NMDA-Dependent GABAA-Mediated Polysynaptic Potentials in the Neonatal Rat Hippocampal CA3 Region

Evoked inhibitory postsynaptic potentials (IPSPs) were studied in CA3 hippocampal neurons from brain slice preparations of rats ranging from 5 to 18 days of age (P5–18) using intracellular recording techniques. With KMeSO₄-filled electrodes the evoked inhibitory response consisted of fast and slow IPSPs mediated by GABA_A and GABA_B receptors respectively. In recordings obtained with electrodes filled with 2-(triethylamino)- N -(2,6-dimethylphenyl) acetamide and KMeSO₄, electrical stimulation evoked monophasic IPSPs in mature slices (P10–18) and biphasic IPSPs with an early and a late phase in neonatal slices (P4–7). In neonates both the early and late phases of the IPSP were mediated by GABA_A receptors. Pharmacological investigation revealed that the early phase arose from both direct and feedforward activation of GABAergic interneurons involving non-NMDA receptors, while the late phase resulted from polysynaptic activation of GABAergic interneurons mediated by NMDA receptors.     

Synchronization of GABAergic interneuronal networks during seizure-like activity in the rat horizontal hippocampal slice

We studied the contribution of GABAergic (gamma-aminobutyric acid) neurotransmission to epileptiform activity using the horizontal hippocampal rat brain slice. Seizure-like (ictal) activity was evoked in the CA1 area by applying high-frequency trains (80 Hz for 2 s) to the Schaffer collaterals. Whole-cell recordings from stratum oriens-alveus interneurons revealed burst firing with superimposed high-frequency spiking which was synchronous with field events and pyramidal cell firing during ictal activity. On the other hand, interictal interneuronal bursts were synchronous with large-amplitude inhibitory postsynaptic potentials (IPSPs) in pyramidal cells. Excitatory and inhibitory postsynaptic potentials were simultaneously received by pyramidal neurons during the ictal afterdischarge, and were synchronous with interneuronal bursting and field potential ictal events. The GABAA receptor antagonist bicuculline greatly reduced the duration of the ictal activity in the CA1 layer, and evoked rhythmic interictal synchronous bursting of interneurons and pyramidal cells. With intact GABAergic transmission, interictal field potential events were synchronous with large amplitude IPSPs (9.8 +/- 2.4 mV) in CA1 pyramidal cells, and with interneuronal bursting. Simultaneous dual recordings revealed synchronous IPSPs received by widely separated pyramidal neurons during ictal and interictal periods, indicative of widespread interneuronal firing synchrony throughout the hippocampus. CA3 pyramidal neurons fired in synchrony with interictal field potential events recorded in the CA1 layer, and glutamate receptor antagonists abolished interictal interneuronal firing and synchronous large amplitude IPSPs received by CA1 pyramidal cells. These observations provide evidence that the interneuronal network may be entrained in hyperexcitable states by GABAergic and glutamatergic mechanisms.     

Giant GABAB-mediated Synaptic Potentials Induced by Zinc in the Rat Hippocampus: Paradoxical Effects of Zinc on the GABAB Receptor

The interaction of zinc with pre- and postsynaptic GABA_B receptors was studied in adult rat hippocampal slices using intracellular recording in CA1 and CA3 pyramidal neurons. Zinc (50 – 300 μM) antagonized baclofen responses with a variable potency, whereas CGP-35348 (100 μM) or barium (300 μM) produced a more substantial and consistent inhibition. Zinc also induced giant GABA_A-mediated depolarizing potentials (GDP) in these neurons. After blocking GABA_A and excitatory synaptic transmission, monosynaptic hyperpolarizing inhibitory postsynaptic potentials (IPSP) mediated by GABA_B receptors (IPSP_B) were inhibited by CGP-35348 or barium; however, zinc increased the latency and prolonged the duration of the IPSP_B and also induced the appearance of spontaneous giant GABA_B-mediated hyperpolarizing potentials (GHP). In some cells, IPSP_Bs in zinc exhibited a multiphasic appearance. The early component was partially inhibited by 300 μM zinc and was followed by a late GHP. CGP-35348 at 100 μM inhibited the early monosynaptic IPSP_B but not the GHP; however, at 300 μM both components were blocked. Paired-pulse inhibition of the IPSP_B was used to assess the effect of zinc on presynaptic GABA_B receptors. Neither the zinc-chelating agent CP94 (400 μM) nor zinc affected this phenomenon. CGP-35348, barium and polyvalent cations, such as cadmium, copper, cobalt, manganese, iron and aluminium, failed to induce giant potentials in hippocampal neurons. It is concluded that zinc is apparently unique in synchronizing the release of GABA to produce GDPs and GHPs.     

