Excitatory and inhibitory transmission from dorsal root afferents to neonate rat motoneurons in vitro

Intracellular recordings were made from antidromically identified motoneurons in neonate (12-22 days) rat transverse spinal cord slices and the transmitters and receptors probably involved in initiating the excitatory (EPSP) and inhibitory (IPSP) postsynaptic potentials were investigated. Stimulation of dorsal roots elicited in motoneurons an EPSP, an IPSP, or an EPSP followed by an IPSP. EPSPs in 70% of motoneurons had a short latency (less than or equal to 1 ms) and in the remaining cells a latency longer than 1 ms. The IPSPs had a long latency (greater than or equal to 1 ms). Short- and long-latency EPSPs were enhanced by the acidic amino acid uptake inhibitor L-aspartic acid-beta-hydroxamate (AAH) and depressed by the non-selective glutamate receptor antagonists gamma-D-glutamylglycine (DGG) and kynurenic acid. Short-latency EPSPs were suppressed by the quisqualate/kainate (QA/KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) but not by the N-methyl-D-aspartate (NMDA) receptor antagonists D-(-)-2-amino-5-phosphonovaleric acid (APV) and ketamine. Long-latency EPSPs were reduced by DNQX as well as by APV and ketamine. Superfusion of the slices with a Mg-free solution increased the EPSPs and unmasked a late, APV-sensitive component. The IPSP was reduced by the glycine antagonist strychnine as well as by APV and ketamine but resistant to DNQX. The results indicate that stimulation of dorsal roots elicited in motoneurons a monosynaptic EPSP mediated by glutamate/aspartate acting predominantly on the QA/KA subtype of glutamate receptors; an NMDA component can be unveiled in Mg-free solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bicuculline- and Phaclofen-Resistant Hyperpolarizations Evoked by Glutamate Applications to Stratum Lacunosum-Moleculare in CA1 Pyramidal Cells of the Rat Hippocampus In Vitro

In the hippocampus, different types of interneurons may mediate distinct gamma-aminobutyric acid (GABA) responses, i.e. the early and late inhibitory postsynaptic potentials (IPSPs). To verify this hypothesis, intracellular recordings were obtained from CA1 pyramidal cells (n=63) in rat hippocampal slices. Glutamate (1 mM) was locally ejected in stratum lacunosum-moleculare to activate interneurons in this region. Glutamate-evoked hyperpolarizing responses were characterized in pyramidal cells and compared to the early IPSP and the late IPSP elicited by stratum radiatum electrical stimulation. Several characteristics were similar for the glutamate-evoked IPSPs and late IPSPs: their amplitude was small (-3.4 versus -4.9 mV, respectively), each was associated with a small conductance increase (5.0 versus 9.3 nS, respectively), their peak latency was slow (124.4 versus 129.8 ms, respectively) and in the majority of cells, each displayed little response reversal. However, the equilibrium potential of the glutamate IPSP (-76.5 mV) was similar to that of the early IPSP (-73.8 mV). Perfusion with a low Ca2+ (0.5 mM)/high Mg2+ (8 mM) medium or with tetrodotoxin (1 microM), which blocked synaptic transmission, also reduced the glutamate IPSP. Therefore the glutamate IPSP may be mediated indirectly by inhibitory interneurons. The GABAA antagonist bicuculline (10 microM), or picrotoxin (10-20 microM), blocked the early IPSP, but not the glutamate IPSP. The GABAB antagonist phaclofen (1 mM) attenuated the late IPSP, but did not affect the glutamate IPSP. The results of these experiments suggest that glutamate stimulation of interneurons in stratum lacunosum-moleculare evokes a slow IPSP different from the GABA-mediated early and late IPSPs in CA1 pyramidal cells of the hippocampus.     

Lateral hypothalamus and local stimulation induced postsynaptic responses in zona incerta neurons in an in vitro slice preparation of the rat.

