimes converted into depolarizing or even excitatory when the postsynaptic Cl- concentration becomes high. Recently we have shown that seizurelike afterdischarge induced by tetanic stimulation in normal extracellular fluid (posttetanic afterdischarge) is mediated through GABAergic excitation in mature hippocampal CA1 pyramidal cells. In this study, we examined the possible contribution of similar depolarizing/excitatory GABAergic input to the CA1 pyramidal cells to the seizurelike afterdischarge induced in a low extracellular Mg2+ condition, another experimental model of epileptic seizure activity (low-Mg2+ afterdischarge). Perfusion of the GABAA antagonist bicuculline abolished the low-Mg2+ afterdischarge, but not the interictal-like activity, in most cases. Each oscillatory response during the low-Mg2+ afterdischarge was dependent on Cl- conductance and contained an F- -insensitive depolarizing component in the pyramidal cells, thus indicating that the afterdischarge response may be mediated through both GABAergic and nonGABAergic transmissions. In addition, local GABA application to the recorded cells revealed that GABA responses were indeed depolarizing during the low-Mg2+ afterdischarge. Furthermore, the GABAergic interneurons located in the strata pyramidale and oriens fired in oscillatory cycles more actively than those in other layers of the CA1 region. These results suggest that the depolarizing GABAergic input may facilitate oscillatory synchronization among the hippocampal CA1 pyramidal cells during the low-Mg2+ afterdischarge in a manner similar to the expression of the posttetanic afterdischarge.     

Glutamatergic propagation of GABAergic seizure-like afterdischarge in the hippocampus in vitro

Previous investigations have suggested that GABA may act actively as an excitatory mediator in the generation of seizure-like (ictal) or interictal epileptiform activity in several experimental models of temporal lobe epilepsy. However, it remains to be known whether or not such GABAergic excitation may participate in seizure propagation into neighboring cortical regions. In our in vitro study using mature rat hippocampal slices, we examined the cellular mechanism underlying synchronous propagation of seizure-like afterdischarge in the CA1 region, which is driven by depolarizing GABAergic transmission, into the adjacent subiculum region. Tetanically induced seizure-like afterdischarge was always preceded by a GABAergic, slow posttetanic depolarization in the pyramidal cells of the original seizure-generating region. In contrast, the slow posttetanic depolarization was no longer observed in the subicular pyramidal cells when the afterdischarge was induced in the CA1 region. Surgical cutting of axonal pathways through the stratum oriens and the alveus between the CA1 and the subiculum region abolished the CA1-generated afterdischarge in the subicular pyramidal cells. Intracellular loading of fluoride ions, a GABAA receptor blocker, into single subicular pyramidal cells had no inhibitory effect on the CA1-generated afterdischarge in the pyramidal cells. Furthermore, the CA1-generated afterdischarge in the subicular pyramidal cells was largely depressed by local application of glutamate receptor antagonists to the subiculum region during afterdischarge generation. The present results indicate that the excitatory GABAergic generation of seizure-like activity seems to be restricted to epileptogenic foci of origin in the seizure-like epilepsy model in vitro.     

Synaptic reorganization of calbindin-positive neurons in the human hippocampal CA1 region in temporal lobe epilepsy

The distribution, morphology, synaptic coverage and postsynaptic targets of calbindin-containing interneurons and afferent pathways have been analyzed in the control and epileptic CA1 region of the human hippocampus. Numerous calbindin-positive interneurons are preserved even in the strongly sclerotic CA1 region. The morphology of individual cells is altered: the cell body and dendrites become spiny, the radially oriented dendrites disappear, and are replaced by a large number of curved, distorted dendrites. Even in the non-sclerotic epileptic samples, where pyramidal cells are present and calbindin-immunoreactive interneurons seem to be unchanged, some modifications could be observed at the electron microscopic level: they received more inhibitory synaptic input, and the calbindin-positive excitatory afferents - presumably derived from the CA1, the CA2 and/or the dentate gyrus - are sprouted. In the strongly sclerotic tissue, with the death of pyramidal cells, calbindin-positive terminals (belonging to interneurons and the remaining excitatory afferents) change their targets. Our data suggest that an intense synaptic reorganization takes place in the epileptic CA1 region, even in the non-sclerotic tissue, before the death of considerable numbers of pyramidal cells. Calbindin-positive interneurons participate in this reorganization: they show plastic changes in response to epilepsy. The enhanced inhibition of inhibitory interneurons may result in the disinhibition of pyramidal cells or in an abnormal synchrony in the output region of the hippocampus.     