Functional Characterization and Modulation of Feedback Inhibitory Circuits in Area CA3 of Rat Hippocampal Slice Cultures

Feedback inhibitory circuits were characterized electrophysiologically in the CA3 region of organotypic rat hippocampal cultures. Pyramidal cells were impaled with sharp microelectrodes and brief depolarizing current pulses were injected intracellularly to elicit single action potentials. An inhibitory postsynaptic potential (I PSP) was observed at fixed latency after the action potential in 27% of impaled cells ( n = 131). These IPSPs were fully blocked by bicuculline, indicating that they were mediated solely by γ-aminobutyric acid type A (GABA_A) receptors. They were also blocked by 6-cyano-7-nitro-quinoxaline-2, 3-dione but not d -2-amino-5- phosphonovalerate, indicating that non- N -methyl- d -asparttate receptors were necessary and sufficient for activating interposed GABAergic interneurons. Adenosine (0.1-5 μM) increased the percentage of action potentials that were not followed by IPSPs by reducing the probability of glutamatergic activation of the interneurons. In 18 of 21 experiments adenosine also decreased the mean amplitude of successfully elicited IPSPs, indicating that more than one interneuron participated in the feedback inhibition of those pyramidal cells. In three experiments the non-failure IPSP amplitude was not affected by adenosine, suggesting that only one interneuron participated. Repetitive stimulation at 2-4 Hz decreased the amplitude of non-failure feedback IPSPs and usually increased the number of failures of transmission. These effects were transient and insensitive to the GABA_B antagonist CGP 35 348. We conclude that both the excitation of interneurons and the release of GABA from interneurons are modulated by repetitive stimulation.     

Excitation of rat prefrontal cortical neurons by dopamine: an in vitro electrophysiological study

The effects of dopamine (DA) on prefrontal pyramidal neurons were studied in vitro on rat cerebral cortex slices using intracellular recordings. Pyramidal neurons were first identified by Lucifer yellow and some of their basic bioelectrical properties were analysed. At resting potential, white matter stimulation mainly evoked depolarizing inhibitory postsynaptic potentials (IPSPs) which reversed between -60 and -50 mV and were almost totally abolished by bicuculline. Furthermore, pyramidal cells often exhibited spontaneous depolarizing IPSPs abolished by bicuculline. Under tetrodotoxin (TTX) this synaptic noise was partly blocked suggesting that it was due both to the spontaneous firing of presynaptic gamma-aminobutyric acid (GABA)ergic neurons and to a spontaneous quantal release from these afferent fibers. In pyramidal cells, DA enhanced the number of spikes evoked by depolarizing current pulses, and the input resistance was increased by 10-20%. DA also clearly increased the inhibitory synaptic noise. This effect was blocked by fluphenazine. In contrast, evoked IPSPs were not consistently affected by DA. Taken altogether, these results suggest, that in the prefrontal cortex, dopamine has a mild excitatory effect on both pyramidal cells and GABAergic interneurons impinging on them.     

Activated microglial cells migrate towards sites of excitotoxic neuronal injury inside organotypic hippocampal slice cultures

The aim of this study was to analyse microglial reactions to excitotoxic N ‐methyl‐ d ‐aspartic acid (NMDA)‐induced degeneration of rat dentate and hippocampal neurons in vitro . We used a migration model combining the techniques of microglial single cell culture and organotypic hippocampal slice culture (OHSC). Site‐specific oxidative damage in OHSCs was induced by pretreatment with 50 μ m NMDA. Neuronal injury determined by propidium iodide (PI) uptake included the hippocampal cell layers of the dentate gyrus (DG) and the cornu ammonis (CA). Fluorescence‐prelabelled microglial cells with ameboid morphology were transferred onto the OHSC and migrated predominantly to the prelesioned cell layers of DG and CA when compared with unlesioned areas of the OHSC. In NMDA pretreated slices, microglial cells clustered around degenerating granule cells in the DG and pyramidal cells in the CA. This effect was significantly inhibited in unlesioned slice cultures and in NMDA‐exposed cultures that were pretreated with the NMDA‐antagonist MK‐801. Our observations suggest that microglia – attracted by the presence of stimuli provided by NMDA‐induced neuronal death – migrate specifically towards these lesioned neurons.     