Postsynaptic potentials evoked in the zona incerta (ZI) neurons were studied in in vitro slice preparations. Lateral hypothalamus (LH) and local stimulation evoked fast IPSPs, fast EPSPs, and slow EPSPs. The amplitude of the slow EPSPs increased when the neuron was hyperpolarized by a low intensity current injection but was blocked when it was hyperpolarized with a strong current. The slow EPSPs were reversibly suppressed by an application of 50 microM DL-2-amino-5-phosphonovaleric acid (APV) and 20 microM 3-[(+/-)-2-carboxypiperazine-4-yl-]-propyl-1-phosphonic acid (CPP). The slow EPSPs were augmented in Mg-free medium and by train pulse stimulation. Pressure application of NMDA induced a depolarization similar to the slow EPSP. On the other hand, the fast EPSPs showed a conventional voltage dependency and were antagonized by kynurenic acid but not by APV or CPP. The fast IPSPs were completely blocked by 10 microM bicuculline methiodide. The results indicate that LH and local stimulation evoked monosynaptic fast EPSPs and slow EPSPs mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors, respectively. The IPSPs appear to be mediated by GABAA receptors and regulate the expression of NMDA receptor-mediated slow EPSPs.     

Inhibitory circuitry in the ventral cochlear nucleus is probably mediated by glycine

Intracellular recordings from brain slice preparations of the ventral cochlear nuclei (VCN) of mice have shown that both the major cell types, stellate and bushy cells, distinguishable by their responses to intracellularly injected current (Oertel, 1983; Wu and Oertel, 1984), receive late inhibitory as well as early excitatory inputs when the auditory nerve is stimulated electrically. When the extracellular Cl- concentration was lowered or when the intracellular Cl- was raised, the reversal potential of IPSPs became more positive; the reversal potentials were independent of the extracellular K+ concentration. Therefore, IPSPs result from increases in Cl- permeability. To determine whether either or both GABA or glycine might mediate the inhibition, the sensitivity of cells to bath-applied putative neurotransmitters was tested. All cells responded to applications of 0.1-10 mM GABA and glycine with large drops in input resistance; these drops were Cl- dependent. To determine which of these 2 substances was more likely to mediate the IPSPs, antagonists specific to GABA and glycine were tested for their ability to block the IPSPs. All IPSPs were eliminated by 1 microM strychnine, a blocker of glycine-mediated inhibition; they were not consistently blocked by 100 microM bicuculline or by 100 microM picrotoxin, blockers of GABA-mediated inhibition. These results indicate that the inhibition is likely to be mediated by glycine. A simple interpretation of the finding that IPSPs have latencies (1.2-4 msec) at least 2X as long as EPSPs (0.6-0.9 msec) is that cells in the VCN are excited monosynaptically by auditory nerve fibers, and that they are inhibited disynaptically through interneurons within the VCN. To test physiologically whether EPSPs and IPSPs are, respectively, monosynaptic and polysynaptic, 500-700 microM sodium pentobarbital was applied to the preparation. Pentobarbital raised the thresholds of all impaled cells and their synaptic inputs. EPSPs could be evoked in the presence of pentobarbital by raising the stimulus strength, as expected when thresholds are raised in a monosynaptic circuit; even if the thresholds of IPSPs were lower than those of EPSPs in normal saline, they were raised above those of EPSPs in the presence of pentobarbital. The finding that the thresholds of IPSPs are raised more than those of EPSPs supports the interpretation that IPSPs are mediated through a polysynaptic pathway, and this may explain why inhibition has been detected inconsistently in vivo.     

Is glycine an inhibitory transmitter in rat lateral horn cells?

Spontaneous inhibitory postsynaptic potentials (IPSPs) and evoked IPSPs were recorded from a portion of lateral horn cells situated in thin transverse thoracolumbar spinal cord slices removed from neonatal rats. The IPSPs were reduced by hyperpolarization and inverted at membrane potentials between -65 and -75 mV. Strychnine but not bicuculline reversibly eliminated the IPSPs. The hyperpolarizations elicited by exogenously applied glycine exhibited electrophysiological and pharmacological characteristics similar to that of IPSPs. The results are consistent with the suggestion that glycine mediates an IPSP in a population of lateral horn cells.     

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.     

Physiological correlates of responses to gamma-aminobutyric acid (GABA) recorded from rat visual cortical neurons in vitro

Responses to focal application of gamma-aminobutyric acid (GABA) were compared to synaptic potentials elicited by afferent stimulation of rat visual cortical neurons, using a slice preparation and conventional intracellular recording techniques. GABA produced three types of responses: a brief hyperpolarization (mean reversal potential, -72 mV), brief depolarization (mean reversal potential, -50 mV), or a prolonged hyperpolarization (mean reversal potential, -80 mV). Synaptic potentials included simple or complex EPSPs and EPSPs followed by mono- or biphasic IPSPs. A comparison of the characteristics of the GABA responses and synaptic potentials indicated that GABA may mediate both phases of the IPSP in these cells. Our results suggest that despite differences in the circuitry of the visual cortex as opposed to other neocortical and allocortical (hippocampal) areas (Mountcastle and Poggio, 1968; Colonnier and Rossignol, 1969; Creutzfeldt, 1978; Kuhlenbeck, 1978), the inhibitory control of cortical pyramidal and nonpyramidal neurons by GABA is quite similar.     