Alterations of perisomatic GABA synapses on hippocampal CA1 inhibitory interneurons and pyramidal cells in the kainate model of epilepsy.

In the kainate model of epilepsy, electrophysiological and anatomical modifications occur in inhibitory circuits of the CA1 region of the rat hippocampus. Using postembedding GABA immunocytochemistry and electron microscopy, we characterized perisomatic GABA and non-GABA synaptic contacts in CA pyramidal cells, and GABAergic interneurons of stratum oriens/alveus and stratum lacunosum-moleculare, and examined if changes occurred at these synapses at two weeks post-kainate treatment. We found that, in control rats, the number and total length of perisomatic GABA synapses were significantly smaller (approximately 40-50%) in lacunosum-moleculare interneurons than in oriens/alveus interneurons and pyramidal cells. Additionally, the number and total length of perisomatic non-GABA synapses were different among all cell types, with these parameters increasing significantly in the following order: pyramidal cells相似文献   

Activity-dependent induction of multitransmitter signaling onto pyramidal cells and interneurons of hippocampal area CA3

The granule cells of the dentate gyrus (DG) are considered to be glutamatergic, but they contain glutamic acid decarboxylase, gamma-amino butyric acid (GABA), and the vesicular GABA transporter mRNA. Their expression is regulated in an activity-dependent manner and coincides with the appearance of GABAergic transmission from the mossy fibers (MF) to pyramidal cells in area CA3. These data support the hypothesis that MF are able to release glutamate and GABA. Following the principle that a given neuron releases the same neurotransmitter(s) onto all its targets, we here demonstrate the emergence, after a generalized convulsive seizure, of MF GABAergic signaling sensitive to activation mGluR-III onto pyramidal cells and interneurons of CA3. Despite this, excitation overrides inhibition in interneurons, preventing disinhibition. Furthermore, on blockade of GABA and glutamate ionotropic receptors, an M1-cholinergic depolarizing signal is also revealed in both targets, which postsynaptically modulates the glutamatergic and GABAergic fast neurotransmission. The emergence of these nonglutamatergic signals depends on protein synthesis. In contrast to cholinergic responses evoked by associational/commissural fibers activation, cholinergic transmission evoked by DG stimulation is only observed after seizures and is strongly depressed by the activation of mGluR-II, whereas both are depressed by M2-AChR activation. With immunohistological experiments, we show that this cholinergic pathway runs parallel to the MF. Thus seizures compromise a delicate balance of excitation and inhibition, on which a complex interaction of different neurotransmitters emerges to counteract excitation at pre- and postsynaptic sites. Particularly, MF GABAergic inhibition emerges to exert an overall inhibitory action on CA3.     

Origin of phasic synaptic inhibition in myotomal motoneurons during fictive locomotion in the lamprey

The periodic membrane potential fluctuations in motoneurons during fictive locomotion in the lamprey, a primitive vertebrate, involve phasic synaptic excitation and inhibition. This paper investigates the origin of the phasic synaptic input to lamprey myotomal motoneurons in the in vitro spinal cord preparation with regard to the relative contribution of descending propriospinal input from interneurons in the local segment. The synaptic drive to myotomal motoneurons in the most rostral and the most caudal part of the spinal cord preparation are compared before and after selective spinal cord lesions. Current clamp recordings of the same cell before and after lesion showed that neither the excitatory phase nor the inhibitory phase was abolished after interruption of the descending or the ascending ipsilateral input, or after interrupting crossing segmental input by a local longitudinal midline incision. None of these sources thus appears to be alone responsible for the phasic synaptic drive. To quantitatively evaluate these effects, and in particular the contribution from the descending propriospinal fibres to the inhibitory phase, voltage clamp recordings were made in combination with a spinal cord hemisection just rostral to the motoneuron. The input from propriospinal interneurons in approximately 15 rostral segments may be responsible for as much as 70% of the phase of inhibitory current during the locomotor cycle. In accordance with these findings, a similar voltage clamp analysis of rostrally and caudally located motoneurons showed that the average peak-to-peak amplitude of the current fluctuations in rostral cells was approximately 50% of that in caudal cells.     

