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PubMed2001

Dopamine D(2)-like receptors selectively block N-type Ca(2+) channels to reduce GABA release onto rat striatal cholinergic interneurones.

1. The modulatory roles of dopamine (DA) in inhibitory transmission onto striatal large cholinergic interneurones were investigated in rat brain slices using patch-clamp recording. 2. Pharmacologically isolated GABA(A) receptor-mediated IPSCs were recorded by focal stimulation within the striatum. Bath application of DA reversibly suppressed the amplitude of evoked IPSCs in a concentration-dependent manner (IC(50), 10.0 microM). 3. A D(2)-like receptor agonist, quinpirole (3-30 microM), also suppressed the IPSCs, whereas a D(1)-like receptor agonist, SKF 81297, did not affect IPSCs. Sulpiride, a D(2)-like receptor antagonist, blocked the DA-induced suppression of IPSCs (apparent dissociation constant (K(B)), 0.36 microM), while a D(1)-like receptor antagonist, SCH 23390 (10 microM), had no effect. 4. DA (30 microM) reduced the frequency of spontaneous miniature IPSCs (mIPSCs) without changing their amplitude distribution, suggesting that GABA release was inhibited, whereas the sensitivity of postsynaptic GABA(A) receptors was not affected. The effect of DA on the frequency of mIPSCs was diminished when extracellular Ca(2+) was replaced by Mg(2+) (5 mM), indicating that DA affected the Ca(2+) entry into the presynaptic terminal. 5. An N-type Ca(2+) channel selective blocker, omega-conotoxin GVIA (omega-CgTX, 3 microM), suppressed IPSCs by 65.4 %, whereas a P/Q-type Ca(2+) channel selective blocker, omega-agatoxin IVA (omega-Aga-IVA, 200 nM), suppressed IPSCs by 78.4 %. Simultaneous application of both blockers suppressed IPSCs by 95.9 %. Assuming a 3rd power relationship between Ca(2+) concentration and transmitter release, the contribution of N-, P/Q- and other types of Ca(2+) channels to presynaptic Ca(2+) entry is estimated to be, respectively, 29.8, 40.0 and 34.5 % at this synapse. After the application of omega-CgTX, DA (30 microM) no longer affected IPSCs. In contrast, omega-Aga-IVA did not alter the level of suppression by DA, suggesting that the action of DA was selective for N-type Ca(2+) channels. 6. A G protein alkylating agent, N-ethylmaleimide (NEM), significantly reduced the DA-induced suppression of IPSCs. 7. These results suggest that presynaptic D(2)-like receptors are present on the terminals of GABAergic afferents to striatal cholinergic interneurones, and down-regulate GABA release by selectively blocking N-type Ca(2+) channels through NEM-sensitive G proteins.

Animals↗
PubMed1995

Presynaptic metabotropic glutamate receptors modulate omega-conotoxin-GVIA-insensitive calcium channels in the rat medulla.

We have previously demonstrated that the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1 aminocyclopentane-1,3-dicarboxylate (ACPD) presynaptically inhibits evoked glutamatergic EPSCs and GABAergic IPSCs in patch clamped rat nucleus tractus solitarius (NTS) neurons recorded in this slices. The present study investigated the pharmacology of the presynaptic mGluRs, the the voltage dependent Ca2+ channel (VDCC) subtypes supporting neurotransmitter release, and possible interactions between the two. Monosynaptic EPSCs or IPSCs were evoked by electrical stimulation in the region of the tractus solitarius (TS). The effects of the mGluR agonists ACPD, (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I) and L-2-amino-4-phosphonobutyrate (AP4) were examined upon EPSCs. The effects of the above compounds and quisqualate (QUIS) were examined upon IPSCs. L-CCG-I proved the most potent inhibitor of EPSCs and IPSCs. The VDCC blockers omega-AGA-IVA (AGA), omega-conotoxin GVIA (GVIA), omega-conotoxin MVIIC (MVIIC) and nimodipine (NIM) were assessed for their ability to inhibit monosynaptic EPSCs and IPSCs. EPSCs were inhibited by GVIA >> AGA > or = MVIIC. IPSCs were inhibited by AGA > or = MVIIC >> GVIA. NIM was without effect on the EPSC or IPSC. The potency of mGluR inhibition of evoked synaptic transmission was assessed in the absence and following treatment with VDCC blockers. mGluR agonists blocked a greater percentage of the EPSC or IPSC following treatment with GVIA, but not the other VDCC antagonists, than under control conditions. We have previously demonstrated that the postsynaptic inhibitory effects of mGluR activation upon GABAA mediated currents can be mimicked by cyclic guanosine monophosphate (cGMP) analogs. The cGMP-dependent protein kinase (PKG) inhibitors H8 and Rp-8-4-chlorophenylthio-guanosine-3',5'-cyclic monophosphorothioate (Rp-cG) blocked mGluR inhibition of GABAA mediated currents without blocking the ability of mGluR agonists to inhibit the IPSC. The effect of L-CCGI was enhanced following treatment with GVIA in the presence of Rp-cG, confirming a presynaptic locus of mGluR mediated inhibition of the IPSC. In contrast, cGMP analogues potentiate postsynaptic responses to glutamate agonists but depress the EPSC. As with the mGluR agonists, the inhibition of the EPSC by cGMP was potentiated following treatment with GVIA. These results suggest that presynaptic mGluR reduce both glutamate release from afferent fibers and GABA release from inhibitory interneurons following electrical stimulation in the region of the TS. Although different VDCCs support the majority of glutamate and GABA release and mGluR effects on release appear to utilize differing intracellular pathways, presynaptic GVIA-insensitive VDCCs are favorably targeted for inhibition by mGluR agonists.