Norepinephrine decreases synaptic inhibition in the rat hippocampus

The effects of norepinephrine (NE) on inhibitory synaptic potentials were studied on CA1 pyramidal neurons in the hippocampal slice in vitro. Norepinephrine caused the appearance of multiple population spikes in the CA1 region of the hippocampal slice, reminiscent of the actions of gamma-aminobutyric acid (GABA) antagonists. Intracellular recording revealed that NE causes a marked and reversible reduction in inhibitory postsynaptic potentials (IPSPs) recorded in CA1 pyramidal cells. This reduced IPSP results in a larger intracellular excitatory postsynaptic potential (EPSP), which can cause the cell to fire more than one action potential. This disinhibitory effect of NE appears to be mediated by an alpha-receptor, and occurs at a site presynaptic to the pyramidal cell, since NE does not change the reversal potential of the IPSP nor does it affect the amplitude or the reversal potential of iontophoretic GABA responses. In addition to reducing evoked IPSPs, NE causes an increase in the frequency of spontaneous IPSPs, suggesting that inhibition of interneuronal firing may not account for this disinhibitory action of NE.     

Dissociation of mu and delta opioid receptor-mediated reductions in evoked and spontaneous synaptic inhibition in the rat hippocampus in vitro.

Modulation of gamma-aminobutyric acid (GABA)-mediated inhibition, and glutamate-mediated excitation by highly selective mu and delta opioid agonists was studied using intracellular recordings of CA1 pyramidal neuron synaptic responses in superfused hippocampal slices. Equimolar concentrations of the selective mu agonist, [Tyr-(D-Ala)-Gly-(N-Me-Phe)-Gly-ol]-enkephalin (DAGO), or the delta selective agonist, [D-Pen2,D-Pen5]-enkephalin (DPDPE), reversibly increased the amplitudes of excitatory post-synaptic potentials (EPSPs), evoked by Schaffer collateral/commissural stimulation, without altering the input resistance or resting membrane potential of these CA1 pyramidal neurons. The increased EPSP amplitudes resulting from superfusion with the enkephalin analogs were qualitatively similar to those caused by the GABAA receptor antagonist, bicuculline methiodide (BMI). Specific stimulation/recording protocols and micro-lesions of the slices were used to evoke relatively pure forms of recurrent and feed-forward GABA-mediated inhibitory post-synaptic potentials (IPSPs). The mu opioid agonist DAGO reduced both recurrent and feed-forward IPSPs, while the delta agonist DPDPE had no effect upon these responses. To test the hypothesis that the enhancement of pyramidal neuron EPSPs by delta (and mu) opioids was due to the reduction of an inhibitory potential that was coincident with the EPSP, DPDPE or the mu agonist, DAGO, were applied while recording monosynaptic IPSPs following the elimination of EPSPs by the glutamate receptor antagonists, D,L-2-amino-5-phosphonovalerate (APV) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). The mu agonist, DAGO, reversibly reduced these pharmacologically isolated IPSPs, while the delta agonist, DPDPE, had no effect upon these responses. Despite the fact that the delta agonist, DPDPE, had no effect on recurrent, feed-forward or monosynaptic evoked IPSPs, this enkephalin did reversibly reduce the frequency of spontaneously occurring IPSPs, measured using whole-cell recordings with pipettes containing 65 mM KCl. The mu agonist, DAGO, and the GABAA antagonist, BMI, similarly reduced spontaneous IPSP rates. We conclude from these data that mu and delta opioid receptor activation increases EPSPs via the reduction of a form of GABAergic inhibition that is difficult to characterize, and which may be distinct from conventional feed-forward and recurrent inhibition. Furthermore, delta opioids seem to reduce this form of GABAergic inhibition selectively, while mu opioids reduced this inhibition, and conventional feed-forward and recurrent IPSPs as well.     

