Modulation by GABAB receptors of spontaneous synchronous activities induced by 4-aminopyridine in the rat hippocampus.

Field potential recordings were made in the CA3 and/or CA1 subfields of rat hippocampal slices maintained in vitro during perfusion with medium containing the convulsant drug 4-aminopyridine (4AP, 50 microM). In this experimental condition, spontaneous interictal epileptiform discharges caused by the activation of excitatory amino acid receptors and synchronous, GABA-mediated potentials occurred regularly in both areas. Interictal discharges and GABA-mediated potentials were reduced and eventually abolished in both subfields by bath application of baclofen (0.5-100 microM), which is a selective agonist for the GABAB receptor. However, interictal epileptiform events disappeared in the presence of baclofen concentrations (i.e., 4.75 +/- 0.7 microM; IC50 = 3.4 microM; n = 9) that were lower than those required for abolishing the GABA-mediated potential (i.e., 96.1 +/- 19.4 microM; IC50 = 9.8 microM; n = 12). The effects of baclofen were antagonized by the GABAB receptor antagonists saclofen (1 mM; n = 3 slices) or CGP 35348 (0.2-1 mM; IC50 = 240 microM; n = 12 slices). Our findings indicate that activation of GABAB receptors by baclofen inhibits both types of 4AP-induced activities in the rat hippocampus although epileptiform discharges appear to be more sensitive than GABA-mediated potentials to this pharmacological procedure. Although our data do not indicate whether the action of baclofen is pre- or postsynaptic, they demonstrate that this mechanism is sensitive to available GABAB receptor antagonists.     

Differential activation of GABAA and GABAB receptors by spontaneously released transmitter.

1. Whole-cell patch-clamp techniques were used to record from dentate gyrus granule cells in adult rat brain slices when N-methyl-D-aspartate (NMDA) and non-NMDA type glutamate receptors were blocked by D-2-amino-5-phosphonovaleric acid (D-AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. Spontaneous inhibitory postsynaptic currents (sIPSCs), each presumably due to vesicular release of gamma-aminobutyric acid (GABA), selectively activated GABAA-type receptors. None of the individual sIPSCs showed a slow-onset potassium current characteristic of GABAB receptor activation. 2. In contrast, stimulation in the molecular layer with a bipolar stimulating electrode or bath application of the convulsant drug 4-aminopyridine (4-AP, 10-30 microM) elicited fast GABAA IPSCs followed by slower outward currents that were sensitive to the selective GABAB antagonist CGP 35348 (0.1-1 mM) and that reversed polarity near the potassium equilibrium potential. 3. CGP 35348 (0.5-1 mM) or the GABAB agonist (-)baclofen (1 microM) had no significant effect on the frequency or average amplitude of sIPSCs. However, either bath application of (-)baclofen (1 microM) or a preceding conditioning stimulus caused large reductions in the amplitude of stimulus-evoked IPSCs, suggesting a strong GABAB-mediated presynaptic inhibition of stimulus-evoked GABA release. 4. We conclude that under normal conditions spontaneous transmitter release does not activate GABAB receptors in dentate gyrus slices. These findings are consistent with either of two general possibilities. Separate groups of interneurons with different basal firing rates may selectively form GABAA and GABAB synapses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different subtypes of GABAB receptors are present at pre- and postsynaptic sites within the rat dorsolateral septal nucleus.