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.     

Sustained and selective block of IPSPs in brain slices from rats made epileptic by intrahippocampal tetanus toxin.

A small dose of tetanus toxin (2-5 ng; 10 mouse LD50) injected into the rat hippocampus produces a chronic epileptic syndrome in which epileptic discharges recur intermittently for 6-8 weeks. Hippocampal slices prepared during this period and maintained in vitro generate both evoked and spontaneous epileptic discharges. The present study used slices prepared 8-18 days after injection of tetanus toxin or vehicle solution into both hippocampi to test whether or not synaptic inhibition was selectively impaired in this experimental epilepsy. Intracellular recordings were made from CA3 pyramidal layer neurones within the tetanus toxin focus, which was identified by field potential recordings of synchronous bursts evoked by afferent stimulation. The intrinsic properties of these neurones did not differ from comparable cells in control-injected rats. All cells generated excitatory postsynaptic potentials (EPSPs) following stimulation of stratum radiatum in CA3. In control slices EPSPs were followed by a 'fast' inhibitory postsynaptic potential (IPSP), peaking at 25-30 ms, with a mean amplitude (+/- SEM) of -6.7 mV (+/- 0.66). In the epileptic slices these were absent, and the EPSP prolonged so that the potential at 30 ms was a depolarisation of +6.6 mV (+/- 2.75). The slow IPSP at 120 ms dropped to -0.27 mV (+/- 0.18) from -3.97 mV (+/- 1.43) (11 cells in each group). The loss of IPSPs cannot be attributed to a shift in reversal potentials in the toxin-injected group because no IPSPs were unmasked by current injection (n = 11). IPSPs also occurred spontaneously in the neurones in control slices, with a mean amplitude of -1.30 mV. Their frequency decreased by a factor of 13 in cells from the chronic focus induced by tetanus toxin (P less than 0.0001, analysis of variance), but their amplitude did not change significantly (mean of -1.22 mV). Spontaneous EPSPs were significantly more frequent and slightly smaller in the toxin-injected group (mean amplitudes 1.35 and 1.13 mV respectively). Together these studies support the hypothesis that the chronically recurring seizures induced by low doses of tetanus toxin can be attributed to a substantial, persistent and selective reduction of inhibitory neurotransmission in the hippocampus.     

Decreased GABA and increased glutamate receptor-mediated activity on inferior colliculus neurons in vitro are associated with susceptibility to ethanol withdrawal seizures

Cessation of ethanol administration in ethanol-dependent rats results in an ethanol withdrawal (ETX) syndrome, including audiogenic seizures (AGS). The inferior colliculus (IC) is the initiation site for AGS, and membrane properties of IC neurons exhibit hyperexcitability during ETX. Previous studies observed that ETX alters GABA and glutamate neurotransmission in certain brain sites. The present study evaluated synaptic properties and actions of GABA or glutamate antagonists during ETX in IC dorsal cortex (ICd) neurons in brain slices from rats treated with ethanol intragastrically 3 times daily for 4 days. A significant increase of spontaneous action potentials (APs) was observed during ETX. The width, area and rise time of excitatory postsynaptic potentials (EPSPs) evoked by stimulation in the commissure of IC were significantly elevated during ETX. A fast EPSP was sensitive to block by the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and a slow EPSP was sensitive to the NMDA receptor antagonist, 2-amino-5-phosphonovalerate (AP5). However, during ETX the concentration of CNQX or AP5 needed to block these EPSPs was elevated significantly. Inhibitory postsynaptic potentials (IPSPs) in ICd neurons evoked in both normal and ETX rats were blocked by the GABA(A) antagonist, bicuculline. However, IPSPs during ETX displayed a significantly greater sensitivity to bicuculline. These data indicate that decreased GABA(A)-mediated inhibition and increased glutamate-mediated excitability in IC may both be critical mechanisms of AGS initiation during ETX, which is similar to observations in a genetic form of AGS. The common changes in IC neurotransmission in these AGS forms may be general mechanisms subserving AGS and other forms of auditory system pathophysiology in which the IC is implicated.     