Nicotinic acetylcholine receptor alpha7 and alpha4beta2 subtypes differentially control GABAergic input to CA1 neurons in rat hippocampus.

The hippocampus, a limbic brain region involved in the encoding and retrieval of memory, has a well-defined structural network assembled from excitatory principal neurons and inhibitory interneurons. Because the GABAergic interneurons form synapses onto both pyramidal neurons and interneurons, the activation of nicotinic acetylcholine receptors (nAChRs) present on certain interneurons could induce either inhibition or disinhibition in the hippocampal circuitry. To understand the role of nAChRs in controlling synaptic transmission in the hippocampus, we evaluated the magnitude of nAChR-modulated GABAergic postsynaptic currents (PSCs) in pyramidal neurons and various interneurons of the CA1 region. Using whole cell patch-clamp recording and post hoc identification of neuronal types in rat hippocampal slices, we show that brief (12-s) nAChR activation by ACh (1 mM) or choline (10 mM) enhances the frequency of GABAergic PSCs in both pyramidal neurons and CA1 interneurons. The magnitude of alpha7 nAChR-mediated GABAergic inhibition, as assessed by the net charge of choline-induced PSCs, was highest in stratum lacunosum moleculare interneurons followed by pyramidal neurons and s. radiatum interneurons. In contrast, the magnitude of alpha4beta2 nAChR-mediated GABAergic inhibition, as assessed by the difference between the net charge of PSCs induced by ACh and choline, was highest in pyramidal neurons followed by s. lacunosum moleculare and s. radiatum interneurons. The present results suggest that cholinergic cues transmitted via specific subtypes of nAChRs modify the synaptic function in the hippocampus by inducing a differential degree of GABAergic inhibition in the target neurons.     

Action of tachykinins in the hippocampus: facilitation of inhibitory drive to GABAergic interneurons

By acting on neurokinin 1 (NK1) receptors, neuropeptides of the tachykinin family can powerfully excite rat hippocampal GABAergic interneurons located in the CA1 region and by this way indirectly inhibit CA1 pyramidal neurons. In addition to contact pyramidal neurons, however, GABAergic hippocampal interneurons can also innervate other interneurons. We thus asked whether activation of tachykinin-sensitive interneurons could indirectly inhibit other interneurons. The study was performed in hippocampal slices of young adult rats. Synaptic events were recorded using the whole-cell patch clamp technique. We found that substance P enhanced GABAergic inhibitory postsynaptic currents in a majority of the interneurons tested. Miniature, action potential-independent inhibitory postsynaptic currents were unaffected by substance P, as were evoked inhibitory synaptic currents. This suggests that the peptide acted at the somatodendritic membrane of interneurons, rather than at their axon terminals. The effect of substance P was mimicked by a selective NK1 receptor agonist, but not by neurokinin 2 (NK2) or neurokinin 3 (NK3) receptor agonists, and was suppressed by a NK1 selective receptor antagonist. In contrast to substance P, oxytocin, another peptide capable of activating hippocampal interneurons, had no effect on the inhibitory synaptic drive onto interneurons. We conclude that tachykinins, by acting on NK1 receptors, can influence the hippocampal activity by indirectly inhibiting both pyramidal neurons and GABAergic interneurons. Depending on the precise balance between these effects, tachykinins may either activate or depress hippocampal network activity.     

