Dicarboxylic Amino Acids Block Epileptiform Activity in Hippocampal Slice

Effects of prolonged (5-10 min) continuous perfusion of excitatory amino acids on penicillin (PEN)-evoked epileptiform activity in hippocampal slices were examined with extracellular and intracellular recordings. L-glutamate (GLU), L-aspartate (ASP), quisqualate (QUIS), and N-methyl-D,L-aspartate reversibly depressed multiple (epileptiform) population spikes elicited by PEN (1.7 mM). Intracellularly recorded, PEN-evoked paroxysmal depolarization shifts (PDS) were partially blocked by 1 mM GLU and largely eliminated by 2 mM GLU or ASP. In the presence of PEN, perfusion with both GLU and ASP induced a transient 4 to 6-mV depolarization, usually followed by spontaneous return of membrane potential to control levels. During the amino acid (AA)-induced block of epileptiform activity, there was no significant change in resting membrane potential, input resistance, or the ability to fire action potentials in response to depolarization, indicating that the decreased responsiveness is not a consequence of nonspecific pyramidal cell overdepolarization. The observed depression of epileptiform activity by continued exposure to GLU and its analogues may reflect desensitization or another regulatory mechanism to limit overexcitation.     

GABA-activated Cl- channels in astrocytes of hippocampal slices

We used kainic acid-lesioned hippocampal slices to examine glial responses to the inhibitory neurotransmitter GABA in a neuron-free environment. Slices were prepared from rats which received intracerebroventricular injections of kainic acid 1 month prior to experiments. Astrocytes (membrane potential averaged 81.4 +/- 5.5 mV; n = 46; mean +/- SD) were impaled in the CA3 region of the slice, which was completely depleted of neurons. GABA (1 mM) application by bath perfusion depolarized membrane potential from 1 to 5 mV. The GABA-induced depolarization was not affected by a tetrodotoxin (1 microM)/high-Mg2+/low-Ca2+ solution. Changing the Cl- equilibrium potential by reducing extracellular Cl- greatly increased the GABA-induced depolarization. Muscimol mimicked the GABA response, while picrotoxin (0.1 mM), an antagonist of the GABA-activated Cl- channel, resulted in a 60% blockade. The barbiturate, pentobarbital (0.1 mM), and the benzodiazepine agonist, flunitrazepam (1 mM), enhanced the depolarization by 60 and 40%, respectively. A blocker of glial GABA uptake, beta-alanine (1 mM), did not affect the GABA-induced membrane depolarization, indicating that the depolarization is not caused by electrogenic uptake of the amino acid. The pharmacological properties of the GABA response of astrocytes from the hippocampal slice is similar to that previously described for cultured astrocytes from rat cerebral hemispheres. Our data suggest that GABA receptors, which are coupled to Cl- channels, are also expressed by astrocytes in an intact tissue.     

Striatal neuronal activity and responsiveness to dopamine and glutamate after selective blockade of D1 and D2 dopamine receptors in freely moving rats.

Although striatal neurons receive continuous dopamine (DA) input, little information is available on the role of such input in regulating normal striatal functions. To clarify this issue, we assessed how systemic administration of selective D1 and D2 receptor blockers or their combination alters striatal neuronal processing in freely moving rats. Single-unit recording was combined with iontophoresis to monitor basal impulse activity of dorsal and ventral striatal neurons and their responses to glutamate (GLU), a major source of excitatory striatal drive, and DA. SCH-23390 (0.2 mg/kg), a D1 antagonist, strongly elevated basal activity and attenuated neuronal responses to DA compared with control conditions, but GLU-induced excitations were enhanced relative to control as indicated by a reduction in response threshold, an increase in response magnitude, and a more frequent appearance of apparent depolarization inactivation. In contrast, the D2 antagonist eticlopride (0.2 mg/kg) had a weak depressing effect on basal activity and was completely ineffective in blocking the neuronal response to DA. Although eticlopride reduced the magnitude of the GLU response, the response threshold was lower, and depolarization inactivation occurred more often relative to control. The combined administration of these drugs resembled the effects of SCH-23390, but whereas the change in basal activity and the GLU response was weaker, the DA blocking effect was stronger than SCH-23390 alone. Our data support evidence for DA as a modulator of striatal function and suggest that under behaviorally relevant conditions tonically released DA acts mainly via D1 receptors to provide a continuous inhibiting or restraining effect on both basal activity and responsiveness of striatal neurons to GLU-mediated excitatory input.     

