Guinea Pig Hippocampal Slices Research Articles

Periodic orbit analysis reveals subtle effects of atropine on epileptiform activity in the guinea-pig hippocampal slice

Epileptiform activity is a state often induced in vitro in order to study seizures and antiepileptic/anticonvulsant drugs. Traditional methods of evaluating drug effects have commonly relied upon measuring changes in the frequency and duration of such events. We have used a recently developed mathematical technique based on periodic orbit analysis to investigate the effect of atropine (a muscarinic antagonist) on epileptiform activity induced by soman (an irreversible acetylcholinesterase inhibitor), 4-aminopyridine (a K + channel blocker) and 8-cyclopentyl-1,3-dipropylxanthine (an adenosine A 1 receptor antagonist) in the guinea-pig hippocampal slice. This technique showed that significant changes in periodic orbits can occur without an accompanying change in burst rate. These results suggest that periodic orbit analysis may be useful in detecting and predicting novel actions of anticonvulsant drugs.

A chemical LTP induced by co-activation of metabotropic and N-methyl- d-aspartate glutamate receptors in hippocampal CA1 neurons

In CA1 neurons of guinea pig hippocampal slices, long-term depression (LTD) was induced in the field EPSP response in the absence of test synaptic inputs (one stimulus every 20 s) by application of the metabotropic glutamate receptor (mGluR) agonist, aminocyclopentane-1S, 3R-dicarboxylic acid (ACPD). This effect was blocked and long-term potentiation (LTP) was induced by co-application of N-methyl- d-aspartate (NMDA) during ACPD perfusion (ACPD/NMDA-induced LTD). These results indicate that the state of NMDA receptor activation during ACPD perfusion determines whether LTP or LTD is induced in hippocampal CA1 neurons. Co-application of an inositol 1, 4, 5-trisphosphate (IP3) receptor inhibitor, 2-aminotheoxydiphenyl borate, during ACPD application had no effect on the ACPD/NMDA-induced LTP, but increased the magnitude of the ACPD-induced LTD, suggesting that the ACPD/NMDA-induced LTP involves NMDA receptors, but not IP3 receptors, whereas the converse applies to the ACPD-induced LTD.

Evidence that phospholipase D activation prevents group I mGluR-induced persistent prolongation of epileptiform bursts.

Selective activation of group I metabotropic glutamate receptors (mGluRs) with (S)-3,5-dihydroxyphenylglycine (DHPG) in guinea pig hippocampal slices converts 275- to 475-ms picrotoxin-induced interictal bursts into persistent seizure-length discharges typically over 1 s in duration. Here we report that l-cysteine sulfinic acid (CSA), a sulfur-containing amino acid, prevented the induction of this persistent group I mGluR-mediated epileptiform burst prolongation. However, CSA had no effect on baseline interictal bursting activity and failed to suppress the expression of the group I mGluR-induced persistent prolonged bursts once they were fully induced. (2R,1'S,2'R,3'S)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-13), a selective antagonist at the phospholipase D (PLD)-coupled mGluR, had no effect of its own on DHPG-induced burst prolongation; however, CSA applied in the presence of PCCG-13 could no longer fully block the burst prolongation induced by DHPG, suggesting that CSA's antiepileptogenic effect is mediated by agonist action at this PLD-coupled receptor. These data parallel our previous data revealing that protein synthesis inhibitors prevent induction but not expression of group I mGluR-mediated persistent seizure-length discharges. Hence, PLD activation with CSA may prevent the synthesis of a protein critical for the induction of group I mGluR-mediated epileptogenesis.