Specific subtypes of GABAA receptors mediate phasic and tonic forms of inhibition in hippocampal pyramidal neurons

The main inhibitory neurotransmitter in the mammalian brain, GABA, mediates multiple forms of inhibitory signals, such as fast and slow inhibitory postsynaptic currents and tonic inhibition, by activating a diverse family of ionotropic GABA(A) receptors (GABA(A)Rs). Here, we studied whether distinct GABA(A)R subtypes mediate these various forms of inhibition using as approach mice carrying a point mutation in the alpha-subunit rendering individual GABA(A)R subtypes insensitive to diazepam without altering their GABA sensitivity and expression of receptors. Whole cell patch-clamp recordings were performed in hippocampal pyramidal cells from single, double, and triple mutant mice. Comparing diazepam effects in knock-in and wild-type mice allowed determining the contribution of alpha1, alpha2, alpha3, and alpha5 subunits containing GABA(A)Rs to phasic and tonic forms of inhibition. Fast phasic currents were mediated by synaptic alpha2-GABA(A)Rs on the soma and by synaptic alpha1-GABA(A)Rs on the dendrites. No contribution of alpha3- or alpha5-GABA(A)Rs was detectable. Slow phasic currents were produced by both synaptic and perisynaptic GABA(A)Rs, judged by their strong sensitivity to blockade of GABA reuptake. In the CA1 area, but not in the subiculum, perisynaptic alpha5-GABA(A)Rs contributed to slow phasic currents. In the CA1 area, the diazepam-sensitive component of tonic inhibition also involved activation of alpha5-GABA(A)Rs and slow phasic and tonic signals shared overlapping pools of receptors. These results show that the major forms of inhibitory neurotransmission in hippocampal pyramidal cells are mediated by distinct GABA(A)Rs subtypes.     

Inhibition and disinhibition of pyramidal neurons by activation of nicotinic receptors on hippocampal interneurons

Nicotinic acetylcholine receptors (nAChRs) are expressed in the hippocampus, and their functional roles are beginning to be delineated. The effect of nAChR activation on the activity of both interneurons and pyramidal neurons in the CA1 region was studied in rat hippocampal slices. In CA1 stratum radiatum with muscarinic receptors inhibited, local pressure application of acetylcholine (ACh) elicited a nicotinic current in 82% of the neurons. The majority of the ACh-induced currents were sensitive to methyllycaconitine, which is a specific inhibitor of alpha7-containing nAChRs. Methyllycaconitine-insensitive nicotinic currents also were present as detected by a nonspecific nAChR inhibitor. The ACh-sensitive neurons in the s. radiatum were identified as GABAergic interneurons by their electrophysiological properties. Pressure application of ACh induced firing of action potentials in approximately 70% of the interneurons. The ACh-induced excitation of interneurons could induce either inhibition or disinhibition of pyramidal neurons. The inhibition was recorded from the pyramidal neuron as a burst of GABAergic synaptic activity. That synaptic activity was sensitive to bicuculline, indicating that GABA(A) receptors mediated the ACh-induced synaptic currents. The disinhibition was recorded from the pyramidal neuron as a reduction of spontaneous GABAergic synaptic activity when ACh was delivered onto an interneuron. Both the inhibition and disinhibition were sensitive to either methyllycaconitine or mecamylamine, indicating that activation of nicotinic receptors on interneurons was necessary for the effects. These results show that nAChRs are capable of regulating hippocampal circuits by exciting interneurons and, subsequently, inhibiting or disinhibiting pyramidal neurons.     