In vitro electrophysiology of rat subicular bursting neurons

Intracellular recordings were used to study the electrophysiological properties of rat subicular neurons in a brain slice preparation in vitro. Cells were classified as bursting neurons (n = 102) based on the firing pattern induced by depolarizing current pulses. The bursting response recorded at resting membrane potential (−66.1 ± 6.2 mV, mean ± SD n = 94) was made up of a cluster of fast action potentials riding on a slow depolarization and was followed by an afterhyperpolarization. Tonic firing occurred at a membrane potential of approximately −55 mV. A burst also occurred upon termination of a hyperpolarizing current pulse. Tetrodotoxin (TTX, 1 μM) blocked the burst and decreased or abolished the underlying slow depolarization. These effects were not induced by the concomitant application of the Ca²⁺ channel blockers Co²⁺ (2 mM) and Cd²⁺ (1 mM). Subicular bursting neurons displayed voltage- and time-dependent inward rectifications of the membrane during depolarizing and hyperpolarizing current pulses. The inward rectification in the depolarizing direction was abolished by TTX, while that in the hyperpolarizing direction was blocked by extracellular Cs⁺ (3 mM), but not modified by Ba²⁺ (0.5–1 mM), TTX, or Co²⁺ and Cd²⁺. Tetraethylammonium (10 mM)-sensitive, outward rectification became apparent in the presence of TTX. These results suggest that neurons in the rat subiculum can display voltage-dependent bursts of action potentials as well as membrane rectification in the depolarizing and hyperpolarizing directions. These results also indicate that activation of a voltage-gated Na⁺ conductance may be instrumental in the initiation of bursting activity. Hippocampus 7:48–57, 1997. © 1997 Wiley-Liss, Inc.     

Evidence that excitatory amino acids not only activate the receptor channel complex but also lead to use-dependent block

The effects of fast application of excitatory amino acids N-methyl-D-aspartate (NMDA), L-aspartate (ASP), L-glutamate (GLU), quisqualate (QU) and kainate (KAIN) were studied in neurons from the embryonic spinal cord of the chick in monolayer cultures by employing the 'patch clamp' technique in the 'whole cell' mode. It was found that NMDA, ASP, GLU and QU, but not KAIN, induced responses that exhibited several components. The early component decayed with a time constant of 2 s to a lower level of membrane current and discontinuation of the application was followed by an after-current which returned to the base-line with a time constant of about 7 s. It is suggested that NMDA, ASP, GLU and QU, but not KAIN, not only activate the receptor channel complex but also induce use-dependent block.     

Transient in vivo membrane depolarization and glutamate release before anoxic depolarization in rat striatum.

Increased extracellular glutamate ([GLU]e), under the condition of cerebral ischemia, anoxia or hypoxia, has been recognized as being associated with neuronal cell damage and death. We performed real-time monitoring of [GLU]e dynamics in vivo in the rat striatum during systemic acute anoxia or hypoxia, as well as monitoring the direct current potential (DC) and cerebral blood flow (CBF). Adult Wistar rats were orotracheally intubated and artificially ventilated with room air. A microdialysis electrode, temperature sensor probe, DC microelectrode and laser Doppler probe were then implanted. The inspired gas was changed to 100% N(2) (anoxia), or to 3, 5 or 8% O(2) (remainder N(2)) (hypoxia). With 100% N(2), distinct biphasic [GLU]e elevations were observed. With 3% O(2), a transient [GLU]e increase was seen before anoxic depolarization (AD). With 5% O(2), however, the start of the transient [GLU]e increase was significantly delayed. Anoxia-induced depolarization started at about 100 s. The 3% O(2)-induced transient depolarization and AD began at nearly the same time as the transient and AD-induced increase in [GLU]e. Similarly, the responses to 5% O(2) showed significant delays in the transient depolarization and AD-induced increase in [GLU]e. CBF during 3 or 5% O(2) hypoxic insult was consistently maintained above the control level, i.e., prior to cardiac arrest. Our new dialysis electrode method employing both GOX and ferrocene-conjugated bovine serum albumin allowed evaluation of transient [GLU]e dynamics in the early phase of severe hypoxia in vivo.     