Animals↗
PubMed2004

Nootropic agents enhance the recruitment of fast GABAA inhibition in rat neocortex.

It is widely believed that nootropic (cognition-enhancing) agents produce their therapeutic effects by augmenting excitatory synaptic transmission in cortical circuits, primarily through positive modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors (AMPARs). However, GABA-mediated inhibition is also critical for cognition, and enhanced GABA function may be likewise therapeutic for cognitive disorders. Could nootropics act through such a mechanism as well? To address this question, we examined the effects of nootropic agents on excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) recorded from layer V pyramidal cells in acute slices of somatosensory cortex. Aniracetam, a positive modulator of AMPA/kainate receptors, increased the peak amplitude of evoked EPSCs and the amplitude and duration of polysynaptic fast IPSCs, manifested as a greater total charge carried by IPSCs. As a result, the EPSC/IPSC ratio of total charge was decreased, representing a shift in the excitation-inhibition balance that favors inhibition. Aniracetam did not affect the magnitude of either monosynaptic IPSCs (mono-IPSCs) recorded in the presence of excitatory amino acid receptor antagonists, or miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin. However, the duration of both mono-IPSCs and mIPSCs was prolonged, suggesting that aniracetam also directly modulates GABAergic transmission. Cyclothiazide, a preferential modulator of AMPAR function, enhanced the magnitude and duration of polysynaptic IPSCs, similar to aniracetam, but did not affect mono-IPSCs. Concanavalin A, a kainate receptor modulator, had little effect on EPSCs or IPSCs, suggesting there was no contribution from kainate receptor activity. These findings indicate that AMPAR modulators strengthen inhibition in neocortical pyramidal cells, most likely by altering the kinetics of AMPARs on synaptically connected interneurons and possibly by modulating GABA(A) receptor responses in pyramidal cells. This suggests that the therapeutic actions of nootropic agents may be partly mediated through enhanced cortical GABAergic inhibition, and not solely through the direct modification of excitation, as previously thought.

Animals↗
PubMed1993

Inhibitory synaptic currents in stellate cells of rat cerebellar slices.

1. In thin cerebellar slices of rats aged 14-21 days, voltage-gated currents, synaptic currents and GABA responses were studied with the tight-seal whole-cell recording technique from stellate cells (8-9 micrograms soma diameter) located in the outer two-thirds of the molecular layer. 2. In symmetrical Cl- conditions, stellate cells voltage-clamped at -60 mV showed spontaneous inhibitory postsynaptic currents (IPSCs). As were the GABAA responses of the same cells, the IPSCs were blocked by bicuculline. The frequency of occurrence of IPSCs ranged from 0.2 to 1.9 events per second (21 cells). The mean amplitude of the events ranged from 61 to 226 pA (mean +/- S.E.M.: 132 +/- 11; n = 21). 3. The temporal course of IPSCs was characterized by a rapid rise (mean +/- S.E.M. of the time to peak: 1.1 +/- 0.1 ms, n = 7) and a slow decay. The decay phase was described by a double exponential function with time constants of 8.7 +/- 0.6 ms, and 40.9 +/- 3.7 ms respectively (means +/- S.E.M.; n = 7). 4. A minor fraction (15 to 20%) of the spontaneous synaptic events recorded in control saline had a faster onset than that of the IPSCs and decayed with a rapid mono-exponential decay (time constant of 1.0-1.3 ms). These were excitatory postsynaptic currents (EPSCs) unaffected by bicuculline and blocked by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). 5. Bath application of TTX (0.5-1 microM), which blocked voltage-gated Na+ currents in stellate cells, induced a variable decrease in the frequency of IPSCs (mean +/- S.E.M. of the frequency ratio in TTX over control: 0.47 +/- 0.09; n = 12). However, the toxin had no significant effect either on the mean amplitude or on the kinetics of the IPSCs. The mean amplitude of the miniature IPSCs was 141 +/- 13 pA (mean +/- S.E.M.; n = 22). 6. In TTX-containing solutions, the frequency of the IPSCs was unaffected when Ca2+ currents were eliminated either by removal of extracellular Ca2+ and addition of EGTA, or by addition of Cd2+. Miniature IPSCs of 200-300 pA were still observed. 7. In symmetrical Cl- conditions, local application of GABA to stellate cells induced an inward current and an increase in membrane noise. Responses to prolonged applications of GABA showed desensitization in both whole-cell mode and somatic outside-out patches. The chord conductance estimated from recording single GABA channel events in somatic outside-out patches was 28 pS.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1993

Discrimination of post- and presynaptic GABAB receptor-mediated responses by tetrahydroaminoacridine in area CA3 of the rat hippocampus.