Depression of early and late monosynaptic inhibitory postsynaptic potentials in hippocampal CA1 neurons following prolonged benzodiazepine administration: Role of a reduction in Cl driving force

GABAergic synaptic responses were studied by direct, monosynaptic activation of GABAergic interneurons in the CA1 region of in vitro hippocampal slices from rats made tolerant to the benzodiazepine, flurazepam. Monosynaptic IPSPs were elicited in CA1 pyramidal neurons, following 1 week oral flurazepam administration, by electrical stimulation at the stratum oriens/stratum pyramidale or stratum radiatum/ stratum-lacanosum border ≤ 0.5 mm from the recording electrode plane. Excitatory input to pyramidal cells and interneurons was eliminated by prior superfusion of the glutamate receptor antagonists, APV (50 μM) and DNQX (10 μM). GABA_A receptor-mediated early IPSPs were further isolated by perfusion of the GABA_B antagonist, CGP 35348 (25 μM) or by diffusion of Cs⁺ from the recording electrode. GABA_B receptor-mediated late IPSPs were pharmacologically isolated by perfusion of the GABA_A antagonist, picrotoxin (50 μM). There was a significant decrease in the amplitude of pharmacologically isolated early and late IPSPs in FZP-treated neurons without a change in passive membrane properties. A shift of the early IPSP, but not the late IPSP, reversal potential in FZP-treated neurons suggested that a change in the driving force for anions, presumably Cl, in CA1 neurons was one important factor related to the decreased early IPSP amplitude after prolonged activation of GABA_A receptors by flurazepam. A decreased early IPSP amplitude accompanied by a decreased late IPSP amplitude suggested that presynaptic GABA release onto FZP-treated pyramidal cells may also be reduced. We conclude from these data that an impairment of GABAergic transmission in CA1 pyramidal neurons associated with the development of tolerance during chronic benzodiazepine treatment may be related to the regulation of both pre- and postsynaptic mechanisms at the GABA synapse. Synapse 25:125–136, 1997. © 1997 Wiley-Liss, Inc.     

Reduction of GABAB inhibitory postsynaptic potentials by serotonin via pre- and postsynaptic mechanisms in CA3 pyramidal cells of rat hippocampus in vitro.

The action of serotonin (5-HT) on GABAergic synaptic transmission was investigated with intracellular recordings in CA3 pyramidal cells of rat hippocampal slices. Local application of 5-HT (500 microM) hyperpolarized CA3 pyramidal cells, decreased cellular input resistance, and reduced slow afterhyperpolarizations. Serotonin attenuated the late (GABAB) component of polysynaptic inhibitory postsynaptic potentials (IPSPs; 47% of control) without affecting the early (GABAA) component. During bath application of the excitatory amino acid antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (20 microM) and 2-amino-5-phosphonovalerate (AP-5) (40 microM), 5-HT similarly decreased the amplitude of the late (GABAB) component (17% of control) of monosynaptic IPSPs but did not affect the early (GABAA) component. The mean reversal potentials of poly- and monosynaptic IPSPs were unaffected by 5-HT. The conductance increases associated with the late component of poly- and monosynaptic IPSPs were reduced by 5-HT. Hyperpolarizing responses evoked in CA3 pyramidal cells by somatic applications of gamma-aminobutyric acid (GABA) were unaffected by 5-HT. During bath application of bicuculline (20-50 microM), hyperpolarizing responses elicited by dendritic GABA application were reduced by 5-HT (71% of control). The effect of 5-HT on these direct GABAB hyperpolarizations (29% decrease in response) does not appear sufficient to fully account for the effect of 5-HT on late GABAB IPSPs (53-83% decrease in response). Therefore, 5-HT may reduce GABAB IPSPs in CA3 pyramidal cells 1) by a postsynaptic action on pyramidal cells and 2) by a selective presynaptic action on GABAergic interneurons mediating the GABAB IPSP. This presynaptic action of 5-HT does not appear to involve excitatory afferents onto inhibitory interneurons.     

In Vivo Modulation of Sigma Receptor Sites by Calcitonin Gene-related Peptide in the Mouse and Rat Hippocampal Formation: Radioligand Binding and Electrophysiological Studies