Characterization of a Barium-sensitive Outward Current following Glutamate Application on Rat Midbrain Dopaminergic Cells

Using intracellular electrophysiological recordings in dopaminergic (principal) neurons of the rat mesencephalon maintained in vitro, we studied a postexcitatory amino acid response (PEAAR). Under current-clamp mode, bath application of glutamate produced a depolarization that was followed by a hyperpolarization when the perfusion of the excitatory amino acid was discontinued. Under single-microelectrode voltage-clamp mode, an outward current followed the glutamate-induced inward current. The PEAAR was associated with an increase in membrane conductance and reversed polarity at about -85 mV (2.5 mM extracellular K⁺). The null potential for the PEAAR was independent of the intracellular loading of chloride ions and was shifted towards less negative values (?23 mV) by increasing extracellular K⁺ from 2.5 to 8.5 mM. The PEAAR was present in neurons treated with tetraethylammonium (5–10 mM), apamin (1 μM) or glibenclamide (1–300 μM). However, it was strongly depressed or blocked by extracellular barium (300 μM to 1 mM), by low-calcium (0.5 mM) plus cadmium (100 μM) or magnesium (10 mM), and by low-sodium solutions. An outward response was also generated after an inward current induced by the perfusion of the specific agonists for the ionotropic excitatory amino acid receptors NMDA, a-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) and kainate. The PEAAR was not affected by tetrodotoxin (1 μM), saclofen (100–300 μM), bicuculline (30 μM), sulpiride (1 μM) or strychnine (1 μM). In addition, the inhibition of the ATP-dependent Na⁺-K⁺ pump by ouabain and strophanthidin (1–10 μM) prolonged the glutamate-induced membrane depolarization/inward current while the subsequent PEAAR was reduced or not observed. Our data indicate that the PEAAR mainly results from the activation of a barium-sensitive potassium current. This response might limit the excitatory and eventually neurotoxic effects of glutamate.     

Inhibition of energy metabolism by 3-nitropropionic acid activates ATP-sensitive potassium channels.

3-Nitropropionic acid (1 mM), which inhibits succinate dehydrogenase activity and reduces cellular energy, produces in the pyramidal cell layer of the hippocampal region CA1 a hyperpolarization for variable lengths of time before evoking an irreversible depolarization. Hyperpolarization is caused by an increased potassium conductance that is attenuated by glibenclamide (1-10 microM), a selective antagonist of ATP-sensitive potassium channels; in contrast, diazoxide (0.5 mM), an agonist at this channel, induces a hyperpolarization in CA1 neurons of rat hippocampal slices. The transient hyperpolarization after prolonged (ca. 1 h) application of 3-NPA is followed by a depolarization that is incompletely reversed by brief application of the glutamate antagonists (D-2-amino-5-phosphonopentanoic acid (APV), 6,7-dichloroquinoxaline-2,3-dione (CNQX), 3-(+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), 7-chloro-kynurenic acid (7Cl-KYN)). Early application of glibenclamide (within the initial 5 min) blocked or reduced hyperpolarization and accelerated the depolarization. These data suggest that metabolic inhibition by 3-NPA initially activates ATP-sensitive potassium channels. Events other than activation of glutamate receptors participate in the final depolarization resulting from uncoupling of oxidative phosphorylation.     

Release of glutamate and GABA in the amygdala of conscious rats by acute stress and baroreceptor activation: differences between SHR and WKY rats

To reveal the functional importance of amino acid neurotransmission in the amygdala (AMY) of conscious spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, the in vivo release of glutamate (GLU) and GABA in this brain structure was studied using the push-pull superfusion technique. Basal GLU and GABA release rates in the AMY were comparable in SHR and WKY rats, although arterial blood pressure (BP) in SHR (152+/-6 mmHg) was higher than in WKY rats (102+/-4 mmHg). Neuronal depolarization by superfusion with veratridine enhanced the release of GLU and GABA to a similar extent in both rat strains. On the other hand, exposure to noise stress (95 dB) for 3 min led to a tetrodotoxin-sensitive increase in GLU release in the AMY of SHR, but not WKY rats. The concurrent pressor response to noise was enhanced in SHR as compared to WKY rats. A rise in BP induced by intravenous infusion of phenylephrine for 9 min had no effect on amino acid release in the AMY of both strains. The data suggest an exaggerated stress response of glutamatergic neurons in the AMY of SHR as compared with WKY rats, which might be of significance for the strain differences in the cardiovascular and behavioural responses to stress. The results also show that, in both rat strains, glutamatergic and GABAergic neurons in the AMY are not modulated by baroreceptor activation. Moreover, hypertension in adult SHR does not seem to be linked to a disturbed synaptic regulation of glutamatergic or GABAergic transmission in the AMY.     