GABAB receptor activation modulates neuronal activity mediated by multiple CNS transmitters and can occur at pre- and postsynaptic sites. In low concentrations, baclofen acts presynaptically to diminish transmitter release via both hetero- and autoreceptors, whereas at increasing concentrations, the same compound alters postsynaptic membrane excitability by inducing a membrane hyperpolarization. We have utilized electrophysiological techniques in vitro to focus on the possibility that pharmacologically different subtypes of GABAB receptors are present on presynaptic sites of glutamatergic terminals when compared with GABAB receptors on postsynaptic sites within the dorsolateral septal nucleus (DLSN). The glutamatergic terminal within the DLSN originates from a pyramidal cell body located within the hippocampus and most likely terminates on a GABAergic neuron from which recordings were made. Whole cell patch voltage-clamp methods were employed to record pharmacologically isolated excitatory postsynaptic currents (EPSCs) from DLSN neurons as an index of glutamatergic transmission. Using a modified internal pipette solution containing QX-314 and in which CsGluconate and GDPbetaS replaced Kgluconate and GTP, respectively, we recorded isolated monosynaptic EPSCs. The GABAA receptor antagonists bicuculline and picrotoxin were included in the external standard superfusion solution. Application of the GABAB receptor agonists, (+/-)-baclofen, CGP44533, and CGP35024 (10 nM to 10 microM) depressed glutamate-mediated EPSCs in a concentration-dependent manner. With the use of this combination of solutions, CGP44533 did not produce postsynaptic membrane property changes. Under these conditions, both (+/-)-baclofen and CGP35024 still induced increases of postsynaptic membrane conductance associated with an outward current. The GABAB receptor antagonist CGP55845A (1 microM) blocked the presynaptic CGP44533-mediated depressant effects of EPSCs, whereas CGP35348 (100 microM) or barium (2 mM) was ineffective. Furthermore, both CGP35348 (100 microM) and CGP55845A (1 microM) were effective in blocking the postsynaptic conductance changes associated with baclofen and CGP35024, whereas barium was ineffective. Our results demonstrate a distinct pharmacology for GABAB agonists acting at putative subtypes of GABAB receptors located on presynaptic sites of a glutamatergic terminal versus GABAB receptors on postsynaptic sites of a DLSN neuron. Furthermore, our results also suggest a different pharmacology and/or coupling of a GABAB receptor to different effectors at postsynaptic sites within the DLSN. Thus there may be three or more pharmacologically distinct GABAB receptors or receptor complexes associated with DLSN neurons: at least one pre- and two postsynaptic. If this distinct pharmacology and GABAB receptor distribution also extends to other CNS structures, such differences could provide development of selective drugs to act at these multiple sites.     

Role of synaptic metabotropic glutamate receptors in epileptiform discharges in hippocampal slices

Application of group I metabotropic glutamate receptor (mGluR) agonists elicits seizure discharges in vivo and prolonged ictal-like activity in in vitro brain slices. In this study we examined 1) if group I mGluRs are activated by synaptically released glutamate during epileptiform discharges induced by convulsants in hippocampal slices and, if so, 2) whether the synaptically activated mGluRs contribute to the pattern of the epileptiform discharges. The GABA(A) receptor antagonist bicuculline (50 microM) was applied to induce short synchronized bursts of approximately 250 ms in mouse hippocampal slices. Addition of 4-aminopyridine (4-AP; 100 microM) prolonged these bursts to 0.7-2 s. The mGluR1 antagonist (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY 367385; 25-100 microM) and the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP; 10-50 microM), applied separately, significantly reduced the duration of the synchronized discharges. The effects of these antagonists were additive when applied together, suggesting that mGluR1 and mGluR5 exert independent actions on the epileptiform bursts. In phospholipase C beta1 (PLCbeta1) knockout mice, bicuculline and 4-AP elicited prolonged synchronized discharges of comparable duration as those observed in slices from wild-type littermates. Furthermore, mGluR1 and mGluR5 antagonists reduced the duration of the epileptiform discharges to the same extent as they did in the wild-type preparations. The results suggest that mGluR1 and mGluR5 are activated synaptically during prolonged epileptiform discharges induced by bicuculline and 4-AP. Synaptic activation of these receptors extended the duration of synchronized discharges. In addition, the data indicate that the synaptic effects of the group I mGluRs on the duration of epileptiform discharges were mediated by a PLCbeta1-independent mechanism.     