Cooperativity between activation of metabotropic glutamate receptors and NMDA receptors in the induction of LTP in hippocampal CA1 neurons

In CA1 neurons of guinea pig hippocampal slices, long-term potentiation (LTP) was induced by 10 min application of 10 μM aminocyclopentane-1S, 3R-dicarboxylic acid (ACPD), the metabotropic glutamate receptor (mGluR) agonist, in the presence of test synaptic inputs (once every 20 s). In contrast, long-term depression (LTD) was induced by application of 10 μM ACPD in the absence of test inputs. When 10 μM ACPD was applied in the presence of test inputs, co-application of the N-methyl- d-aspartate (NMDA) receptor antagonist, d, l-2-amino-5-phosphonovalerate resulted in LTD induction when used at 50 μM. In ACPD-induced LTP, the delivery of test synaptic inputs to CA1 neurons could be replaced by co-application of NMDA (100 nM) during ACPD perfusion. These results suggest that, in CA1 neurons, a co-operative effect involving the activation of both mGluRs and NMDA receptors is required to trigger the process involved in ACPD-induced LTP. In addition, ACPD-induced LTD was blocked by co-application of an inositol 1,4,5-trisphosphate (IP3) receptor inhibitor, 2-aminotheoxydiphenyl borate (10 μM), which had no effect on ACPD-induced LTP. The results of the present study, therefore, indicate that ACPD-induced LTP involves NMDA receptors, but not IP3 receptors, whereas the converse applies to ACPD-induced LTD.

Partial adenosine A 1 receptor agonists inhibit sarin-induced epileptiform activity in the hippocampal slice

Organophosphate poisoning can result in seizures and subsequent neuropathology. One possible therapeutic approach would be to employ adenosine A 1 receptor agonists, which have already been shown to have protective effects against organophosphate poisoning. Using an in vitro model of organophosphate-induced seizures, we have investigated the ability of several adenosine A 1 receptor agonists to inhibit epileptiform activity induced by the organophosphate sarin, in the CA1 stratum pyramidale of the guinea pig hippocampal slice. Application of the adenosine A 1 receptor agonist N 6-cyclopentyladenosine (CPA) or the partial adenosine A 1 receptor agonists 2-deoxy- N 6-cyclopentyladenosine (2-deoxy-CPA) and 8-butylamino- N 6-cyclopentyladenosine (8-butylamino-CPA) abolished epileptiform activity in a concentration-related manner. The rank order of potency was CPA (IC 50 4–5 nM)>2-deoxy-CPA (IC 50 113–119 nM)=8-butylamino-CPA (IC 50 90–115 nM). These data suggest that partial adenosine A 1 receptor agonists, which have fewer cardiovascular effects, should be further evaluated in vivo as potential treatments for organophosphate poisoning.

Bioactivity of a peptide derived from acetylcholinesterase: electrophysiological characterization in guinea-pig hippocampus.

Acetylcholinesterase is well known to have noncholinergic functions. Only recently, however, has the salient part been identified of the molecule responsible for these nonclassical actions, a peptide of 14 amino acids towards the C-terminus of acetylcholinesterase. The aim of this study was to test the bioactivity of this 'acetylcholinesterase-peptide' using intracellular recordings in guinea-pig hippocampal slices. In the presence of tetrodotoxin, acetylcholinesterase-peptide alone affected neither the membrane potential nor the input resistance of CA1 neurons; however, a modulatory action was observed, as a concentration-dependent decrease of N-methyl-d-aspartic acid-induced depolarization. When calcium potentials were elicited by a depolarizing current pulse, application of acetylcholinesterase-peptide increased or reduced the degree of calcium spike firing in, respectively, the presence or absence of the N-methyl-d-aspartic acid antagonist d(-)-2-amino-5-phosphonopentanoic acid. In contrast, a peptide derived from the equivalent region of butyrylcholinesterase, which also hydrolyses acetylcholine, had no effect. In conclusion, acetylcholinesterase-peptide has a selective bioactivity in the hippocampus; it could thus offer new ways of targeting mechanisms of calcium-induced neurotoxicity.

Dual effects of 5-HT4 receptor activation on GABA release from guinea pig hippocampal slices.