Possible interactions between sigma (σ) receptor sites and calcitonin gene-related peptides (CGRP) were investigated using receptor subtype-related analogues and fragments in in vivo [³H](+)SKF 10 047/σ binding in the hippocampus, and electrophysiological recording of the N -methyl-D-aspartate (NMDA)-induced activation of CA3 pyramidal neurons, two well-established σ assays. In both paradigms, CGRP and the agonist [Cys(ACM)^2,7]hCGRPα modulated σ systems. In vivo binding experiments demonstrated that CGRP and [Cys(ACM)^2,7]hCGRPα inhibited 25–40% of specific [³H](+)SKF 10 047 labelling in the mouse hippocampal formation while the purported antagonist hCGRP_8–37 was inactive. The specificity of this modulation was demonstrated further by the lack of effect of other vasoactive peptides, including the atrial natriuretic peptide, substance P, and its N-terminal fragment, substance P_1–7. In the CA3 subfield of the rat dorsal hippocampus, hCGRPα decreased (up to 61%) the NMDA-induced activation of the pyramidal neurons. Conversely, the linear analogue [Cys(ACM)^2,7]hCGRPα enhanced (by 85%) the NMDA-induced activation of CA3 pyramidal neurons, while the antagonistic fragment hCGRP_8–37 had no effect. Haloperidol, a high-affinity σ receptor ligand, inhibited by 90% the in vivo [3H](+)SKF 10 047 labelling, and prevented the modulation of the NMDA-induced activation by hCGRPα and [Cys(ACM)^2,7]hCGRPα. It thus appears that CGRP can modulate σ-related systems in the hippocampal formation.     

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.     

A subset of local interneurons generate slow inhibitory postsynaptic potentials in hippocampal neurons

Topical application of 4-aminopyridine (4-AP) onto hippocampal slices produced spontaneous repetitive large hyperpolarizing potentials in CA1 neurons. This effect of 4-AP was blocked by a new GABAB receptor antagonist, 2-hydroxy-saclofen. 2-Hydroxy-saclofen also blocked slow IPSPs evoked by stimulation of stratum radiatum. It is suggested that 4-AP-evoked slow hyperpolarizing potentials are in fact slow IPSPs evoked by activation of a selective subset of interneurons which do not produce fast IPSPs.     

Effects of Substance P on Medial Vestibular Nucleus Neurons in Guinea-pig Brainstem Slices

The undecapeptide substance P (SP) has been recently implicated in the control of vestibular function. In particular, it seems to be co-localized with glutamate in approximately half of the primary vestibular afferents in mammals. Using intracellular recordings in guinea-pig brainstem slices, we have investigated the effects of SP and of several agonists of the three known tachykinin receptor subtypes (NK₁, NK₂ and NK₃) on the three main types (A, B and B+LTS) of guinea-pig medial vestibular nucleus neurons (MVNn) that we had previously described. SP could induce two distinct kinds of effects on all types of MVNn. Whereas around half of them were depolarized and had their membrane resistance increased by SP, ∼ 10% of all MVNn were in contrast hyperpolarized and inhibited while their membrane resistance was decreased. Both responses persisted under conditions of blockade of synaptic transmission, and were thus due to the activation of postsynaptic binding sites. The SP-induced membrane depolarization could not be reproduced with any one of the specific agonists of the three tachykinin receptor subtypes, nor was it blocked by the specific NK₁ receptor antagonists GR 82334 and CP 99994. This effect might therefore be due to the activation of a new, pharmacologically distinct, 'NK₁-like' receptor. Only the hyperpolarizing effects, which were in contrast mimicked by the specific NK₁ receptor agonists GR 73632 and [Sar⁹, Met (O₂)¹¹]-SP, would be mediated by the few typical NK₁ receptors which have been demonstrated in the medial vestibular nucleus.     

Effects of phenytoin on pyramidal neurons of the rat hippocampus

The effectsof phenytoin (35 μg/ml) on membrane properties and inhibitory postsynaptic potentials (IPSPs) in CA1 and CA3 pyramidal neurons of the in vitro rat hippocampus were examined. No significant change was observed on input resistance or resting membrane potential. Action potential amplitude, overshoot, rate of rise and rate of decay were decreased. IPSP conductance increase and reversal potential, evoked in CA3 cells through mossy fiber stimulation and in CA1 cells through recurrent and Schaffer's collateral stimulation, were unaffected.     

Dissociation of μ and δ opioid receptor-mediated reductions in evoked and spontaneous synaptic inhibition in the rat hippocampus in vitro

Modulation of γ-aminobutyric acid (GABA)-mediated inhibition, and glutamate-mediated excitation by highly selective μ and δ opioid agonists was studied using intracellular recordings of CA1 pyramidal neuron synaptic responses in superfused hippocampal slices. Equimolar concentrations of the selective μ agonist,