Low-magnesium epilepsy in rat hippocampal slices: inhibitory postsynaptic potentials in the CA1 subfield

Spontaneous, synchronous epileptiform discharges were recorded in both CA3 and CA1 subfields of rat hippocampal slices perfused with Mg2+-free medium. Surgical separation of the two areas abolished the spontaneous discharges only in the CA1 subfield. However, epileptiform responses in the isolated CA1 subfield could still be evoked by orthodromic stimulation. Intracellularly these stimulus-induced responses were characterized by a depolarization associated with a burst of action potentials. Stimulation of the alveus still evoked a hyperpolarizing potential, presumably a recurrent inhibitory postsynaptic potential (IPSP) in CA1 pyramidal cells. Both spontaneous and stimulus-induced epileptiform discharges were blocked by the selective antagonist of N-methyl-D-aspartate (NMDA) receptors DL-2-amino-phosphonovalerate (APV). APV also reduced the amplitude and duration of the IPSP induced by alveus stimulation. Thus, epileptiform discharges evoked by lowering Mg2+ in the CA1 subfield are associated with a preservation of inhibitory mechanisms. Furthermore the effects exerted by APV upon the IPSP implicate that NMDA receptors might be involved in the neuronal circuit responsible for the hyperpolarizing IPSP generated by CA1 pyramidal neurons.     

Prolonged epileptiform bursting induced by 0-Mg(2+) in rat hippocampal slices depends on gap junctional coupling

The transition from brief interictal to prolonged seizure, or 'ictal', activity is a crucial event in epilepsy. In vitro slice models can mimic many phenomena observed in the electroencephalogram of patients, including transition from interictal to ictaform or seizure-like activity. In field potential recordings, three discharge types can be distinguished: (1) primary discharges making up the typical interictal burst, (2) secondary bursts, lasting several hundred milliseconds, and (3) tertiary discharges lasting for seconds, constituting the ictal series of bursts. The roles of chemical synapses in these classes of burst have been explored in detail. Here we test the hypothesis that gap junctions are necessary for the generation of secondary bursts.In rat hippocampal slices, epileptiform activity was induced by exposure to 0-Mg(2+). Epileptiform discharges started in the CA3 subfield, and generally consisted of primary discharges followed by 4-13 secondary bursts. Three drugs that block gap junctions, halothane (5-10 mM), carbenoxolone (100 microM) and octanol (0.2-1.0 mM), abolished the secondary discharges, but left the primary bursts intact. The gap junction opener trimethylamine (10 mM) reversibly induced secondary and tertiary discharges. None of these agents altered intrinsic or synaptic properties of CA3 pyramidal cells at the doses used. Surgically isolating the CA3 subfield made secondary discharges disappear, and trimethylamine under these conditions was able to restore them.We conclude that gap junctions can contribute to the prolongation of epileptiform discharges.     

Comparison of kynurenic acid and 2-APV suppression of epileptiform activity in rat hippocampal slices

The N-methyl-D-aspartate (NMDA) receptor blocker 2-amino-5-phosphonovaleric acid [+/-)-2-APV) and kynurenic acid both suppressed spontaneous epileptiform burst discharges in the CA3 region of rat hippocampal slices. When the bursts were induced by perfusion with magnesium-free medium (+/-)-2-APV was the more potent inhibitor (ED50 66 microM for (+/-)-2-APV and 110 microM for kynurenate). When bursts were induced by picrotoxin, kynurenate was more potent with an ED50 of 132 microM, compared with 290 microM for (+/-)-2-APV. Both antagonists were selective inhibitors of responses to NMDA when examined against excitations induced by NMDA, kainate and quisqualate applied by microiontophoresis onto CA3 pyramidal cells. The results may indicate a complex receptor profile for endogenous compounds involved in epileptiform bursts, or the existence of non-pyramidal cells bearing non-NMDA receptors sensitive to kynurenic acid.     