Glutamate, kainate and quisqualate enhance GABA-dependent chloride uptake in cortex

Several lines of evidence indicate a possible interaction between the major inhibitory and excitatory cortical neurotransmitters, GABA and glutamate. To assess the neurochemical basis for such an interaction, we examined the effects of glutamate and several analogs on GABA-dependent chloride uptake in a mouse cortical synaptoneurosome preparation. L-Glutamate and the specific receptor subtype ligands kainate and quisqualate led to a small but significant enhancement in chloride uptake in the presence, but not the absence, of the GABA analog muscimol (5 microM). Enhancement was seen at excitatory amino acid (EAA) concentrations of 2-10 microM, but not at higher concentrations. D-Glutamate, NMDA, the NMDA-related antagonists APV and MK801, and the kainate/quisqualate antagonist CNQX, had no effect on chloride uptake. However, CNQX (50 microM) but not APV (50 microM) blocked the increase in chloride uptake due to kainate or quisqualate (10 microM). In addition, depolarization of synaptoneurosomes using high potassium (40 mM KC1) or ouabain pretreatment (5 microM) blocked the effects of kainate and quisqualate. Glutamate, kainate, and quisqualate had no effect on binding at the benzodiazepine, TBPS, or GABA sites on the GABAA receptor complex.     

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.     

Lemniscal Input to Identified Neurons of the Central Nucleus of Mouse Inferior Colliculus: an Intracellular Brain Slice Study

Intracellular recordings were performed in 34 neurons in the central nucleus of the inferior colliculus in brain slice preparations of the mouse. Sixteen neurons recorded were stained intracellularly by injection of biocytin and identified as multipolar. After electrical stimulation of the lateral lemniscus, 32 of 34 neurons exhibited postsynaptic potentials (PSPs). Onset latencies of the PSPs were 5.0±2.8 ms (range 2-12 ms), presumably reflecting the lack of a significant monosynaptic input to most of the neurons recorded. An excitatory PSP (EPSP), often followed by a late inhibitory PSP (IPSP), was present in all neurons which received synaptic input. The IPSPs usually had a reversal potential positive to the cell's resting membrane potential, thus working as shunting inhibitors. Superfusion of the slice with the GABA_A antagonist bicuculline resulted in blockade of the IPSP and pronounced prolongation of the EPSP. In 50% of these cases, paroxysmal depolarizing shifts were observed in the presence of bicuculline. Blocking the non-NMDA glutamate receptors with 6,7-dinitroquinoxaline-2,3-dione resulted not only in the total disappearance of EPSPs but also of late IPSPs, indicating that the latter depend on the glutamatergic EPSPs. Furthermore, all neurons recorded must receive substantial innervation from sources within the inferior colliculus, together constituting a complex neuronal network in the inferior colliculus with an important role of the inhibitory neurotransmitter GABA in controlling network properties.     

Properties of unitary IPSPs evoked by anatomically identified basket cells in the rat hippocampus

Hippocampal pyramidal cells receive GABA-mediated synaptic input from several distinct interneurons. In order to define the effect of perisomatic synapses, intracellular recordings were made with biocytin-containing microelectrodes from synaptically connected inhibitory and pyramidal cell pairs in subfields CA1 and CA3 of the rat hippocampus. Subsequent physiological analysis was restricted to the category of cells, here referred to as basket cells (n= 14), which had an efferent synaptic target profile (n= 282 synaptic contacts) of predominantly somatic (48.2%) and proximal dendritic synapses (45.0%). Electron microscopic analysis revealed that in two instances identified postsynaptic pyramidal cells received a total of 10 and 12 labelled basket cell synapses respectively. At an average membrane potential of -57.8 ± 4.6 mV, unitary inhibitory postsynaptic potentials (IPSPs; n= 24) had a mean amplitude of 450 ± 238 μV, a 10–90% rise time of 4.6 ± 3.2 ms and, measured at half-amplitude, a mean duration of 31.6 ± 18.2 ms. In most instances (n= 19) the IPSP decay could be fitted with a single exponential with a mean time constant of 32.4 ± 18.0 ms. Unitary basket cell-evoked IPSPs fluctuated widely in amplitude, ranging from the level of detectability to <2 mV. The response reversal of IPSPs (n= 5) was extrapolated to be at -74.9 ± 6.0 mV. Averages of unitary IPSPs had a mean calculated conductance of 0.95 ± 0.29 nS, ranging from 0.52 to 1.16 nS. Unitary basket cell IPSPs (n= 3) increased in amplitude by 26.3 ± 19.9% following bath application of the GABA_B receptor antagonist CGP 35845A (1–4 μM), whereas subsequent addition of the GABA^A receptor antagonist bicuculline (10–13 μM) reduced the IPSP amplitude to 13.5 ± 3.1% of the control response. Rapid presynaptic trains of basket cell action potentials resulted in the summation of up to four postsynaptic responses (n= 5). However, any increase in the rate of tonic firing (2- to 10-fold) led to a <50% reduction of the postsynaptic response amplitude. At depolarized membrane potentials, averaged IPSPs could be followed by a distinct depolarizing overshoot or postinhibitory facilitation (n= 4). At firing threshold, pyramidal cells fired postinhibitory rebound-like action potentials, the latter in close temporal overlap with the depolarizing overshoot. In conclusion, hippocampal basket cells have been identified as one source of fast, GABA_A receptor-evoked perisomatic inhibition. Unitary events are mediated by multiple synaptic release sites, thus providing an effective mechanism to avoid total transmission failures.     