1. The effects of the K+ channel blocker 9-amino-1,2,3,4-tetrahydroacridine (THA) on the actions of baclofen and gamma-aminobutyric acid (GABA) at post- and presynaptic GABAB receptors were studied with whole-cell voltage-clamp recording in area CA3 of rat hippocampal slices. 2. The effect of THA on postsynaptic GABAB receptor-mediated responses was studied in neurons perfused internally with potassium gluconate and guanosine triphosphate (GTP). At a holding potential of -70 mV, the GABAB receptor agonist (+/-)-baclofen (30 microM) induced an outward current and increased membrane conductance. In the presence of the excitatory amino acid receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and (+/-)-2-amino-5-phosphonovalerate (APV), stimulation in stratum pyramidale or proximal stratum radiatum evoked GABAA receptor-mediated, fast monosynaptic inhibitory postsynaptic currents (IPSCs) and GABAB receptor-mediated, late monosynaptic IPSCs. THA (0.3 mM) blocked the baclofen-induced current and conductance increase and GABAB receptor-mediated IPSCs. 3. The effect of THA on presynaptic GABAB receptor-mediated responses was studied in neurons perfused internally with Cs+ and lidocaine N-ethyl bromide (QX-314), which blocked post-synaptic GABAB receptor-mediated responses. Stimulation in the presence of DNQX and APV evoked GABAA receptor-mediated IPSCs; when pairs of stimuli were delivered 200 ms apart the second IPSC was depressed. Baclofen reversibly depressed IPSCs, and partially occluded paired-pulse depression of IPSCs. The GABAB receptor antagonist CGP 35348 (0.5-1.0 mM) reversed baclofen-induced depression of IPSCs and partially blocked paired-pulse depression. Baclofen-induced and paired-pulse depression of IPSCs were not by affected by THA (0.3 mM). 4. Baclofen reversibly decreased the amplitude and frequency of spontaneous monosynaptic IPSCs (sIPSCs). Depression of sIPSCs by baclofen was unchanged by THA. 5. These results indicate that THA blocks the actions of baclofen and GABA at post- but not presynaptic GABAB receptors. We conclude that post- and presynaptic GABAB receptors in area CA3 of the rat hippocampus couple to different effector mechanisms; postsynaptic GABAB receptors activate THA-sensitive K+ channels, and presynaptic GABAB receptors decrease neurotransmitter release through a THA-insensitive mechanism.

2-Amino-5-phosphonovalerate↗
PubMed1994

Modulation of high-threshold transmission between heart interneurons of the medicinal leech by FMRF-NH2.

1. We examined high-threshold synaptic transmission between oscillatory pairs of leech heart interneurons. Inhibitory postsynaptic currents (IPSCs) could be reliably evoked by depolarizing the presynaptic neuron in voltage clamp from a holding potential of -35 mV. At this presynaptic potential, the Ca2+ currents underlying graded transmission are completely inactivated, and we conclude that a high-threshold Ca2+ current is extant in heart interneurons. Further evidence for this was that inhibitory postsynaptic currents were blocked when Co2+ replaced Ca2+ in the saline and thus high-threshold transmission was dependent on the presence of external Ca2+. 2. When IPSCs were evoked by a 200-ms duration voltage step from a holding potential of -35 mV in the presynaptic neuron, the time course of turn-on of the IPSC consisted of a fast (time-to-peak = 17.5 +/- 1.93 (SE) ms [n = 7]) and a slow (time-to-peak = 250 +/- 28.5 ms [n = 8]) component. FMRF-NH2 reduced the amplitude of the fast component but did not affect the slow component. When the presynaptic voltage step was ended the IPSC turned off with a single exponential time course. FMRF-NH2 slowed the time course of turn-off of the IPSC. 3. When IPSCs were evoked by a 1500-ms duration voltage step from a holding potential of -35 mV in the presynaptic neuron, these IPSCs peaked around 300 ms. Following the peak, the IPSC decayed with a single exponential time course. FMRF-NH2 accelerated the time course of this decay. At potentials of 0 mV and +5 mV, FMRF-NH2 produced a significant decrease in the peak current and at potentials of -5 mV and 0 mV, produced a significant decrease in the current integral. 4. High-threshold IPSCs could also be evoked by a spike in the presynaptic neuron. Bath application of 1 microM FMRF-NH2 decreased the amplitude of the spike-evoked IPSC and slowed the time course of its falling phase. 5. We examined the effect of FMRF-NH2 on the quantal synaptic transmission. Bath-application of FMRF-NH2 increased binomial p, the probability of release, and decreased binomial n, the number of units available for release. FMRF-NH2 had no effect on q, the unit size, when calculated from the distributions of PSPs, and increased the coefficient of variation (CV). 6. The lack of a change in q and the increase in CV suggested that FMRF-NH2 acted at a presynaptic location.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

GABA-mediated synaptic transmission in neuroendocrine cells: a patch-clamp study in a pituitary slice preparation.