NMDA receptor antagonists CPP and MK-801 partially suppress the epileptiform discharges induced by the convulsant drug bicuculline in the rat neocortex

Intracellular recordings were obtained from neurons located in the superficial layers of rat neocortical slices maintained in vitro. In the presence of 50 microM of bicuculline methiodide, epileptiform discharges were evoked by extracellular local stimuli. Bath applications of the NMDA receptor antagonists CPP or MK-801 (3-5 microM) produced the following effects: (i) prolongation of the burst latency; (ii) attenuation of the burst duration, mainly its late phase; (iii) increase in the threshold of burst activation. These effects were not accompanied by any change in membrane potential, input resistance and repetitive firing evoked by intracellular pulses of depolarizing current. Our results indicate the involvement of conductances mediated through NMDA receptors in the genesis of epileptiform activities recorded in the neocortex upon blockade of GABA receptors.     

Neuropeptide Y suppresses epileptiform activity in rat frontal cortex and hippocampus in vitro via different NPY receptor subtypes.

Neuropeptide Y (NPY) and different NPY receptor (Y) subtype-selective agonists were tested for their effects on spontaneous epileptiform discharges which developed in rat cortical and hippocampal slices in Mg(2+)-free medium. Epileptiform activity, recorded extracellularly, was attenuated by NPY (0.5-1 microM) in both the frontal cortex and hippocampal CA3/CA1 pyramidal cell layers. In the cortex the Y1/5 selective agonist [Leu31 Pro34] NPY was more effective than the Y2 preferring agonist NPY13-36 and the Y2/5 preferring agonist NPY3-36. The suppression of epileptiform discharges induced by NPY in cortical slices was blocked by the selective Y1 receptor antagonist (R)-N2-(diphenylacetyl)-N-((4-hydroxyphenyl)methyl] argininamide (BIBP 3226). In the hippocampus, NPY13-36 and NPY3-36 were more effective than [Leu31 Pro34] NPY. In conclusion, the antiepileptic activity of NPY is mediated predominantly by the Y1 receptor subtype in the frontal cortex and by Y2 and probably Y5 receptors in the hippocampal CA3/CA1 areas.     

Mu-opioid receptors facilitate the propagation of excitatory activity in rat hippocampal area CA1 by disinhibition of all anatomical layers

Hippocampal mu-opioid receptors (MORs) have been implicated in memory formation associated with opiate drug abuse. MORs modulate hippocampal synaptic plasticity acutely, when chronically activated, and during drug withdrawal. At the network level, MORs increase excitability in area CA1 by disinhibiting pyramidal cells. The precise inhibitory interneuron subtypes affected by MOR activation are unknown; however, not all subtypes are inhibited, and specific interneuron subtypes have been shown to preferentially express MORs. Here we investigate, using voltage-sensitive dye imaging in brain slices, the effect of MOR activation on the patterns of inhibition and on the propagation of excitatory activity in rat hippocampal CA1. MOR activation augments excitatory activity evoked by stimulating inputs in stratum oriens [i.e., Schaffer collateral and commissural pathway (SCC) and antidromic], stratum radiatum (i.e., SCC), and stratum lacunosum-moleculare (SLM; i.e., perforant path and thalamus). The augmented excitatory activity is further facilitated as it propagates through the CA1 network. This was observed as a proportionately larger increase in amplitudes of excitatory activity at sites distal from where the activity was evoked. This facilitation was observed for excitatory activity propagating from all three stimulation sites. The augmentation and facilitation were prevented by GABAA receptor antagonists (bicuculline, 30 microM), but not by GABAB receptor antagonists (CGP 55845, 10 microM). Furthermore, MOR activation inhibited IPSPs in all layers of area CA1. These findings suggest that MOR-induced suppression of GABA release onto GABAA receptors augments all inputs to CA1 pyramidal cells and facilitates the propagation of excitatory activity through the network of area CA1.     