The effects of BIMU-8, a 5-HT4 receptor agonist, were studied on GABA release in guinea pig hippocampal slices. BIMU-8 did not modify GABA outflow at rest but did display a complex action in electrically stimulated slices: at low concentrations it increased, and at higher concentrations inhibited, GABA release. These responses were competitively counteracted by GR 125487, a selective 5-HT4 receptor antagonist. The dual effects of BIMU-8 are consistent with its indirect cholinergic action since the M1 and M3 antagonist, 4-DAMP, prevented BIMU-8-elicited GABA facilitation, whereas the M2 antagonist AFDX-116 cancelled GABA inhibition. These results provide evidence that serotonin exerts a complex modulation on the GABAergic system, via 5-HT4 receptors, and suggest that the amine releases acetylcholine which, in turn, bidirectionally modulates GABA release.

Pre- rather than co-application of vigabatrin increases the efficacy of tiagabine in hippocampal slices.

The antiepileptic drug vigabatrin (VGB) increases intracellular availability of the inhibitory transmitter gamma-aminobutyric acid (GABA) by inhibition of GABA-transaminase. A blockade of the GABA uptake is the main mechanism of action of tiagabine (TGB). Based on this, the two antiepileptic drugs (AEDs) can be speculated to act synergistically so that their combined antiepileptic efficacy is supraadditive. To test this, experiments were performed on hippocampal slices of guinea-pigs. As an epilepsy model, epileptiform field potentials (EFPs) were induced by omission of Mg2+ from the bath solution and recorded in stratum pyramidale of the CA3 region. VGB (7.5 microM) and TGB (0.75 microM) were added to the superfusate. VGB, given alone, failed to decrease the repetition rate of EFPs. Similarly, TGB applied alone only transiently led to a nonsignificant reduction of the EFP frequency. Combining VGB and TGB, their suppressive efficacy increased, yielding a significant reduction of EFP frequency, which, however, again did not persist. Pretreatment of the preparations with VGB for 2 h, followed by additional application of TGB, or TGB alone, drastically and persistently potentiated the effects. These results demonstrate that VGB and TGB show favorable pharmacodynamic interactions, provided VGB is allowed to block intracellular GABA degradation before GABA uptake block by TGB.

Epileptogenic effect of cyclosporine in guinea-pig hippocampal slices

Cyclosporine A (CsA) neurotoxicity is a common cause of seizures in transplant patients and others receiving immunosuppressive therapy. CsA at concentrations higher than the levels estimated for cerebrospinal fluid of the patients suffering from seizure attacks was ineffective to induce epileptiform field potentials (EFP) in in vitro brain-slice preparation. The aim of this study was to test the effect of CsA at lower concentrations on neuronal activity. Guinea-pig hippocampal slices were exposed to artificial cerebrospinal fluid containing CsA (0.1–2 μM). Furthermore, the effects of CsA (0.25–10 μM) were tested on EFP elicited by omission of Mg 2+ from superfusate. Low concentrations of CsA (0.1–0.25 μM) induced EFP while higher doses (0.5–2 μM) failed to decrease the seizure threshold. CsA at concentrations of 0.25 and 1 μM had no significant effect on the low Mg 2+-induced EFP. Higher CsA concentration (10 μM) strongly suppressed EFP. The results indicate that CsA at doses that are probably clinically relevant increases the neuronal excitability.

Background potassium concentrations and epileptiform discharges: II. Involvement of calcium channels

Potassium- and calcium conductances regulate neuronal excitability and epileptiform activity. In this study, the effects of different extracellular potassium concentrations ([K +] o) were investigated on the modulatory effect of the L-type transmembranous calcium currents on epileptiform discharges. The in vitro brain slice technique was used to examine the effects of calcium channel blockers, verapamil and nifedipine, on the repetition rate, amplitude, and duration of epileptiform field potentials (EFP) in the presence of low, physiological, and high background [K +] o in guinea pig hippocampal slices. Epileptiform activity was induced by omission of Mg 2+ from artificial cerebrospinal fluid contained 2, 4, and 8 mM [K +] o. Both verapamil and nifedipine suppressed EFP after a transient increase in repetition rate. The extent of EFP frequency rate acceleration significantly increased with reduction of [K +] o. The increase in EFP frequency rate induced by application of verapamil and nifedipine was accompanied by a reduction in the EFP amplitude and a reversible increase in the burst discharge duration. The extent of burst discharge prolongation was also significantly higher with decreasing [K +] o. Further application of verapamil and nifedipine suppressed the epileptiform burst activity in the presence of different [K +] o. The latency of EFP depression was significantly diminished both with increased and decreased background potassium concentrations. The data indicate the importance of the effect of the L-type transmembranous calcium currents on the regulatory effect of background [K +] o on epileptiform burst discharge frequency and duration.