Effect of inhibitory amino acid antagonists on IPSPs induced in lumbar motoneurons upon stimulation of the nucleus reticularis gigantocellularis during active sleep

The present study was performed to generate data implicating glycine or γ-aminobutyric acid as neurotransmitter candidates mediating the IPSPs which are recorded in lumbar motoneurons following electrical stimulation of the nucleus reticularis gigantocellularis (NRGc) during the atonia of active sleep. Accordingly, intracellular records were obtained from lumbar motoneurons in unanesthetized, normally respiring cats during naturally occurring states of active sleep, while inhibitory amino acid antagonists were microiontophoretically released next to the recorded cell. Electrical stimuli, applied to the NRGc during active sleep under drug-free conditions, evoked inhibitory postsynaptic potentials (IPSPs) in all of the lumbar motoneurons which were examined. These NRGc-induced IPSPs exhibited an average latency-to-onset of 26.6 ± 1.3 ms, a latency-to-peak of 42.5 ± 1.3 ms, an average amplitude of 3.9 ± 0.4 mV and a duration of 34.4 ± 2.1 ms. Strychnine, when applied microiontophoretically, abolished or markedly suppressed these NRGc-induced IPSPs. In contrast, the microiontophoretic application of picrotoxin or bicuculline methiodide failed to block these IPSPs. To the extent that strychnine may be considered to be a specific antagonist of glycine, the present results suggest that glycine (or a structurally related amino acid) participates in the generation of NRGc-induced IPSPs during the atonia of active sleep.     

GABA mediated excitation in immature rat CA3 hippocampal neurons

Intracellular recordings from rat hippocampal neurons in vitro during the first postnatal week revealed the presence of spontaneous giant depolarizing potentials (GDPs). These were generated by the synchronous discharge of a population of neurons. GDPs reversed polarity at -27 and -51 mV when recorded with KCl or K-methylsulphate filled electrodes, respectively. GDPs were blocked by the GABAA receptor antagonist bicuculline (10 microM). Iontophoretic or bath applications of GABA (10-300 microM) in the presence of tetrodotoxin (1 microM), induced a membrane depolarization or in voltage clamp experiments an inward current which reversed polarity at the same potential as GDPs. The response to GABA was blocked in a non-competitive manner by bicuculline (10 microM) and did not desensitize. GABA mediated GDPs were presynaptically modulated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Their frequency was reduced or blocked by NMDA receptor antagonists and by the rather specific non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The frequency of GDPs was enhanced by glycine and D-serine (10-30 microM) in a strychnine insensitive manner. This effect was blocked by AP-5, suggesting that it was mediated by the allosteric modulatory site of the NMDA receptor. These observations suggest that most of the 'excitatory' drive in immature neurons is mediated by GABA acting on GABAA receptors; furthermore excitatory amino acids modulate the release of GABA by a presynaptic action on GABAergic interneurons.     