Patch-clamp recording techniques were applied to thin slices of the rat pituitary gland in order to study synaptic transmission between hypothalamic nerve terminals and neuroendocrine cells of the intermediate lobe. Inhibitory postsynaptic currents (IPSCs) could be evoked by electrical stimulation of afferent neuronal fibres in the surrounding tissue of the slice. The IPSCs could be evoked in an all-or-nothing mode depending on the stimulus intensity, suggesting that single afferent fibres were stimulated. They had a chloride-dependent reversal potential and were blocked by bicuculline (Kd = 0.1 microM), indicating that they were mediated by gamma-aminobutyric acid A (GABAA) receptors. In symmetrical chloride solutions the current/voltage relation of the IPSC peak amplitudes was linear. The IPSCs were characterized by a fast (1-2 ms) rise time and a biexponential decay, with time constants of 21 +/- 4 ms and 58 +/- 14 ms at a holding potential of -60 mV (n = 6 cells). Both decay time constants increased with depolarization in an exponential manner. Spontaneously occurring IPSCs had a time course that was similar to that of evoked IPSCs. These miniature IPSCs, recorded in 1 microM tetrodotoxin, displayed an amplitude distribution that was well fitted by single Gaussian functions, with a mean value of its maxima of 18.1 +/- 2.3 pA (n = 4 cells). Amplitude histograms of evoked IPSCs were characterized by multiple peaks with a modal amplitude of about 18 pA (n = 6 cells). These findings indicate the quantal nature of GABAergic synaptic transmission in this system, with a quantal conductance step of about 280 pS. Single-channel currents underlying the IPSCs were studied by bath application of GABA to outside-out patches excised from intermediate lobe cells. Such GABA-induced currents revealed two conductance levels of 14 pS and 26 pS. In conclusion, GABAergic synaptic transmission in neuroendocrine cells of the pituitary has properties that are quite similar to those observed in neurones of the central nervous system.

Animals↗
PubMed2004

Depression of GABAergic input to identified hippocampal neurons by group III metabotropic glutamate receptors in the rat.

The release of GABA in synapses is modulated by presynaptic metabotropic glutamate receptors (mGluRs). We tested whether GABA release to identified hippocampal neurons is influenced by group III mGluR activation using the agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) on inhibitory postsynaptic currents (IPSCs) evoked in CA1 interneurons and pyramidal cells. In interneurons, characterized with biocytin and immunolabelling for somatostatin, evoked IPSCs were depressed by 50 micro m L-AP4 (activating mGluR4 and 8) to 68 +/- 6% of control, but they were rarely depressed in pyramidal cells (96 +/- 4% of control). At 300-500 micro m concentration (activating mGluR4, 7 and 8), L-AP4 depressed IPSCs in both interneurons (to 70 +/- 6%) and pyramidal cells (to 67 +/- 4%). The change in trial-to-trial variability and in paired-pulse depression indicated a presynaptic action. In interneurons, the degree of IPSC depression was variable (to 9-87%), and a third of IPSCs were not affected by L-AP4. The L-AP4-evoked IPSC depression was blocked by LY341495. The depression of IPSCs was similar in O-LM cells and other interneurons. The lack of cell-type selectivity and the similar efficacy of different concentrations of L-AP4 suggest that several group III mGluRs are involved in the depression of IPSCs. Electron microscopic immunocytochemistry confirmed that mGluR4, mGluR7a and mGluR8a occur in the presynaptic active zone of GABAergic terminals on interneurons, but not on those innervating pyramidal cells. The high variability of L-AP4-evoked IPSC suppression is in line with the selective expression of presynaptic mGluRs by several distinct types of GABAergic neuron innervating each interneuron type.

2-Amino-5-phosphonovalerate↗
PubMed2000

Presynaptic dopamine D2 and muscarine M3 receptors inhibit excitatory and inhibitory transmission to rat subthalamic neurones in vitro.

Whole-cell patch-clamp recordings were made from subthalamic nucleus (STN) neurones in brain slices from rats. Stimulation with bipolar electrodes evoked synaptic currents mediated by glutamate (EPSCs) and GABAA (IPSCs) receptors. Dopamine reversibly reduced the amplitude of GABAA IPSCs by up to 48 % with an IC50 value of 3.4 +/- 0.8 microM. The dopamine D2 receptor agonist quinpirole, but not the D1 receptor agonist SKF 82958, also inhibited GABAA IPSCs. This effect was completely reversed by the D2 receptor antagonist sulpiride but not by SCH 23390, a D1 antagonist. Muscarine reversibly reduced the amplitude of GABAA IPSCs by up to 70 % with an IC50 value of 0.6 +/- 0.1 microM. Inhibition of IPSCs by muscarine was completely blocked by scopolamine (10 microM), a muscarinic receptor antagonist. The M3 muscarinic receptor antagonist 4-DAMP effectively reversed muscarine-induced inhibition of IPSCs with an IC50 of 0.11 +/- 0.03 microM. Although the M1 receptor antagonist pirenzepine also reversed the inhibition of IPSCs by muscarine, this effect was only observed at relatively high concentrations (IC50 = 21.7 +/- 9.4 microM). Dopamine and muscarine both increased the paired-pulse ratio of GABAA IPSCs. Neither agent produced sustained changes in postsynaptic holding current. Glutamate EPSCs were also inhibited reversibly by dopamine (by up to 29%; IC50 = 16 +/- 3 microM) and muscarine (by up to 41%; IC50 = 1.0 +/- 0.4 microM). However, both agents were more potent and efficacious for reducing GABA IPSCs compared with glutamate EPSCs. These results suggest that the most significant effect of dopamine and muscarine in the STN is to reduce inhibitory synaptic input by acting at presynaptic dopamine D2 and muscarinic M3 receptors, respectively.