Glycine-gated chloride channels depress synaptic transmission in rat hippocampus

An inhibitory role for strychnine-sensitive glycine-gated chloride channels (GlyRs) in mature hippocampus is beginning to be appreciated. We have reported previously that CA1 pyramidal cells and GABAergic interneurons recorded in 3- to 4-wk-old rat hippocampal slices express functional GlyRs, dispelling previous misconceptions that GlyR expression ceases in early development. However, the effect of GlyR activation on cell excitability and synaptic circuits in hippocampus has not been fully explored. Using whole cell current-clamp recordings, we show that activation of strychnine-sensitive GlyRs through exogenous glycine application causes a significant decrease in input resistance and prevents somatically generated action potentials in both CA1 pyramidal cells and interneurons. Furthermore, GlyR activation depresses the synaptic network by reducing suprathreshold excitatory postsynaptic potentials (EPSPs) to subthreshold events in both cell types. Blockade of postsynaptic GlyRs with the chloride channel blocker 4, 4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS) or altering the chloride ion driving force in recorded cells attenuates the synaptic depression, strongly indicating that a postsynaptic mechanism is responsible. Increasing the local glycine concentration by blocking reuptake causes a strychnine-sensitive synaptic depression in interneuron recordings, suggesting that alterations in extracellular glycine will impact excitability in hippocampal circuits. Finally, using immunohistochemical methods, we show that glycine and the glycine transporter GlyT2 are co-localized selectively in GABAergic interneurons, indicating that interneurons contain both inhibitory neurotransmitters. Thus we report a novel mechanism whereby activation of postsynaptic GlyRs can function to depress activity in the synaptic network in hippocampus. Moreover, the co-localization of glycine and GABA in hippocampal interneurons, similar to spinal cord, brain stem, and cerebellum, suggests that this property is likely to be a general characteristic of inhibitory interneurons throughout the CNS.     

Use-dependent shift from inhibitory to excitatory GABAA receptor action in SP-O interneurons in the rat hippocampal CA3 area

Cortical inhibitory interneurons set the pace of synchronous neuronal oscillations implicated in synaptic plasticity and various cognitive functions. The hyperpolarizing nature of inhibitory postsynaptic potentials (IPSPs) in interneurons has been considered crucial for the generation of oscillations at beta (15-30 Hz) and gamma (30-100 Hz) frequency. Hippocampal basket cells and axo-axonic cells in stratum pyramidale-oriens (S-PO) play a central role in the synchronization of the local interneuronal network as well as in pacing of glutamatergic principal cell firing. A lack of conventional forms of plasticity in excitatory synapses onto interneurons facilitates their function as stable neuronal oscillators. We have used gramicidin-perforated and whole cell clamp recordings to study properties of GABAAR-mediated transmission in CA3 SP-O interneurons and in CA3 pyramidal cells in rat hippocampal slices during electrical 5- to 100-Hz stimulation and during spontaneous activity. We show that GABAergic synapses onto SP-O interneurons can easily switch their mode from inhibitory to excitatory during heightened activity. This is based on a depolarizing shift in the GABAA reversal potential (EGABA-A), which is much faster and more pronounced in interneurons than in pyramidal cells. We also found that the shift in interneuronal function was frequency dependent, being most prominent at 20- to 40-Hz activation of the GABAergic synapses. After 40-Hz tetanic stimulation (100 pulses), GABAA responses remained depolarizing for approximately 45 s in the interneurons, promoting bursting in the GABAergic network. Hyperpolarizing EGABA-A was restored >60 s after the stimulus train. Similar but spontaneous GABAergic bursting was induced by application of 4-aminopyridine (100 microM) to slices. A shift to depolarizing IPSPs by the GABAAR permeant weak acid anion formate provoked interneuronal population bursting, supporting the role of GABAergic excitation in burst generation. Furthermore, depolarizing GABAergic potentials and synchronous interneuronal bursting were enhanced by pentobarbital (100 microM), a positive allosteric modulator of GABAARs, and were blocked by picrotoxin (100 microM). Intriguingly, GABAergic bursts displayed short (<1 s) oscillations at 15-40 Hz, even though only depolarizing GABAA responses were seen in the SP-O interneurons. This beta-gamma rhythmicity in the interneuron network was dependent on electrotonic coupling, and was abolished by blockade of gap junctions with carbenoxolone (200 microM). Results here implicate the rapid activity-dependent degradation of hyperpolarizing IPSPs in SP-O interneurons in setting the temporal limits for a given interneuron to participate in beta-gamma oscillations synchronized by GABAergic synapses. Furthermore, they imply that mutual GABAergic excitation provided by interneurons may be an integral part in the function of neuronal networks. We suggest that the use-dependent change in EGABA-A could represent a form of short-term plasticity in interneurons promoting coherent and sustained activation of local GABAergic networks.     