Nicotinic acetylcholine receptor-mediated synaptic potentials in rat neocortex

In the neocortex, fast excitatory synaptic transmission can typically be blocked by using excitatory amino acid (EAA) receptor antagonists. In recordings from layer II/III neocortical pyramidal neurons, we observed an evoked excitatory postsynaptic potential (EPSP) or current (EPSC) in the presence of EAA receptor antagonists (40-100 microM D-APV+20 microM CNQX, or 5 mM kynurenic acid) plus the GABA(A)-receptor antagonist bicuculline (BIC, 20 microM). This EAA-antagonist resistant EPSC was observed in about 70% of neurons tested. It had a duration of approximately 20 ms and an amplitude of 61.5+/-6.8 pA at -70 mV (n=35). The EAA-antagonist resistant EPSC current-voltage relation was linear and reversed near 0 mV (n=23). The nonselective nicotinic acetylcholine receptor (nAChR) antagonists dihydro-beta-erythroidine (DH beta E, 100 microM) or mecamylamine (50 microM) reduced EPSC amplitudes by 42 (n=20) and 33% (n=9), respectively. EPSC kinetics were not significantly changed by either antagonist. Bath application of 10 microM neostigmine, a potent acetylcholinesterase inhibitor, prolonged the EPSC decay time. EAA-antagonist resistant EPSCs were observed in the presence of antagonists of metabotropic glutamate, serotonergic (5-HT(3)) and purinergic (P2) receptors. The EAA-antagonist resistant EPSC appears to be due in part to activation of postsynaptic nAChRs. These results suggest the existence of functional synaptic nAChRs on pyramidal neurons in rat neocortex.     

Modulation of bistratified cell IPSPs and basket cell IPSPs by pentobarbitone sodium, diazepam and Zn2+: dual recordings in slices of adult rat hippocampus

Simultaneous intracellular recordings from presynaptic Stratum pyramidale interneurons and postsynaptic pyramidal cells in adult rat hippocampal slices were performed to investigate the strength of the modulation of single-axon inhibitory postsynaptic potentials (IPSPs) by the GABAA receptor modulators pentobarbitone, diazepam and zinc. The processing of biocytin-filled interneurons for light microscopy revealed that these single-axon IPSPs were generated by basket cells (n = 33), bistratified cells (n = 18) and axo-axonic cells (n = 2). The IPSPs generated by these three groups of interneurons had amplitudes and widths at half amplitude with similar ranges, but when bistratified cell IPSPs were compared with basket cell IPSPs with similar half widths their rise times were slower. Pentobarbitone sodium (250 microM) powerfully enhanced 13 tested IPSPs generated by all three cell types. Amplitudes were enhanced by 82 +/- 56%, 10-90% rise times by 150 +/- 101% and the widths at half amplitude by 71 +/- 29%. Diazepam (1-2 microM) also increased all IPSPs tested, although the changes were more moderate in basket cell IPSPs (amplitudes increased by 19 +/- 11%, n = 8) than in bistratified cell IPSPs (amplitudes increased by 66 +/- 48%, n = 5). Basket cell IPSP 10-90% rise times and widths at half amplitude were not significantly increased. Bistratified cell IPSP 10-90% rise times were increased by 44 +/- 24% and the widths at half amplitude by 32 +/- 35%. The one tested IPSP generated by an axo-axonic cell was also diazepam-sensitive. Zinc, 250 microM, decreased four out of 10 IPSPs generated by basket cells and four out of five IPSPs generated by bistratified cells. The one tested axo-axonic cell IPSP was zinc-insensitive. These data suggest that IPSPs generated in CA1 pyramidal cells by basket and bistratified cells display different pharmacologies and may be mediated by different receptors or receptor combinations.     

N-methyl-D-aspartate receptor antagonists reduce synaptic excitation in the hippocampus

The hypothesis that synaptic excitation in the CA1 region of the hippocampus is mediated in part by N-methyl-D-aspartate (NMDA) receptors was tested using intra- and extracellular recording techniques. Synaptic potentials elicited by stratum radiatum stimulation were examined in individual neurons before and after bath application of the NMDA receptor antagonist, DL-2-amino-5-phosphonovalerate (APV). This antagonist reduced both excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs). When IPSPs were suppressed by the addition of picrotoxin, EPSPs were seen in isolation. APV reduced these EPSPs but did not block synaptic transmission. This antagonist demonstrated anticonvulsant actions when tested against picrotoxin-induced epileptiform activity. These results suggest that, as in the spinal cord and neocortex, synaptic excitation in the CA1 region of the hippocampus is partially mediated by APV-sensitive NMDA receptors. The fact that synaptic activity is not blocked by NMDA antagonists indicates that EPSPs in CA1 neurons are not mediated solely by this receptor.