Animals↗
PubMed1988

Outward rectification of inhibitory postsynaptic currents in cultured rat hippocampal neurones.

1. Inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) were recorded from cultured hippocampal neurones of the embryonic rat at 22 degrees C, using the whole-cell patch-clamp technique with a low-Cl-, 145 mM-potassium gluconate solution in the patch pipette. Individual synaptic events were elicited at low frequency (0.05-0.1 Hz) by stimulating a presynaptic neurone either by direct intracellular current injection, or by applying a brief pulse of L-glutamate. 2. In target neurones voltage clamped at -40 mV, outwardly directed IPSCs of mean amplitude 0.23 nA were recorded. The IPSCs were depressed by the GABA antagonist bicuculline, and reversed polarity between -50 and -80 mV (mean -64 mV), as did current responses to gamma-aminobutyric acid. The IPSPs and IPSCs reversed as a single phase; no bicuculline-resistant 'late' synaptic event was observed. 3. The IPSCs had variable kinetics, with rise times between 1 and 5 ms (mean 2.9 ms) at -40 mV, and slower, monoexponential, decay phases (decay time constant, tau IPSC, 10-40 ms at -40 mV). In some cells, tau IPSC clearly increased with depolarization. 4. The IPSC reversal potential was -64 +/- 9 mV (n = 23) under the experimental conditions used; this suggests that the synaptically activated channels are approximately 25 times more permeable to Cl- than to the gluconate anion. 5. The peak conductance associated with the IPSC showed outward rectification. The synaptic conductance measured at -40 mV was 1.7 times greater than that measured at -100 mV; at -20 mV, synaptic conductance was 2.5 times greater than at -100 mV. This outward rectification can be explained by a constant field model under these experimental conditions of asymmetric Cl- concentrations.

Action Potentials↗
PubMed1996

Postsynaptic mechanism of depression of GABAergic synapses by oxytocin in the supraoptic nucleus of immature rat.

1. Oxytocin is known to act on autoreceptors of oxytocin neurones in the supraoptic nucleus (SON). We investigated whether oxytocin modulates putative oxytocin neurones by suppressing the GABAA receptor-mediated synaptic inputs on these cells. 2. GABAergic inhibitory postsynaptic currents (IPSCs) were recorded from SON neurones in hypothalamic slices from young rats. Oxytocin specifically reduced the amplitude of both spontaneous and evoked IPSCs, without altering their current kinetics. 3. The effect of oxytocin was observed in 70% of the magnocellular neurones recorded from the dorsomedial part of the SON. d(CH2)5OVT, a specific antagonist of oxytocin receptors, blocked the effect of oxytocin on the IPSCs. Vasopressin had no effect on oxytocin-sensitive SON neurones. 4. The intervals between spontaneous IPSCs were not affected by oxytocin. This suggested that oxytocin had a postsynaptic effect on SON neurones. 5. This postsynaptic origin was further substantiated by application of TTX, which blocked all evoked release but did not prevent the suppressive effect of oxytocin on the amplitude of the spontaneous IPSCs still present in the recording. The selective effect of oxytocin on IPSC amplitude was also maintained in nominally zero extracellular calcium. 6. Intracellular perfusion of SON neurones with GTP gamma S mimicked the effect of oxytocin on IPSCs, while GDP beta S, similarly applied, abolished the effect of oxytocin. 7. Application of calcium mobilizers such as thapsigargin and caffeine also reduced the amplitude of spontaneous IPSCs without significantly altering the frequency at which IPSCs occurred. 8. Thus, oxytocin depresses GABAergic synapses in the SON via modulation of the postsynaptic GABAA receptors. This would lead to disinhibition of SON neurones sensitive to oxytocin and could, therefore, be a powerful means of controlling the firing of oxytocin neurones.

Age Factors↗
PubMed2003

Frequency-dependent properties of inhibitory synapses in the rostral nucleus of the solitary tract.