Background potassium concentrations and epileptiform discharges: I. Electrophysiological characteristics of neuronal activity

Intra- and extracellular recording techniques were used to study the epileptiform activity generated by guinea pig hippocampal slices perfused with free-magnesium artificial cerebrospinal fluid in the presence of physiologic (4 mM), reduced (2 mM) or elevated (8 mM) extracellular potassium concentrations ([K +] o). Extracellular field potentials along with intracellular recordings were recorded in CA1 or CA3 region. Reduction of [K +] o significantly increased the latency of epileptiform field potential (EFP) appearance as well as burst discharge duration and decreased EFP repetition rate. Depending on different background [K +] o, epileptiform burst discharges appeared in different patterns including varied types of paroxysmal depolarisation shifts and burst activity in CA1 and CA3 subfields. Comparison with physiological and increased [K +] o, reduction of [K +] o significantly increased the mean duration of bursts, mean amplitude of depolarisation, mean after-hyperpolarisation duration, and inter-spike intervals in both CA1 and CA3 areas. Three distinct patterns were distinguished on the basis of their evoked firing pattern in response to application of depolarising current pulses in the interval of epileptiform burst discharges. Neurons superfused with 2 mM [K +] o presented fast adapting pattern while cells washed with 4 or 8 mM [K +] o exhibited intrinsically bursting or slow adapting patterns. Comparing the groups with different background [K +] o, there is a more severe form of discharges in low K + and a subtle difference between 4 and 8 mM K +. The data indicate the importance of background [K +] o on epileptiform burst discharge pattern and characteristics.

Adenosine triphosphate accelerates recovery from hypoxic/hypoglycemic perturbation of guinea pig hippocampal neurotransmission via a P 2 receptor

The present study was designed to assess the effects of adenosine triphosphate (ATP) on hippocampal neurotransmissions under the normal and hypoxic/hypoglycemic conditions. ATP reversely depressed population spikes (PSs), which were monitored in the dentate gyrus of guinea pig hippocampal slices, in a dose-dependent manner at concentrations ranged from 0.1 μM to 1 mM. A similar depression was obtained with the P 2 receptor agonist, α,β-methylene ATP (α,β-MeATP), and the effect was inhibited by the P 2 receptor antagonists, suramin and PPADS. The inhibitory action of ATP or α,β-MeATP was inhibited by the γ-aminobutyric acid A (GABA A) receptor antagonist, bicuculline, but it was not affected by theophylline, a broad inhibitor of adenosine (P 1) receptors, tetraethylammonium, a broad inhibitor of K + channels, or ecto-protein kinase inhibitors. ATP or α,β-MeATP enhanced GABA release from guinea pig hippocampal slices, that was inhibited by deleting extracellular Ca 2+ or in the presence of tetrodotoxin, while ATP had no effect on GABA release from cultured rat hippocampal astrocytes or postsynaptic GABA-gated channel currents in cultured rat hippocampal neurons. Twenty-minutes deprivation of glucose and oxygen from extracellular solution abolished PSs, the amplitude recovering to about 30% of basal levels 50 min after returning to normal conditions. ATP or α,β-MeATP accelerated the recovery after hypoxic/hypoglycemic insult (approximately 80% of basal levels). Adenosine diphosphate and adenosine monophosphate accelerated the recovery, but to a much lesser extent, and adenosine had no effect. The results of the present study thus suggest that ATP inhibits neuronal activity by enhancing neuronal GABA release via a P 2 receptor, perhaps a P2X receptor, thereby protecting against hypoxic/hypoglycemic perturbation of hippocampal neurotransmission.