Synaptic triggering of epileptiform discharges in CA1 pyramidal cells in vitro

Intra- and extracellular recordings were made in the transverse hippocampal slice in vitro to study the requirements for the triggering of epileptiform discharges of CA1 cells. Spontaneous and induced epileptiform discharges were produced by adding small amounts of sodium benzyl penicillin. Recorded intracellularly, the epileptiform activity consisted of a burst of action potentials superimposed on a depolarizing wave. Extracellular recordings demonstrated a marked synchronization. The epileptiform activity of the CA1 cells appeared without changes in the passive membrane properties or in the spike generating mechanism. Spontaneous epileptiform discharges of the CA1 cells depended upon a synaptic activation from the CA3 region. Stimulation of afferent fibres evoked an early and a late burst response in the CA1 cells. The long latency burst was caused by a re-excitation from the CA3 region. The early burst response seems to be an intrinsic property of the CA1 cells and may be induced by synaptic activation of either apical or basal dendrites. The findings suggest that synaptic depolarization is necessary for the generation of epileptiform discharges of the CA1 cells.     

Antagonistic effect of delta-aminovaleric acid on bicuculline-insensitive gamma-aminobutyric acid B (GABA B) sites in the rat's brain

The effect of delta-aminovaleric acid (delta-AV) on bicuculline-insensitive gamma-aminobutyric acid B (GABA B) sites in the central nervous system (CNS) was investigated by binding studies and experiments on slices in vitro. delta-AV inhibited [3H]GABA (10 nM) binding to GABA B sites in a rat brain membrane preparation with an IC50 value of 10(-4) M. It also inhibited [3H]baclofen (20 nM) binding with an IC50 value of 10(-4) M. In preparations of hippocampal slices, (-)-baclofen (5 microM) reduced the population spikes evoked by stimulating the Schaffer collaterals in CA1 pyramidal cells in the presence of 100 microM bicuculline. delta-AV (1 mM) antagonized this inhibitory action of baclofen. Since baclofen is an agonist of GABAB sites, our results indicate that delta-AV has an antagonistic effect on GABAB sites in the CNS.     

Characterization of an N-methyl-D-aspartate receptor component of synaptic transmission in rat hippocampal slices

The involvement of N-methyl-D-aspartate receptors in synaptic transmission from Schaffer collateral-commissural fibres to CA1 neurons has been investigated in rat hippocampal slices. When the perfusion medium was changed from one containing 1 mM Mg2+ to one with no added Mg2+ there was a pronounced increase in the amplitude of the population spike, the appearance of secondary population spikes and in some slices spontaneous epileptiform discharges developed. The secondary and spontaneous population spikes were abolished by the selective N-methyl-D-aspartate antagonist, D-2-amino-5-phosphonovalerate. The effects on the primary population spike depended on the strength of synaptic activation. At low intensities, the N-methyl-D-aspartate antagonist reduced or abolished this response whereas at high intensities the primary population spike was slightly increased in amplitude by this compound. Mg2+ had dose-dependent (20-500 microM) effects on synaptic responses which were identical to those of D-2-amino-5-phosphonovalerate. Increasing the Ca2+ concentration over a range of 1-3 mM also reduced or abolished secondary population spikes and, at low stimulus intensities, the primary population spike. At higher stimulus intensities, however, the primary population spike was insensitive to the Ca2+ concentration over this range. These results demonstrate the major extent to which N-methyl-D-aspartate receptors can contribute to synaptic transmission and epileptiform activity in the CA1 region of the hippocampus. They also show that an important role of Mg2+ in this region is to prevent significant activation of this receptor system during low-frequency synaptic transmission.     

Epileptiform activity induced by changes in extracellular potassium in hippocampus