To explore the parameters that define the characteristics of either inhibitory postsynaptic potentials (IPSP) or currents (IPSC) in the gustatory nucleus of the solitary tract (rNST), whole cell patch-clamp recordings were made in horizontal brain stem slices of newborn rats. Neurons were labeled with biocytin to confirm both their location and morphology. IPSPs or IPSCs were evoked by delivering either single, paired-pulse, or tetanic stimulus shocks (0.1-ms duration) via a bipolar stimulating electrode placed on the rNST. Pure IPSP/IPSCs were isolated by the use of glutamate receptor antagonists. For 83% of the single-stimulus-evoked IPSCs, the decay time course was fitted with two exponentials having average time constants of 38 and 181 ms, respectively, while the remainder could be fitted with one exponential of 59 ms. Paired-pulse stimulation resulted in summation of the amplitude of the conditioning and test-stimulus-evoked IPSCs. The decay time course of the test-stimulus-evoked IPSC was slower when compared to the decay time of the conditioning stimulus IPSC. Repeated stimulation resulted in an increase in the decay time of the IPSP/Cs where each consecutive stimulus contributed to prolongation of the decay time constant. Most of the IPSP/Cs resulting from a 1-s >/= 30-Hz tetanic stimulus exhibited an S-shaped decay time course where the amplitude of the IPSP/Cs after termination of the stimulus was initially sustained before starting to decay back to the resting membrane potential. Elevation of extracellular Ca(2+) concentration 10 mM resulted in an increase in the amplitude and decay time of single-stimulus shock-evoked IPSP/Cs. The benzodiazepine GABA(A) receptor modulator diazepam increased the decay time of single-stimulus shock-evoked IPSCs. However, application of diazepam did not affect the decay time of tetanic-stimulation-evoked IPSP/Cs. These results suggest that the decay time of single-stimulus-evoked IPSCs is defined either by receptor kinetics or neurotransmitter clearance from the synaptic cleft or both, while the decay time course of the tetanic stimulus evoked IPSP/Cs is defined by neurotransmitter diffusion from the synaptic cleft. During repetitive stimulation, neurotransmitter accumulates in the synaptic cleft prolonging the decay time constant of the IPSCs. High-frequency stimulation elevates the GABA concentration in the synaptic cleft, which then oversaturates the postsynaptic receptors, and, as a consequence, after termination of the tetanic stimulus, the amplitude of IPSP/Cs is sustained resulting in an S shaped decay time course. This activity-dependent plasticity at GABAergic synapses in the rNST is potentially important in the encoding of taste responses because the dynamic range of stimulus frequencies that result in synaptic plasticity (0-70 Hz) corresponds to the breadth of frequencies that travels via afferent gustatory nerve fibers in response to taste stimuli.

Animals↗
PubMed1989

Activity-dependent disinhibition. III. Desensitization and GABAB receptor-mediated presynaptic inhibition in the hippocampus in vitro.

1. Single-electrode voltage-clamp recordings were made from CA3 pyramidal cells in organotypic hippocampal slice cultures for measurement of membrane currents underlying both the gamma-aminobutyric acid (GABA)-mediated, Cl- -dependent inhibitory postsynaptic potential (IPSC), evoked in response to stimulation of the mossy fiber pathway, and responses to iontophoretically applied GABA. Pre- and postsynaptic mechanisms mediating activity-dependent reductions in the conductance underlying the IPSC (gIPSC) were investigated. 2. During 99-s applications of GABA, the mean evoked conductance (gGABA) decreased 43% with an initial time constant of 51 s. Desensitization was never complete. 3. Ca2+-influx, activated with depolarizing voltage commands of 100-ms to 15-s duration in the presence of intracellular Cs+, had no effect on GABA responses. 4. Iontophoretic application of the GABAA-receptor agonist muscimol caused a rapid decrease of 80-100% in the amplitude of IPSCs evoked at depolarized membrane potentials (Vm). Recovery was 80% complete in 30 s. The second of two paired applications of muscimol, delivered at the same iontophoretic intensity, was reduced in amplitude 35%. This was shown to result from a decrease in driving force rather than from desensitization. We conclude that muscimol decreases IPSCs by causing an increase in the intracellular Cl- concentration. 5. Iontophoretic application of the GABAB-receptor agonist (+/-)-baclofen caused a decrease of only 30% in the amplitude of IPSCs evoked at depolarized Vms. This effect outlasted the post-synaptic effects of baclofen; recovery was 80% complete between 60 and 90 s. 6. Bath application of (-)-baclofen was found to decrease gIPSC without affecting the IPSC reversal potential. This effect was rapid in onset, could be observed at concentrations as low as 1 X 10(-7) M, and recovered quickly. The EC50 was roughly 5 X 10(-7) M and appeared similar to that for the baclofen-activated increase in postsynaptic conductance. No effect on responses to iontophoretically applied GABA was observed, demonstrating that baclofen decreases gIPSC by reducing presynaptic release via GABAB receptors. 7. Iontophoretic application of GABA reduced IPSCs in a dose-dependent manner. At low iontophoretic intensities, IPSCs were reduced only 30% and recovered slowly, as with baclofen iontophoresis. At higher iontophoretic intensities, IPSCs were more completely blocked. Recovery was initially fast, but took 60-90 s to be complete.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Mechanism of early anoxia-induced suppression of the GABAA-mediated inhibitory postsynaptic current.