Effects of methohexital on bioelectrical reactions in guinea pig hippocampal slices during hypoxia

The barbiturate methohexital (42 and 140 μmol/l) was tested for an acute effect on anoxic depolarization (AD) and evoked potentials (EP) in hippocampal slices of guinea pigs exposed to repeated hypoxic conditions ( n=78). The dosages of methohexital resemble the range of plasma levels measured in patients with an intraoperative burst suppression electroencephalogram. Direct current potential and EP were recorded in the CA1 region. Hypoxia was terminated either when AD had reached its peak, or 2 min after maximum AD. Excluding the actions of methohexital on cerebral blood flow, reperfusion phase and the delayed mechanisms of cellular protection, a dose-dependent direct effect on EP even after repeated hypoxic conditions could be observed.

Cooperativity between extracellular adenosine 5′-triphosphate and activation of N-methyl- D-aspartate receptors in long-term potentiation induction in hippocampal CA1 neurons

The mechanism of ATP-induced long-term potentiation (LTP) was studied pharmacologically using guinea-pig hippocampal slices. LTP, induced in CA1 neurons by 10 min application of 10 μM ATP, was blocked by co-application of the N-methyl- D-aspartate (NMDA) receptor antagonist, D, L-2-amino-5-phosphonovalerate (5 or 50 μM). In ATP-induced LTP, the delivery of test synaptic inputs (once every 20 s) to CA1 neurons could be replaced by co-application of NMDA (100 nM) during ATP perfusion. These results suggest that, in CA1 neurons, a co-operative effect between extracellular ATP and activation of NMDA receptors is required to trigger the process involved in ATP-induced LTP. In addition, ATP-induced LTP was blocked by co-application of an ecto-protein kinase inhibitor, K-252b (40 or 200 nM), whereas a P2X purinoceptor antagonist, pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid 4-sodium (50 μM), or a P2Y purinoceptor antagonist, basilen blue (10 μM), had no effect. The results of the present study, therefore, indicate that the mechanisms of ATP-induced LTP involve the modulation of NMDA receptors/Ca 2+ channels and the phosphorylation of extracellular domains of synaptic membrane proteins, one of which could be the NMDA receptor/Ca 2+ channel.

Glutamatergic modulation of GABAergic signaling among hippocampal interneurons: novel mechanisms regulating hippocampal excitability.

Because interneurons play a central role in regulating the excitability of the hippocampal formation, it is important to understand the mechanisms that modulate gamma-aminobutyric acid (GABA)ergic signaling among them. This study addresses the modulation of GABA release from interneuron terminals by presynaptic glutamate receptors. Whole-cell recordings were obtained from CA1 stratum radiatum interneurons in guinea pig hippocampal slices. Selective agonists and blockers of glutamate receptors were used to study modulation of GABAergic transmission by group III metabotropic receptors or kainate receptors. Antidromic action-potential initiation also was analyzed by stimulating the axons of interneurons. Agonists of group III metabotropic glutamate receptors attenuated monosynaptic GABAergic signals in interneurons, but not in pyramidal neurons, in agreement with anatomic evidence on the distribution of these receptors. Submicromolar kainate enhanced the frequency and amplitude of spontaneous GABAergic signals in interneurons. Kainate also depolarized the axons of hippocampal interneurons, and triggered spontaneous ectopic action potentials in axons. Synaptically released glutamate reproduced many of the effects of both agonists, implying that these receptors can sense the ambient glutamate concentration, and therefore indirectly respond to the excitatory traffic in the hippocampus. When the two classes of receptors were stimulated simultaneously, complex interactions were obtained. Group III metabotropic receptors and kainate receptors profoundly affect GABAergic signaling among interneurons of the hippocampus. Glutamatergic modulation of GABAergic signaling among interneurons represents a novel class of mechanisms that potentially plays a major role in determining the initiation, propagation, and termination of seizures.

Metabotropic Glutamate Receptors and Epileptiform Bursting.