Hypocretin/orexin depolarizes and decreases potassium conductance in locus coeruleus neurons

Recent studies demonstrated that noradrenergic locus coeruleus (LC) neurons are a particularly strong target of the novel neuropeptide, hypocretin (orexin). The present study sought to elucidate the action of hypocretin-B (HCRT) on LC neurons recorded intracellularly in rat brain slices. Bath (1.0 microM) or local puff application (50-100 microM in pipette) of HCRT depolarized LC neurons in rat brain slices and increased their spontaneous discharge rate. Depolarization evoked by HCRT was persistent in the presence of tetrodotoxin (TTX, 1 microM) and Co2+ (1 mM), indicating that HCRT directly activated LC neurons, and that its effect on the postsynaptic cell was not due to activation of TTX-sensitive sodium channels or Co2+-sensitive calcium channels. The apparent input resistance was significantly increased in the majority of LC neurons during the HCRT-evoked depolarization. Moreover, the HCRT-evoked depolarization was decreased in amplitude with hyperpolarization of membrane. The present results indicate that decreased potassium conductance is involved in the effect of HCRT on LC neurons.     

Seasonal differences and protection by creatine or arginine pretreatment in ischemia of mammalian and molluscan neurons in vitro

We investigated the dose-response relationship of protection by creatine against ischemic damage, and we asked whether or not such protection may be observed in invertebrate neurons that might provide a simpler experimental model. Rat isolated pyramidal neurons from the CA3 region of hippocampus subjected to ischemia ("in vitro ischemia") showed anoxic depolarization (AD) after 3-7 min of incubation in anoxic medium. Membrane potential (MP) was reduced 25-78% from preanoxic value. Inward current was decreased by an average 49%. Supplementation with creatine protected against these changes, with 1 mM being the minimal effective concentration, 2 mM providing a near-maximal protection, a maximal effect being obtained with 5 mM creatine. No additional protection was provided by up to 20 mM creatine. Isolated giant neurons of Lymnaea stagnalis showed a similar response to in vitro ischemia. However, a clear seasonal dependence of sensitivity of these cells was detected. In cells obtained during summertime (May-August), AD latency ranged from 3 to 10 min; during wintertime (December-March), this response did not occur even after 25-50 min. The addition of creatine to the medium did not cause changes in AD latency, probably because these neurons rely on a phosphoarginine/arginine energy system. However, treatment of the cells, harvested during summertime, with 2 mM arginine did provide clear protection against anoxic-aglycaemic changes. Summing up, besides confirming previous findings on creatine protection in mammalian neurons, we (1) better characterized their dose-response relationship and extended the findings to the CA3 region and to isolated neurons, (2) found that invertebrate neurons are not protected by creatine but by arginine supplementation and (3) reported a novel mechanism of seasonal dependence in sensitivity of in vitro ischemia by invertebrate neurons.     

Effect of calcium ions on the sensitivities of cultured cerebellar neurons to glutamate and aspartate

Iontophoretically applied glutamate and aspartate induced depolarizations in immature (6-13 days in culture) and mature (25-45 days) cultured chick cerebellar neurons, immature neurons being less sensitive. The input resistances of the neurons were variously changed by these amino acids. Reversal potentials of the depolarizations induced by both amino acids were similar in either immature or mature neurons. The population of amino acid-sensitive neurons increased with maturation. In mature neurons, the amplitude of glutamate- or aspartate-induced depolarization was decreased by addition of 10 mM Ca2+ to normal Tyrode's solution, aspartate responses being decreased more greatly. In low-Na+ solution (2.7 mM), however, high Ca2+ significantly enhanced amino acid-induced depolarizations. In immature neurons, on the other hand, the amplitude of glutamate- or aspartate-induced depolarization was drastically and consistently increased when 10 mM Ca2+ was added either to normal solution or to the low-Na+ solution. These enhancing actions of Ca2+ were abolished by Mn2+, but only partially by 10 mM glutamic acid diethylester or 1 mM D-alpha-aminoadipate, though responses to both amino acids in normal solution were blocked by these antagonists at the same concentrations. These results suggest that calcium ions enhance the effect of glutamate and aspartate in immature neurons, possibly by interacting with the ionophores involved in amino acid responses.     