Using extra- and intracellular recording techniques, we investigated the induction and frequency modulation of spontaneous epileptiform activity produced by changes in the concentration of extracellular potassium ([K+]o). This paper describes a quantitative relationship between [K+]o and the frequency of spontaneously occurring epileptiform events. Recordings were made from the CA3 subfield of the rat in vitro hippocampal slice preparation. Intracellular microelectrodes were filled with 2 M Cs2SO4 and connected to a 3-kHz, time-share, single-electrode current- and voltage-clamp device. The frequency of spontaneous epileptiform (interictal) discharges was determined from extracellular recordings as a function of [K+]o. Current- and voltage-clamp techniques were used to characterize the intracellular correlate of these epileptiform events. The frequency of bicuculline-induced spontaneous epileptiform discharges was dependent on [K+]o. Below 4 mM [K+]o, spontaneous discharges occurred sporadically in the presence of 10 microM bicuculline. Increasing [K+]o from 5 to 10 mM caused a fivefold increase in the rate of spontaneous discharges. Spontaneous epileptiform discharges also occurred in the absence of bicuculline when [K+]o was increased above 6.5 mM. The rate of these discharges was dependent on [K+]o in much the same way as the discharges induced by bicuculline. For any given [K+]o concentration greater than 6.5 mM, however, the resultant discharge rate was faster than that obtained when bicuculline was present in the bathing solution. Simultaneous intra- and extracellular recordings revealed that the spontaneous high-[K+]o-induced interictal discharge was accompanied by a large depolarization of the membrane potential that appeared similar to the paroxysmal depolarizing shift (PDS) seen with other convulsants. The intracellularly recorded event fulfilled the criteria for a synaptically mediated PDS. The waveform of the PDS was complex and dependent on the membrane potential. When the membrane potential was held at 0 mV, spontaneously occurring hyperpolarizing potentials were noted during the inter-PDS interval. These events were blocked by picrotoxin or bicuculline and were probably spontaneous inhibitory postsynaptic potentials. The complexity of the PDS waveform suggested that more than one synaptic conductance was involved in the generation of the PDS. The mean measured reversal potential of the depolarizing phase was -10.7 mV. Voltage-clamp techniques were used to measure the conductance underlying the depolarizing phase of the high-[K+]o-induced PDS. The mean measured conductance was 51.5 nS, with a reversal potential of -7.9 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sharp wave-like activity in the hippocampus in vitro in mice lacking the gap junction protein connexin 36

Bath application of kainate (100-300 nM) induced a persistent gamma-frequency (30-80 Hz) oscillation that could be recorded in stratum radiatum of the CA3 region in vitro. We have previously described that in knockout mice lacking the gap junction protein connexin 36 (Cx36KO), gamma-frequency oscillations are reduced but still present. We now demonstrate that in the Cx36KO mice, but not in wild-type (WT), large population field excitatory postsynaptic potentials, or sharp wave-burst discharges, also occurred during the on-going gamma-frequency oscillation. These spontaneous burst discharges were not seen in WT mice. Burst discharges in the Cx36KO mice occurred with a mean frequency of 0.23 +/- 0.11 Hz and were accompanied by a series of fast (approximately 60-115 Hz) population spikes or "ripple" oscillations in many recordings. Intracellular recordings from CA3 pyramidal cells showed that the burst discharges consisted of a depolarizing response and presumed coupling potentials (spikelets) could occasionally be seen either before or during the burst discharge. The burst discharges occurring in Cx36KO mice were sensitive to gap junctions blockers as they were fully abolished by carbenoxolone (200 microM). In control mice we made several attempts to replicate this pattern of sharp wave activity/ripples occurring with the on-going kainate-evoked gamma-frequency oscillation by manipulating synaptic and electrical signaling. Partial disruption of inhibition, in control slices, by bath application of the gamma-aminobutyric acid-A (GABA(A)) receptor antagonist bicuculline (1-4 microM) completely abolished all gamma-frequency activity before any burst discharges occurred. Increasing the number of open gap junctions in control slices by using trimethylamine (TMA; 2-10 mM), in conjunction with kainate, failed to elicit any sharp wave bursts or fast ripples. However, bath application of the potassium channel blocker 4-aminopyridine (4-AP; 20-80 microM) produced a pattern of activity in control mice (13/16 slices), consisting of burst discharges occurring in conjunction with kainate-evoked gamma-frequency oscillations, that was similar to that seen in Cx36KO mice. In a few cases (n = 9) the burst discharges were accompanied by fast ripple oscillations. Carbenoxolone also fully blocked the 4-AP-evoked burst discharges (n = 5). Our results show that disruption of electrical signaling in the interneuronal network can, in the presence of kainate, lead to the spontaneous generation of sharp wave/ripple activity similar to that observed in vivo. This suggests a complex role for electrically coupled interneurons in the generation of hippocampal network activity.     