1. We investigated the mechanism of hypoxia-induced depression of gamma-aminobutyric acid-A (GABAA)-mediated inhibitory postsynaptic currents (IPSCs) in CA1 neurons of hippocampal slices from 21- to 28-day-old rats. Cells were examined by whole-cell patch-clamp recording and hypoxia was induced by switching perfusion of the slice from oxygenated artificial cerebral spinal fluid (ACSF) to ACSF saturated with 95% N2-5% CO2. 2. Synaptic responses evoked by stimulation of the Schaffer collateral-commissural projection at a fixed holding potential (VH = -60 mV) during anoxia revealed that the IPSC appeared more sensitive than the excitatory postsynaptic current to anoxia-induced depression. All subsequent studies examined the GABAA-mediated IPSC synaptic responses in isolation by direct stimulation of GABA interneurons in the stratum radiatum in the presence of extracellular 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP) (20 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (50 microM) to block glutamatergic currents and intracellular QX-314 (lidocaine N-ethyl bromide, 1 mM) to block GABAB-mediated currents. When studied in this manner (VH = -60 mV) the GABAA-mediated IPSC appeared to change from an outward to inward current after exposure to anoxia. 3. The current-voltage relationship of GABAA-mediated IPSCs revealed that these changes resulted from a positive shift in the IPSC reversal potential without a significant change in the conductance. Thus under patch clamp apparent IPSC inhibition may result from a decrease in the extracellular concentration of chloride ions. Similar findings were observed with micropipettes that contained high intracellular chloride concentrations. 4. Miniature spontaneous IPSCs were examined in the presence of tetrodotoxin (1 microM) with micropipettes containing high intracellular chloride concentrations. The miniature IPSCs (mIPSCs) appeared as spontaneous transient inward currents. Consistent with an anoxia-induced decrease in extracellular chloride, the mean amplitude of the mIPSCs increased after the onset of anoxia. A significant decrease in rise and decay time was also noted during anoxia. The frequency of the mIPSCs also increased by approximately 300%. 5. The resting input resistance of the cells was examined by measuring the current resulting from a 20-mV hyperpolarizing pulse. A significant reduction in resistance was observed 2 min after the onset of anoxia. This still occurred, although to a lesser degree, in the presence of glutamatergic blockers (20 microM CPP plus 50 microM CNQX). In the presence of both GABAergic (picrotoxin, 100 microM) and glutamatergic blockers no significant reduction in resting input resistance was apparent after 2 min of anoxia.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1999

GABAB-Receptor-mediated currents in interneurons of the dentate-hilus border.

GABA(B)-receptor-mediated inhibition was investigated in anatomically identified inhibitory interneurons located at the border between the dentate gyrus granule cell layer and hilus. Biocytin staining was used to visualize the morphology of recorded cells. A molecular layer stimulus evoked a pharmacologically isolated slow inhibitory postsynaptic current (IPSC), recorded with whole cell patch-clamp techniques, in 55 of 63 interneurons. Application of the GABA(B) receptor antagonists, CGP 35348 (400 microM) or CGP 55845 (1 microM) to a subset of 25 interneurons suppressed the slow IPSC by an amount ranging from 10 to 100%. In 56% of these cells, the slow IPSC was entirely GABA(B)-receptor-mediated. However, in the remaining interneurons, a component of the slow IPSC was resistant to GABA(B) antagonists. Subtraction of this antagonist resistant current from the slow IPSC isolated the GABA(B) component (IPSC(B)). This IPSC(B) had a similar onset and peak latency to that recorded from granule cells but a significantly shorter duration. The GABA(B) agonist, baclofen (10 microM), produced a CGP 55845-sensitive outward current in 19 of 27 interneurons. In the eight cells that lacked a baclofen current, strong or repetitive ML stimulation also failed to evoke an IPSC(B), indicating that these cells lacked functional GABA(B) receptor-activated potassium currents. In cells that expressed a baclofen current, the amplitude of this current was approximately 50% smaller in interneurons with axons that projected into the granule cell dendritic layer (22.2 +/- 5.3 pA; mean +/- SE) than in interneurons with axons that projected into or near the granule cell body layer (46.1 +/- 10.0 pA). Similarly, the IPSC(B) amplitude was smaller in interneurons projecting to dendritic (9.4 +/- 2.7 pA) than perisomatic regions (34.3 +/- 5.1 pA). These findings suggest that GABA(B) inhibition more strongly regulates interneurons with axons that project into perisomatic than dendritic regions. To determine the functional role of GABA(B) inhibition, we examined the effect of IPSP(B) on action potential firing and synaptic excitation of these interneurons. IPSP(B) and IPSP(A) both suppressed depolarization-induced neuronal firing. However, unlike IPSP(A), suppression of firing by IPSP(B) could be easily overcome with strong depolarization. IPSP(B) markedly suppressed N-methyl-D-aspartate but not AMPA EPSPs, suggesting that GABA(B) inhibition may play a role in regulating slow synaptic excitation of these interneurons. Heterogeneous expression of GABA(B) currents in hilar border interneurons therefore may provide a mechanism for the differential regulation of excitation of these cells and thereby exert an important role in shaping neuronal activity in the dentate gyrus.

Animals↗
PubMed2000

Mechanisms underlying LTP of inhibitory synaptic transmission in the deep cerebellar nuclei.