Analysis of preganglionic nerve evoked cholinergic contractions of the guinea pig bronchus.

We compared cholinergic bronchial muscle contractions induced by vagus nerve (preganglionic) stimulation (VNS) with those induced by electrical field (postganglionic) stimulation (EFS). When normalized to their respective maximum response, the frequency-response curves (10 s trains) between 4 and 16 Hz were similar between VNS and EFS; however, at frequencies of 0.1-2 Hz, and at frequencies greater than 32 Hz, the VNS contractions were significantly less than EFS. When contractions elicited by 100 pulses were examined, it was found that the responses to VNS were maximal at 10-30 Hz then declined significantly to 82-35% of maximal between 40 and 200 Hz, whereas the response to EFS was essentially unchanged at frequencies up to 60 Hz and declined only to 72% of maximal up to 200 Hz. At frequencies as low as 20 Hz, the contractions evoked by VNS faded to 45 +/- 9% of the peak contraction during 60 sec of continuous stimulation, whereas those evoked by 60 sec continuous EFS remained constant. This fade observed during prolonged VNS was not blocked by the antagonists, pirenzepine and AFDX-116, at concentrations selective for M1 and M2 muscarinic receptors, respectively; nor was the fade blocked by pre-treatment with indomethacin, propranolol, phentolamine, or choline. At frequencies greater than 10 Hz, the amplitude of the preganglionic compound action potential also faded during repetitive stimulation. The results support the hypothesis that the airway ganglion neurons innervating guinea pig bronchial smooth muscle effectively filter preganglionic stimuli, especially at low and relatively high frequencies. During continuous vagus nerve stimulation, preganglionic mechanisms may also play a role in limiting the ultimate output of airway ganglia.     

Regulation of parasympathetic neurons by mast cells and histamine in the guinea pig heart

The potential interaction between the immune system and the autonomic nervous system was examined in the cardiac ganglia of guinea pigs. Intracellular voltage recordings were used to determine the effects of mast cell degranulation on the membrane properties of parasympathetic neurons in animals actively sensitized to ovalbumin. Stimulation of mast cell degranulation by perfusion with ovalbumin (10 micrograms/ml) produced a depolarization and increase in the excitability of intracardiac neurons. These effects could be mimicked by histamine application, either by perfusion (10 microM) or by local pressure application (100 microM, 1-2 s application). In either case, histamine application resulted in a similar membrane depolarization and increase in excitability. Immunohistochemical experiments demonstrated that histamine-immunoreactive mast cells are located in close proximity to parasympathetic postganglionic neurons. The histamine response was not due to release of other neurotransmitters from adjacent nerve terminals and both the depolarization and increase in excitability were inhibited by the H1 antagonist, pyrilamine (300 nM), and were unaffected by the H2 antagonist cimetidine (5 microM). Incubation of cardiac ganglion preparations from sensitized animals with pyrilamine prior to ovalbumin perfusion resulted in the inhibition of both the depolarization and increase in excitability. These results demonstrate that mast cell degranulation, and the subsequent release of histamine, results in the stimulation of intracardiac neurons via the activation of H1 receptors. Thus, local inflammatory reactions in the cardiac tissue can lead to the rapid activation of parasympathetic neurons, thereby altering cardiac function.     

Electrophysiological and pharmacological actions of N-acetylaspartylglutamate intracellularly studied in cultured chick cerebellar neurons

The electrophysiological and pharmacological actions of N-acetylaspartylglutamate (NAAG) in cultured chick cerebellar neurons were intracellularly investigated in comparison with L-aspartate (ASP) and L-glutamate (GLU). Iontophoretically applied NAAG dose-dependently induced depolarizations associated with increases in spike discharge and changes in membrane conductance. Relative excitatory potencies seemed to be GLU greater than ASP greater than or equal to NAAG. The voltage-dependent increase in input resistance observable in the presence of Mg ions was most notable for ASP, moderate for NAAG and least for GLU. The reversal potential of NAAG-induced depolarization was at about 0 mV and similar to that for ASP or GLU, indicating primary concern of Na+/K+-conductances to the NAAG action. Mg ions depressed the actions of ASP and NAAG more strongly than the GLU action. 2-Amino-5-phosphonovalerate (APV) and D-alpha-aminoadipate antagonized the actions of ASP and NAAG more effectively than the GLU action. 2-Amino-4-phosphonobutyrate (APB) and glutamic acid diethylester showed rather non-selective antagonisms to NAAG, ASP and GLU. These results suggest that NAAG is excitatory to cultured chick cerebellar neurons and functionally resembles ASP or is intermediate between ASP and GLU, and may also support the suggested candidacy of NAAG for a neurotransmitter in the CNS.     

Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death

The pathophysiology of hypoxic neuronal death, which is difficult to study in vivo, was further defined in vitro by placing dispersed cultures of rat hippocampal neurons into an anoxic atmosphere. Previous experiments had demonstrated that the addition of high concentrations of magnesium, which blocks transmitter release, protected anoxic neurons. These more recent experiments have shown that gamma-D-glutamylglycine (DGG), a postsynaptic blocker of excitatory amino acids, was highly effective in preventing anoxic neuronal death. DGG also completely protected the cultured neurons from the toxicity of exogenous glutamate (GLU) and aspartate (ASP). In parallel physiology experiments, DGG blocked the depolarization produced by GLU and ASP, and dramatically reduced EPSPs in synaptically coupled pairs of neurons. These results provide convincing evidence that the synaptic release of excitatory transmitter, most likely GLU or ASP, mediates the death of anoxic neurons. This result has far-reaching implications regarding the interpretation of the existing literature on cerebral hypoxia. Furthermore, it suggests new strategies that may be effective in preventing the devastating insults produced by cerebral hypoxia and ischemia in man.     

Synaptic potentials in the rat neostriatum in dissociated embryonic cell culture

Neostriatal cells of embryonic days 19-21 were grown in dissociated cell culture. To test whether the cultures contained predominantly neostriatal cells, a glyoxylic acid staining procedure was used which, after dopamine loading, stained neostriatal cells but not neurons of embryonic neocortical tissue. Whole cell current clamp recording was performed in the neurons after 1-2 weeks in cell culture. Although cells could be driven to discharge by direct depolarization, spontaneous activity was low. All cells responded to gamma-aminobutyric acid (GABA) (0.1-0.5 mM), and the majority of them responded to glutamate (Glu) (0.1 mM). Only about 50% were depolarized by acetylcholine (ACh) (0.1-0.5 mM). Atropine (1-10 microM) did not block this depolarization. Barrages of postsynaptic potentials (PSPs) were induced by applications of Glu or ACh, even if the neuron under observation was not depolarized. All PSPs were depressed by bicuculline (50 microM), indicating their mediation by GABAergic receptors. Exclusively GABAergic PSPs were also observed in cultures raised in the presence of nerve growth factor. The study indicates that neostriatal cells form GABAergic, but not excitatory cholinergic synapses when cultured at this embryonic age under our conditions, resembling the pattern of development observed in slices obtained from neonatal rats.     

Effects of vigabatrin on epileptiform abnormal discharges in hippocampal CA3 neurons of spontaneously epileptic rats (SER)

Vigabatrin, a γ-amino butyric acid (GABA) transaminase inhibitor, is known to inhibit partial epilepsy in humans. The spontaneously epileptic rat (SER), a double mutant (zi/zi, tm/tm), exhibits both tonic convulsion and absence-like seizures from the age of 8 weeks. Hippocampal CA3 pyramidal neurons in SER show a long-lasting depolarization shift with accompanying repetitive firing when a single stimulus is delivered to the mossy fibers in slice preparations. The effects of vigabatrin on the abnormal excitability of hippocampal CA3 pyramidal neurons in SER were examined to elucidate the mechanism underlying the antiepileptic action of the drug. Intracellular recordings were performed in 24 hippocampal slice preparations of 20 SER aged 8–17 weeks old. Bath application of vigabatrin (1 mM) inhibited the depolarizing shifts with repetitive firing induced by mossy fiber stimulation in 15 min without affecting the first spike and resting membrane potentials in hippocampal CA3 neurons of SER. A higher dose of vigabatrin (10 mM) sometimes inhibited the first spike. However, vigabatrin at doses up to 10 mM did not significantly affect the single action potential elicited by stimulation of the mossy fibers in the hippocampal CA3 neurons of age-matched Wistar rats. In addition, application of vigabatrin (10 mM) did not significantly affect the firing induced by depolarizing pulse applied in the CA3 neurons of the SER, nor the miniature excitatory postsynaptic potential (mEPSP) recorded in the CA3 neurons of SER. The inhibitory effect of vigabatrin (1 mM) on the mossy fiber stimulation-induced depolarization shift with repetitive firing was blocked by concomitant application of bicuculline (10 μM), a GABA_A receptor antagonist. These findings strongly suggested that GABA increased by inhibition of GABA transaminase with vigabatrin inhibits abnormal excitation of hippocampal CA3 neurons of SER via GABA_A receptors, although the possibility that the drug acted directly on the GABA_A receptors of CA3 neurons could not be completely excluded.     