Cesium induces spontaneous epileptiform activity without changing extracellular potassium regulation in rat hippocampus

Cesium has been widely used to study the roles of the hyperpolarization-activated (I(h)) and inwardly rectifying potassium (K(IR)) channels in many neuronal and nonneuronal cell types. Recently, extracellular application of cesium has been shown to produce epileptiform activity in brain slices, but the mechanisms for this are not known. It has been proposed that cesium blocks the K(IR) in glia, resulting in an abnormal accumulation of potassium in the extracellular space and inducing epileptiform activity. This hypothesis has been tested in hippocampal slices and cultured hippocampal neurons using potassium-sensitive microelectrodes. In the present study, application of cesium produced spontaneous epileptiform discharges at physiological extracellular potassium concentration ([K(+)](o)) in the CA1 and CA3 regions of hippocampal slices. This epileptiform activity was not mimicked by increasing the [K(+)](o). The epileptiform discharges induced by cesium were not blocked by the N-methyl-D- aspartate (NMDA) receptor antagonist AP-5, but were blocked by the non-NMDA receptor antagonist CNQX. In the dentate gyrus, cesium induced the appearance of spontaneous nonsynaptic field bursts in 0 added calcium and 3 mM potassium. Moreover, cesium increased the frequency of field bursts already present. In contrast, ZD-7288, a specific I(h) blocker, did not cause spontaneous epileptiform activity in CA1 and CA3, nor did it affect the field bursts in the dentate gyrus, suggesting that cesium induced epileptiform activity is not directly related to blockade of the I(h). When potassium-sensitive microelectrodes were used to measure [K(+)](o), there was no significant increase in [K(+)](o) in CA1 and CA3 after cesium application. In the dentate gyrus, cesium did not change the baseline level of [K(+)](o) or the rate of [K(+)](o) clearance after the field bursts. In cultured hippocampal neurons, which have a large and relatively unrestricted extracellular space, cesium also produced cellular burst activity without significantly changing the resting membrane potential, which might indicate an increase in [K(+)](o). Our results suggest that cesium causes epileptiform activity by a mechanism unrelated to an alteration in [K(+)](o) regulation.     

Anticonvulsant action of GABA in the high potassium-low magnesium model of ictogenesis in the neonatal rat hippocampus in vivo and in vitro

Previous developmental studies in vitro suggested that the inhibitory neurotransmitter GABA exerts depolarizing and excitatory actions on the immature neurons and that depolarizing GABA is causally linked to ictal activity during the first weeks of postnatal life. However, remarkably little is known on the role of GABA in the generation of neonatal seizures in vivo. Here, using extracellular recordings from CA3 hippocampus, we studied the effects of GABA(A)-acting drugs on electrographic seizures induced by local intrahippocampal injection of the epileptogenic agents (high K(+)/low Mg(2+)) in the nonanesthetized rats in vivo and in the hippocampal slices in vitro during the second postnatal week (postnatal days P8-12). We found that in vivo, the induction of ictal-like events was facilitated by co-infusion of high-K(+)/low Mg(2+) together with the GABA(A) antagonist bicuculline or gabazine. Moreover, the infusion of bicuculline alone caused ictal-like activity in approximately 30% of cases. Co-infusion of the GABA(A) receptor agonist isoguvacine or the GABA(A)-positive allosteric modulator diazepam completely prevented high-K(+)/low Mg(2+)-induced seizures. In in vitro studies using hippocampal slices, we also found that high-K(+)/low Mg(2+) produced ictal activity that was exacerbated by bicuculline and gabazine and reduced by isoguvacine. Thus in the model of high-K(+)/low Mg(2+)-induced seizures both in in vivo and in vitro conditions, GABA, acting via GABA(A) receptors, has an anticonvulsant effect during the critical developmental period of enhanced excitability.     