Rapid activation of GABAergic interneurons and possible calcium independent GABA release in the mormyrid electrosensory lobe

The primary afferent fibers from the electroreceptors of mormyrid electric fish terminate centrally in the granular layer of the electrosensory lobe (ELL). This study examines the excitatory and inhibitory processes that take place in this layer using an in vitro slice preparation and field potentials evoked by stimulation of primary afferent fibers in the deep fiber layer of ELL. The postsynaptic response to stimulation of the afferent fibers was still present after blocking chemical transmission in three different ways: by adding glutamate receptor antagonists to the medium, by substituting a nominally calcium-free medium for normal medium, and by blocking calcium channels with cadmium. Blockade of chemical transmission was demonstrated by disappearance of control responses to parallel fiber stimulation. The continued presence of a postsynaptic response in the absence of chemical excitation is consistent with previous anatomic and physiological evidence for electrical synapses between afferent fibers and granular cells in ELL. Granular cell activation by primary afferent fibers was followed by a powerful, short-latency inhibition mediated by GABA and GABA(A) receptors, as indicated by a large increase in the postsynaptic response to afferent fiber stimulation following application of the GABA(A) receptor antagonist, bicuculline. Bicuculline caused a marked increase of the postsynaptic response even after chemical synaptic excitation had been blocked by glutamate receptor antagonists, by a calcium-free medium, or by cadmium. Thus activation of the inhibitory interneurons responsible for GABA release did not require chemical excitation. Nonchemical excitation of the inhibitory interneurons could be mediated either by electrical synapses between afferent fibers and inhibitory interneurons, or by nonsynaptic activation of the large GABAergic terminals that are known to be present on granular cells. The marked increase of the postsynaptic response caused by bicuculline in a calcium-free medium or in the presence of cadmium suggests that the release of GABA by inhibitory terminals was not entirely dependent on calcium influx. This effect of bicuculline on the postsynaptic response in a calcium-free medium or in the presence of cadmium was markedly reduced by prior addition of the GABA transporter antagonist, nipecotic acid. Thus calcium-independent release of GABA may occur in ELL and may be partly dependent on reversal of a GABA transporter. Rapid and powerful inhibition at the first stage in the processing of electrosensory information could serve to enhance the small differences in latency among afferent fibers that appear to encode small differences in stimulus intensity.     

Enhancement of the excitatory actions of GABA by barbiturates and benzodiazepines.

Whole cell recordings from hippocampal CA1 pyramidal neurons using electrode chloride concentrations of 12-80 mM demonstrated that the effect of synaptic activation of GABAA receptors was dependent on the transmembrane chloride gradient. When the chloride reversal potential was positive to action potential threshold, GABAA receptor activation was excitatory, and anticonvulsant barbiturates and benzodiazepines enhanced this excitation. Enhancement of GABAergic excitation of interneurons may contribute to the efficacy of these drugs, while enhancement of GABAergic excitation of principal neurons may be an important mechanism of failure, such as occurs in the treatment of neonatal seizures.     

GABA actions in hippocampal area CA3 during postnatal development: differential shift from depolarizing to hyperpolarizing in somatic and dendritic compartments