Whole-cell recordings were used to investigate long-term potentiation of inhibitory synaptic currents (IPSCs) in neurons of deep cerebellar nuclei (DCN) in slices. IPSCs were evoked by electrical stimulation of the white matter surrounding the DCN in the presence of non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (20 microM). High-frequency stimulation induced a long-term potentation (LTP) of the IPSC amplitude without changing its reversal potential, rise time, and decay-time constant. This LTP did not require the activation of postsynaptic gamma-aminobutyric acid-A (GABA(A)) receptors but depended on the activation of NMDA receptors. LTP of IPSCs in DCN neurons could also be induced by voltage-depolarizing pulses in postsynaptic neurons and appeared to depend on an increase in intracellular calcium as the LTP was blocked when the cells were loaded with a calcium chelator, 1,2-bis-(2-amino-phenoxy)-N,N,N', N'-tetraacetic acid (BAPTA, 10 mM). LTP of IPSCs was accompanied by an increase in the frequency of spontaneous IPSCs and miniature IPSCs (recorded in the presence of tetrodotoxin 1 microM), but there was no significant change in their amplitude. In addition, during the LTP, the amplitude of response to exogenously applied GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride was increased. Intracellular application of tetanus toxin, a powerful blocker of exocytosis, in DCN neuron prevented the induction of LTP of IPSCs. Our results suggest that the induction of LTP of IPSCs in the DCN neurons likely involves a postsynaptic locus. Plasticity of inhibitory synaptic transmission in DCN neurons may play a crucial role in cerebellar control of motor coordination and learning.

2-Amino-5-phosphonovalerate↗
PubMed2006

Presynaptic I1-imidazoline receptors reduce GABAergic synaptic transmission in striatal medium spiny neurons.

Imidazoline receptors are expressed widely in the CNS. In the present study, whole-cell patch-clamp recordings were made from medium spiny neurons in dorsal striatum slices from the rat brain, and the roles of I1-imidazoline receptors in the modulation of synaptic transmission were studied. Moxonidine, an I1-imidazoline receptor agonist, decreased the GABAA receptor-mediated IPSCs in a concentration-dependent manner. However, glutamate-mediated EPSCs were hardly affected. The depression of IPSCs by moxonidine was antagonized by either idazoxan or efaroxan, which are both imidazoline receptor antagonists containing an imidazoline moiety. In contrast, yohimbine and SKF86466 (6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine), which are alpha2-adrenergic receptor antagonists with no affinity for imidazoline receptors, did not affect the moxonidine-induced inhibition of IPSCs. Moxonidine increased the paired-pulse ratio and reduced the frequency of miniature IPSCs without affecting their amplitude, indicating that this agent inhibits IPSCs via presynaptic mechanisms. Moreover, the sulfhydryl alkylating agent N-ethylmaleimide (NEM) significantly reduced the moxonidine-induced inhibition of IPSCs. Thus, the activation of presynaptic I1-imidazoline receptors decreases GABA-mediated inhibition of medium spiny neurons in the striatum, in which NEM-sensitive proteins such as G(i/o)-type G-proteins play an essential role. The adenylate cyclase activator forskolin partly opposed IPSC inhibition elicited by subsequently applied moxonidine. Furthermore, the protein kinase C (PKC) activator phorbol 12,13-dibutyrate attenuated and the PKC inhibitor chelerythrine potentiated the moxonidine-induced inhibition of IPSCs. These results suggest that IPSC inhibition via presynaptic I1-imidazoline receptors involves intracellular adenylate cyclase activity and is influenced by static PKC activity in the striatum.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1993

Patch-clamp analysis of spontaneous synaptic currents in supraoptic neuroendocrine cells of the rat hypothalamus.

Spontaneous synaptic currents were recorded in supraoptic magnocellular neurosecretory cells using whole-cell patch-clamp techniques in the rat hypothalamic slice preparation. Numerous spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were observed in the 27 cells recorded. The rate of occurrence of the spontaneous currents and the relative proportion of EPSCs versus IPSCs varied significantly from cell to cell. Miniature EPSCs and IPSCs were clearly distinguished from background noise in TTX (n = 3 cells at 0.5 micrograms/ml). The frequency of EPSCs and IPSCs decreased by approximately 70% and the largest events were blocked in TTX, but the peaks of the amplitude histograms were shifted by only a few picoamperes. Bicuculline (n = 10 cells at 10 microM and 2 cells at 20 microM) blocked completely all the IPSCs without any detectable effect on the frequency or amplitude of the EPSCs. No slow spontaneous outward currents, indicative of a K+ current from activation of GABAB receptors, were observed. The alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate-type glutamate receptor antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX; n = 7 cells at 10 microM) consistently blocked all EPSCs without any apparent effect on the frequency or amplitude of the IPSCs. No synaptic events could be detected when CNQX was applied in combination with bicuculline (n = 4). The decay phase of averaged spontaneous IPSCs and EPSCs recorded at resting membrane potential could be well fitted by single exponential functions in most cells. The time constants ranged from 0.92 to 3.0 msec for EPSCs (five cells) and from 5.3 to 6.6 msec for IPSCs (four cells). A second, slower time constant of 4-15 msec was found in the largest averaged EPSCs (> or = 40 pA). The amplitude of this slow component was -2 to -4 pA. These results suggest that, in the in vitro slice preparation, glutamate mediates all the spontaneous EPSCs in magnocellular neurosecretory cells by acting primarily on AMPA/kainate-type receptors at resting membrane potential and that activation of GABAA receptors mediates most if not all IPSCs.

6-Cyano-7-nitroquinoxaline-2,3-dione↗