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(DAGO), or the δ selective agonist, [ -Pen², -Pen⁵]-enkephalin (DPDPE), reversibly increased the amplitudes of excitatory post-synaptic potentials (EPSPs), evoked by Schaffer collateral/commissural stimulation, without altering the input resistance or resting membrane potential of these CA1 pyramidal neurons. The increased EPSP amplitudes resulting from superfusion with the enkephalin analogs were qualitatively similar to those caused by the GABA_A receptor antagonist, bicuculline methiodide (BM1). Specific stimulation/recording protocols and micro-lesions of the slices were used to evoke relatively pure forms of recurrent and feed-forward GABA-mediated inhibitory post-synaptic potentials (IPSPs). The μ opioid agonist DAGO reduced both recurrent and feed-forward IPSPs, while the δ agonist DPDPE had no effect upon these responses. To test the hypothesis that the enhancement of pyramidal neuron EPSPs by δ (and μ) opioids was due to the reduction of an inhibitory potential that was coincident with the EPSP, DPDPE or the μ agonist, DAGO, were applied while recording monosynaptic IPSPs following the elimination of EPSPs by the glutamate receptor antagonists, -2-amino-5-phosphonovalerate (APV) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). The μ agonist, DAGO, reversibly reduced these pharmacologically isolated IPSPs, while the δ agonist, DPDPE, had no effect upon these responses. Despite the fact that the δ agonist, DPDPE, had no effect on recurrent, feed-forward or monosynaptic evoked IPSPs, this enkephalin did reversibly reduce the frequency of spontaneously occurring IPSPs, measured using whole-cell recordings with pipettes containing 65 mM KC1. The μ agonist, DAGO, and the GABA_A antagonist. BMI, similarly reduced spontaneous IPSP rates. We conclude from these data that μ and δ opioid receptor activation increases EPSPs via the reduction of a form of GABAergic inhibition that is difficult to characterize, and which may be distinct from conventional feed-forward and recurrent inhibition. Furthermore, δ opioids seem to reduce this form of GABAergic inhibition selectively, while μ opioids reduced this inhibition, and conventional feed-forward and recurrent IPSPs as well.     

Effects of Glutamate Application on the Rhythm of Low Magnesium-induced Epileptiform Activity in Hippocampal Slices of Guinea-pigs

The extracellular concentration of glutamate has previously been reported to increase to more than 10-fold the basal level during seizure activity. In the present study, we tested whether localized increases in extracellular glutamate concentration influence the rhythm of epileptiform discharges in the low-magnesium epilepsy model. In hippocampal slices of guinea-pigs, epileptiform activity was induced by omission of magnesium from the bath fluid. Glutamate and its subreceptor agonists N -methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) were ejected into different strata of the CA3 and CA1 regions using microiontophoretic and micropressure application. Glutamate, NMDA and AMPA applied to the CA3 region, but not to the CA1 region, induced a short-lasting increase in epileptiform discharge frequency, often followed by a transient reduction. The effect was most pronounced with application into the stratum lacunosum-moleculare of the CA3 region and could only be evoked in slices exceeding 400 μm in thickness. The effects on the rhythm of epileptiform discharges induced by NMDA and AMPA were blocked by their specific receptor antagonists. They were not influenced by application of GABA_A and GABA_B receptor antagonists. Changes in somatic membrane potential of CA3 pyramidal neurons did not correlate with changes in the rhythm of epileptiform discharges elicited in this region. The transient suppression of epileptiform discharges that followed the increase in discharge frequency was abolished by an adenosine A₁ receptor antagonist. We propose that localized increases in extracellular glutamate concentration modify the rhythm of epileptiform discharges due to changes in neuronal network activity.     

Lack of feedback inhibition on rat basolateral amygdala following stress or withdrawal from sedative-hypnotic drugs

Previous research has demonstrated that suppression of inhibition in projection neurons of the basolateral complex of the amygdala (BLA) represents an essential mechanism underlying the emergence of negative emotional responses, including exaggerated fear and anxiety. The present work evaluates inhibitory postsynaptic potentials (IPSPs) in pyramidal projection neurons of the BLA in rats subjected to either diazepam or ethanol withdrawal or uncontrollable stress. These are experimental paradigms conducive to a negative emotional state. In slices containing the BLA, IPSPs were studied using whole-cell patch clamp. In control animals, a small IPSP was evoked by sub-threshold stimulation of the external capsule. When an action potential (AP) was evoked by supra-threshold stimuli, IPSPs were considerably larger; these IPSPs were sensitive to blockade of GABA(A) receptors by picrotoxin. However, IPSPs were clearly reduced in diazepam- or ethanol-withdrawn and in stressed rats. Firing of an AP by a depolarizing pulse applied through the patch pipette consistently evoked an inhibitory postsynaptic current (IPSC) in the pyramidal neurons of control animals from all three experimental models; these IPSCs were mediated by GABA(A) receptor activation and were blocked after suppression of glutamatergic transmission. In contrast, no IPSCs were observed in slices from diazepam- or ethanol-withdrawn or stressed animals, although the depolarizing pulse regularly evoked an AP in pyramidal neurons. It is concluded that, in withdrawn or stressed rats, GABAergic disinhibition occurs due to attenuation or suppression of feedback inhibition.     