Differential Roles for mGluR1 and mGluR5 in the Persistent Prolongation of Epileptiform Bursts Merlin LR J Neurophysiol 2002;87:621–625 Purpose Transient activation of group I metabotropic glutamate receptors (mGluRs) with the selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) produces persistent prolongation of epileptiform bursts in guinea-pig hippocampal slices, the maintenance of which can be reversibly suppressed with group I mGluR antagonists. To determine the relative roles of mGluR1 and mGluR5 in these group I mGluR-dependent induction and maintenance processes, subtype-selective antagonists were used. In the presence of picrotoxin, DHPG (50 μ M, 20–45 min) converted interictal bursts into 1- to 3-s discharges that persisted for hours after washout of the mGluR agonist. 2-Methyl-6–(phenylethynyl)-pyridine (MPEP, an mGluR5 antagonist; 25 μ M) and (+)-2-methyl-4-carboxy-phenylglycine (LY367385, an mGluR1 antagonist; 20–25 (μ M) each significantly suppressed the ongoing expression of the mGluR-induced prolonged bursts. However, LY367385 was more effective, reducing the burst prolongation by nearly 90%; MPEP produced only a 64% reduction in burst prolongation. Nevertheless, MPEP was more effective at preventing the induction of the burst prolongation; all 10 slices tested failed to express prolonged bursts both during and after coapplication of DHPG with MPEP. Coapplication of DHPG with LY367385, in contrast, resulted in significant burst prolongation (in 68% of slices tested) that was revealed on washout of the two agents. These results suggest that although both receptor subtypes participate in both the induction and maintenance of mGluR-mediated burst prolongation, mGluR1 activation plays a greater role in sustaining the expression of prolonged bursts, whereas mGluR5 activation may be a more critical contributor to the induction process underlying this type of epileptogenesis.

Metabotropic Glutamate Receptors and Epileptiform Bursting

Differential Roles for mGluR1 and mGluR5 in the Persistent Prolongation of Epileptiform BurstsMerlin LRJ Neurophysiol 2002;87:621–625PurposeTransient activation of group I metabotropic glutamate receptors (mGluRs) with the selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) produces persistent prolongation of epileptiform bursts in guinea-pig hippocampal slices, the maintenance of which can be reversibly suppressed with group I mGluR antagonists. To determine the relative roles of mGluR1 and mGluR5 in these group I mGluR-dependent induction and maintenance processes, subtype-selective antagonists were used. In the presence of picrotoxin, DHPG (50 μM, 20–45 min) converted interictal bursts into 1- to 3-s discharges that persisted for hours after washout of the mGluR agonist. 2-Methyl-6–(phenylethynyl)-pyridine (MPEP, an mGluR5 antagonist; 25 μM) and (+)-2-methyl-4-carboxy-phenylglycine (LY367385, an mGluR1 antagonist; 20–25 (μM) each significantly suppressed the ongoing expression of the mGluR-induced ...

Effects of the mono- and tetrasialogangliosides GM1 and GQ1b on ATP-induced long-term potentiation in hippocampal CA1 neurons.

The effects of the mono- and tetrasialogangliosides, GM1 and GQ1b, on ATP-induced long-term potentiation (LTP) were studied in CA1 neurons of guinea pig hippocampal slices. Application of 5 or 10 microM ATP for 10 min resulted in a transient depression followed by a slow augmentation of synaptic transmission, leading to LTP. LTP induced by treatment with 5 microM ATP was facilitated in hippocampal slices prepared from animals treated for 6 days with a ceramide analog, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propranol, which stimulates ganglioside biosynthesis. In addition, LTP induced by 5 microM ATP was significantly enhanced when naive slices were incubated with GQ1b but not with GM1. These results suggest that a cooperative effect between extracellular ATP and GQ1b enhances ATP-induced LTP in hippocampal CA1 neurons. In addition, the LTP induced by 10 microM ATP was blocked by coapplication of the NMDA antagonist AP5 (5 microM or 50 microM), and this effect was partially inhibited by GQ1b pretreatment of the slices, suggesting that in hippocampal CA1 neurons, the enhancing effect of GQ1b on ATP-induced LTP is mediated by modulation of NMDA receptors/Ca(2+) channels.

GABA application to hippocampal CA3 or CA1 stratum lacunosum-moleculare excites an interneuron network.