The actions of 2-hydroxy-saclofen at presynaptic GABAB receptors in the rat hippocampus

The actions of 2-hydroxy-saclofen (2-OH-S), a recently developed analog of baclofen, were studied at presynaptic GABAB receptors in the rat hippocampal slice. Baclofen (0.5-20 microM) reduces the amplitude of excitatory postsynaptic potentials (EPSPs) recorded from hippocampal CA1 pyramidal neurons. In the presence of 200-500 microM 2-OH-S, the synaptic depressant action of baclofen is significantly reduced. These data show that 2-OH-S is an effective antagonist at presynaptic GABAB receptors on excitatory terminals in the hippocampus.     

An N-methyl-D-aspartate receptor-independent excitatory action of partial reduction of extracellular [Mg2+] in CA1-region of rat hippocampal slices

Partial reduction of [Mg2+]o from 2 to 1 mM markedly enhanced neuronal responses evoked by Schaffer collateral-commissural fiber stimulation in the CA1-region of rat hippocampal slices. The amplitude of extracellular population potentials recorded in the CA1-pyramidal cell layer and maximum dV/dt of extracellular population EPSP's recorded in the CA1-pyramidal apical dendritic layer were both increased. However, unlike findings from slices where Mg2+ was completely removed from the bathing medium, there was no spontaneous or evoked epileptiform activity, and the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (2-APV) did not antagonize the enhancement of evoked responses. These results indicate that, in addition to the participation of NMDA receptors in the epileptiform activity observed when Mg2+ is completely removed from the bathing medium, there is also an NMDA receptor-independent excitatory action of partial reduction of [Mg2+]o in hippocampal slices.     

Epileptiform burst activity induced by potassium in the hippocampus and its regulation by GABA-mediated inhibition

Intracellular and extracellular recordings were made from pyramidal neurons in hippocampal slices in order to study spontaneous paroxysmal bursting induced by raising the extracellular potassium concentration from 3.5 to 8.5 mM. Extracellular recordings from all hippocampal subfields indicated that spontaneous bursts appeared to originate in region CA3c or CA3b as judged by burst onset. Burst intensity was also greatest in regions CA3b and CA3c and became progressively less toward region CA2. Intracellular recordings indicated that in 8.5 mM potassium, large spontaneous excitatory postsynaptic potentials (EPSPs), large burst afterhyperpolarizations, and rhythmic hyperpolarizing-depolarizing waves of membrane potential were invariably present in CA3c neurons. High potassium (8.5 mM) induced a positive shift (+9 mV) in the reversal potential of GABAergic inhibitory postsynaptic potentials (IPSPs) in CA3c neurons without changing input resistance or resting potential. This resulted in a drastic reduction in amplitude of the IPSP. Reduction of IPSP amplitude occurred before the onset of spontaneous bursting and was reversible upon return to normal potassium. A new technique to quantify the relative intensity of interictal-like burst discharges is described. Pentobarbital, diazepam, and GABA uptake inhibitors, which enhance GABA-mediated synaptic inhibition, reduced the intensity of potassium-induced bursts, whereas the GABA antagonist bicuculline increased burst intensity. Diphenylhydantoin and phenobarbital, anticonvulsants that have little effect on GABAergic inhibition, were without effect on spontaneous bursts. Burst frequency was reduced by bicuculline and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol but was unaffected by other drugs. Reduction of slice temperature from 35 to 19 degrees C dramatically reduced burst intensity but did not markedly affect burst frequency. We hypothesize that high potassium induces a rise in intracellular chloride concentration, possibly by activating an inward KCl pump or by a passive Donnan effect, which results in a decreased IPSP amplitude. With inhibition suppressed, the large spontaneous EPSPs that appear in high potassium cause individual CA3c neurons to fire. A combination of synaptic and electrical interactions among CA3c cells then synchronizes discharges into interictal spike bursts.