Effects of BDNF and NT-4/5 on Striatonigral Neuropeptides or Nigral GABA Neurons In Vivo

Supranigral infusions of the TrkB-receptor-preferring neurotrophins BDNF or NT-4/5 augment locomotor behaviours, pars compacta firing rates and striatal dopamine metabolism. However these actions of BDNF or NT-4/5 may involve other neurotransmitter systems in addition to dopamine neurons in the substantia nigra. We thus investigated the effects of 2-week supranigral infusions of BDNF or NT-4/5 on rat peptidergic striatonigral neurons and nigral GABAergic neurons. Radioimmunoassay revealed that BDNF and NT-4/5 elevated substantia nigra levels of substance P (by 46 and 57% respectively) and substance K (by 64 and 81%). In addition, BDNF elevated substance K by 59% in a nigral projection area, the superior colliculus. NT-4/5 elevated dynorphin A in the substantia nigra (by 52%) and met -enkephalin in substantia nigra and globus pallidus (by 89%). None of these neuropeptides were altered in the striatum. Consistent with these findings, supranigral infusions of BDNF elevated the mRNA for preprotachykinin A in striatal neurons. In the same animals, glutamic acid decarboxylase (GAD)₆₇ mRNA was increased by 48% in the substantia nigra. The cross-sectional area of GAD₆₇-positive neuronal somata in the BDNF-infused nigra was increased by 59%, and 70% of nigral GABAergic neurons had a cross-sectional area >550 μm², whereas 95% of the neurons in vehicle-infused animals had cross-sectional areas <550 μm^2. Thus, supranigral infusions of BDNF or NT-4/5 increase tachykinin mRNA and protein levels within striatonigral neurons and increase the size and GAD₆₇ mRNA expression levels of nigral GABAergic neurons. These results suggest that BDNF or NT-4/5 may modify the output of the basal ganglia not only through effects on dopamine neurons but also by increasing neurotransmission in striatonigral peptidergic and nigral GABAergic pathways.     

Inhibition of the bradycardic component of the von Bezold–Jarisch reflex and carotid chemoreceptor reflex by periaqueductal gray stimulation: involvement of medullary receptors

Stimulation of the dorsolateral periaqueductal gray matter (dlPAG) and the B3 cell group inhibits the cardiovagal component of the baroreflex in rats. Our aim was to determine whether the defence reaction induces similar modulatory effects on the cardiac response of the von Bezold–Jarisch reflex and the carotid chemoreceptor reflex. We examined the effects of dlPAG stimulation on the reflex bradycardia triggered by systemic administration of phenylbiguanide or potassium cyanide. Electrical and chemical stimulation of the dlPAG produced marked inhibition of the cardiovagal components of the von Bezold–Jarisch and the carotid chemoreceptor reflexes. In addition, as 5-HT₃, NK₁ and GABA_A receptor activation blocks cardiac reflex responses, we studied whether these receptors were involved in the dlPAG-induced inhibitory effects. We found that, after microinjection of granisetron (a 5-HT₃ receptor antagonist), bicuculline (a GABA_A receptor antagonist) and GR-205171 (an NK₁ receptor antagonist) into the nucleus of the solitary tract (NTS), reflex bradycardic responses were preserved during dlPAG stimulation. Finally, activation of the B3 region also inhibited both reflex bradycardic responses, and these effects were prevented by prior blockade of 5-HT₃ receptors in the NTS. The inhibitory effect of dlPAG stimulation on the cardiac reflex responses was prevented by inhibition of neurons in the medullary B3 region. In conclusion, 5-HT₃, GABA_A and NK₁ receptors in the NTS appear to be involved in the inhibition of the von Bezold–Jarisch reflex and the carotid chemoreceptor reflex bradycardia evoked by activation of neurons in the dlPAG and the raphé magnus.