Whole cell voltage-clamp recording and focal application of the neurotransmitter gamma-aminobutyric acid (GABA) were used to investigate the ability of exogenous GABA applied to different locations within the guinea pig hippocampal slice to trigger a giant GABA-mediated postsynaptic current (GPSC) in pyramidal cells. A GPSC reflects the synchronous release of GABA from a group of interneurons. Recordings were done in the presence of 4-aminopyridine (4-AP) and blockers of ionotropic glutamatergic synaptic transmission. Spontaneous GPSCs occurred rhythmically in pyramidal cells under these conditions. Brief focal pressure application of GABA (500 microM; 30-200 ms) to CA3 stratum lacunosum-moleculare (SLM) or to the border between CA3 s. radiatum (SR) and SLM triggered an "all-or-none" GPSC in CA3 and CA1 pyramidal cells that looked like the spontaneous GPSCs. During the refractory period following a spontaneous GPSC, application of GABA could not trigger a GPSC. Both spontaneous GPSCs and GPSCs triggered by exogenous GABA were blocked by suppressing synaptic transmission with high Mg(2+)/low Ca(2+) bath solution. On the other hand, focal application of GABA to CA3 s. oriens (SO) or to proximal SR did not trigger a GPSC in the CA3 pyramidal cell; instead it produced a graded response. Focal application of GABA to regions other than CA3 was also tested. Focal application of GABA to CA1 SLM always triggered a GPSC in the CA3 pyramidal cell. Focal application of GABA within the outer two-thirds of the dentate molecular layer often elicited a GPSC in the CA3 pyramidal cell. In contrast, focal application of GABA to CA1 SO, to CA1 SR, or to the hilus elicited no current response in the CA3 pyramidal cell. These data indicate that the GPSC recorded in pyramidal cells that was triggered by focal GABA application resulted from the synchronous synaptic release of GABA from activated interneurons rather than from the binding of exogenous GABA to receptors on the pyramidal cell. Furthermore, the "all-or-none" nature of the response to SLM GABA applications of different durations indicates that the exogenous GABA was exciting (directly or indirectly) some members of a network of interneurons, which in turn recruited the rest of the network, rather than individually activating each interneuron that contributed to the GPSC. Interestingly, the effective sites of GABA application--CA3 SLM, CA1 SLM, and the outer two-thirds of the dentate molecular layer--are also the sites which receive direct innervation from the entorhinal cortex in an intact animal.

Synaptic adaptation to repeated hypoglycemia depends on the utilization of monocarboxylates in Guinea pig hippocampal slices.

This report provides in vitro evidence that synaptic activity becomes resistant to repeated hypoglycemia, i.e., hypoglycemic synaptic adaptation occurs. Synaptic function was estimated by the amplitude of the postsynaptic population spike (PS) recorded in the granule cell layer of guinea pig hippocampal slices. ATP, phosphocreatine (PCr), glycogen, and glucose concentrations were measured to investigate energy metabolism homeostasis. Glucose deprivation produced a complete elimination of the PS amplitude, with a 50% inhibition by 10.6 min, and a approximately 15% reduction in ATP and PCr concentrations. Low-glucose (0.5-1 mmol/l) medium gradually depressed the PS. After recovery from glucose depletion, repeated glucose deprivation produced a slowly developing depression of PS, with a 50% inhibition by 36.5 min. However, ATP and PCr concentrations were maintained. Incubation in secondary low-glucose medium maintained PS amplitude. Hippocampal glycogen and glucose concentrations promptly decreased during repeated glucose deprivation, indicating that glycogenolysis does not fuel synaptic adaptation to repeated hypoglycemia. Synaptic function during repeated glucose depletion was reversibly depressed by addition of alpha-cyano-4-hydroxycinnamic acid or 3-isobutyl-1-methylxanthine, inhibitors of the monocarboxylate transporter. Replacement of extracellular glucose with Na-lactate or Na-pyruvate sustained synaptic transmission after transient glucose depletion. These results indicate that synaptic utilization of monocarboxylates sustains hypoglycemic synaptic adaptation.