GABAergic inhibition and epileptiform discharges in the turtle hippocampus in vitro

The effects of excitatory amino acid (EAA) receptor antagonists were examined on intracellularly recorded epileptiform discharges in turtle hippocampal (ventromedial cortical) pyramidal neurons in vitro. Afferent synaptic activation of turtle hippocampal neurons evoked monophasic or biphasic GABAergic inhibitory postsynaptic potentials (IPSPs). In the presence of bicuculline (5 microM) or picrotoxin (100 microM) IPSPs were reduced, and long-lasting ictal-like discharges were transiently observed prior to the establishment of a regular rhythm of discharge of spontaneous paroxysmal depolarization shifts (PDSs). Bicuculline-induced PDSs were reversibly reduced in amplitude and duration, but not abolished by the EAA receptor antagonists kynurenic acid (1 mM), cis-2,3-piperidine dicarboxylic acid (cis-2,3-PDA) (1 mM), or DL-2-amino-5-phosphonovalerate (DL-AP-5) (100 microM), revealing a long-lasting hyperpolarizing afterpotential. These results indicate that the blockade of GABAergic inhibition leads to the genesis of epileptiform discharges, and EAA receptor antagonists (particularly those of the N-methyl-D-aspartate (NMDA) receptor subtype) block the maintained depolarization underlying PDSs, but do not prevent their spontaneous discharge in turtle hippocampus.     

Intrinsic membrane potential oscillations in hippocampal neurons in vitro

Membrane potential oscillations (MPOs) of 2-10 Hz and up to 6 mV were found in almost all stable hippocampal CA1 and CA3 neurons in the in vitro slice preparation. MPOs were prominent for pyramidal cells but less pronounced in putative interneurons. MPOs were activated at threshold depolarizations that evoked a spike and the frequency of the MPOs increased with the level of depolarization. MPOs were distinct from and seemed to regulate spiking, with a spike often riding near the top of a depolarizing MPO wave. Analysis of the periodicity of the oscillations indicate that the period of MPOs did not depend on the afterhyperpolarization (AHP) following a single spike. MPOs persisted in low (0-0.1 mM) Ca2+ medium, with or without Cd2+ (0.2 mM), when synaptic transmission was blocked. Choline-substituted low-Na+ (0-26 mM) medium, 3 microM tetrodotoxin (TTX) or intracellular injection of QX-314 reduced or abolished the fast Na(+)-spike and reduced inward anomalous rectification. About 40% of CA1 neurons had no MPOs after Na+ currents were blocked, suggesting that these MPOs were Na(+)-dependent. In about 60% of the cells, a large depolarization activated Ca(2+)-dependent MPOs and slow spikes. MPOs were not critically affected by extracellular Ba2+ or Cs2+, or by 0.2 mM 4-aminopyridine, with or without 2 mM tetraethylammonium (TEA). However, in 5-10 mM TEA medium, MPOs were mostly replaced by 0.2-3 Hz spontaneous bursts of wide-duration spikes followed by large AHPs. Low Ca2+, Cd2+ medium greatly reduced the spike width but not the spike-bursts. In conclusion, each cycle of an MPO in normal medium probably consists of a depolarization phase mediated by Na+ currents, possibly mixed with Ca2+ currents activated at a higher depolarization. The repolarization/hyperpolarization phase may be mediated by Na+/Ca2+ current inactivation and partly by TEA-sensitive, possibly the delayed rectifier, K+ currents. The presence of prominent intrinsic, low-threshold MPOs in all hippocampal pyramidal neurons suggests that MPOs may play an important role in information processing in the hippocampus.