Gamma-aminobutyric acid type A receptor (GABA(A)-R) activation leads to depolarization of pyramidal cells during the first postnatal week and produces hyperpolarization from the second week. However, immunohistochemical evidence has suggested that during the second and third postnatal weeks the NKCC1 cotransporter relocates from the soma to the dendrites of CA3 pyramidal cells. We hypothesized that this leads to depolarizing responses in apical dendrites. Here we show that the activation of GABA(A)-R in the distal dendrites of CA3 pyramidal cells at P15 by restricted application of muscimol or synaptic activation by stimulation of interneurons in stratum radiatum (SR) causes depolarizing postsynaptic potentials (PSPs), which are blocked by NKCC1 cotransporter antagonists. By contrast, activation of proximal GABA(A)-R by muscimol application or by stimulation of interneurons in s. oriens (SO) leads to hyperpolarizing PSPs. Activation of the dentate gyrus (DG) in the presence of glutamatergic blockers evokes hyperpolarizing responses during the second postnatal week; however, the reversal potential of the DG-evoked inhibitory (I)PSPs is more depolarized than that of IPSPs evoked by activation of SO interneurons. Despite the shift of GABA action from depolarizing to hyperpolarizing, DG-evoked field potentials (f-PSPs) recorded in s. lucidum/radiatum (SL/R) do not change in polarity until the third week. Current source density analysis yielded results consistent with depolarizing actions of GABA in the dendritic compartment. Our data suggest that GABAergic input to apical dendrites of pyramidal cells of CA3 evokes depolarizing PSPs long after synaptic inhibition has become hyperpolarizing in the somata, in the axon initial segments and in basal dendrites.     

ATP contributes to the generation of network-driven giant depolarizing potentials in the neonatal rat hippocampus

In the immature hippocampus, the so-called 'giant depolarizing potentials' (GDPs) are network-driven synaptic events generated by the synergistic action of glutamate and GABA. Here we tested the hypothesis that ATP, a widely distributed neurotransmitter, directly contributes to the network activity during the first postnatal week. We found that in CA3 pyramidal cells, in the presence of the adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), ATP produced a transient facilitation of GDPs followed by a depressant effect. A similar biphasic effect was produced by blockade of the ectoATPase activity with 6- N,N -diethyl- d -β,γ-dibromomethylene ATP (ARL-67156). The effects of exogenous and endogenous ATP on GDPs were prevented by the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). On pyramidal cells, ATP upregulated spontaneous action-potential-dependent GABA_A-mediated synaptic events (GABA-SPSPs), suggesting a network-driven effect. Recordings from interneurones allowed comparison of ATP effects on GABAergic and glutamatergic synaptic activity. While ATP depressed GABA-SPSPs via metabotropic P2Y₁ receptors, it up- and downregulated glutamatergic SPSPs via PPADS-sensitive receptors. Thus, ATP exerts an excitatory action on CA3 pyramidal cells via facilitation of GDPs and SPSPs. This excitatory drive is propagated to pyramidal cells by interneurons that represent the 'common pathway' for generation of GDPs and SPSPs. Our results show that ATP operating via distinct P2X and P2Y receptors directly contributes to modulate network activity at the early stages of postnatal development.     

Synaptically activated Cl- accumulation responsible for depolarizing GABAergic responses in mature hippocampal neurons

It is known that GABA, a major inhibitory transmitter in the CNS, acts as an excitatory (or depolarizing) transmitter transiently after intense GABAA receptor activation in adult brains. The depolarizing effect is considered to be dependent on two GABAA receptor-permeable anions, chloride (Cl-) and bicarbonate (HCO3-). However, little is known about their spatial and temporal profiles during the GABAergic depolarization in postsynaptic neurons. In the present study, we show that the amplitude of synaptically induced depolarizing response was correlated with intracellular Cl- accumulation in the soma of mature hippocampal CA1 pyramidal cells, by using whole cell patch-clamp recording and Cl- imaging technique with a Cl- indicator 6-methoxy-N-ethylquinolinium iodide (MEQ). The synaptically activated Cl- accumulation was mediated dominantly through GABAA receptors. Basket cells, a subclass of fast-spiking interneurons innervating the somatic portion of the pyramidal cells, actually fired at high frequency during the Cl- accumulation accompanying the depolarizing responses. These results suggest synaptically activated GABAA-mediated Cl- accumulation may play a critical role in generation of an excitatory GABAergic response in the mature pyramidal cells receiving intense synaptic inputs. This may be the first demonstration of microscopic visualization of intracellular Cl- accumulation during synaptic activation.