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PubMed1999

Calcium channels in the GABAergic presynaptic nerve terminals projecting to meynert neurons of the rat.

Effects of selective Ca2+ channel blockers on GABAergic inhibitory postsynaptic currents (IPSCs) were studied in the acutely dissociated rat nucleus basalis of Meynert (nBM) neurons attached with nerve endings, namely, the "synaptic bouton" preparation, and in the thin slices of nBM, using nystatin perforated and conventional whole-cell patch recording modes, respectively. In the synaptic bouton preparation, nicardipine (3 x 10(-6) M) and omega-conotoxin-MVIIC (3 x 10(-6) M) reduced the frequency of spontaneous postsynaptic currents by 37 and 22%, respectively, whereas omega-conotoxin-GVIA had no effect. After blockade of L- and P/Q-type Ca2+ channels, successive removal of Ca2+ from external solution had no significant effect on the residual spontaneous activities, indicating that N-, R-, and T-type Ca2+ channels are not involved in the spontaneous GABA release. Thapsigargin, but not ryanodine, increased the frequency of spontaneous IPSCs in both the synaptic bouton and slice preparations, suggesting the partial contribution of the intracellular Ca2+ storage site to the spontaneous GABA release. In contrast, omega-conotoxin-GVIA (3 x 10(-6) M) and omega-conotoxin-MVIIC (3 x 10(-6) M) suppressed the evoked IPSCs by 31 and 37%, respectively, but nicardipine produced no significant effect. The residual evoked currents were abolished in Ca2+-free external solution but not in the external solution containing 10(-5) M Ni2+, suggesting the involvement of N-, P/Q-, and R-type Ca2+ channels but not L- and T-type ones in the evoked IPSCs. Neither thapsigargin nor ryanodine had any significant effects on the evoked IPSCs. It was concluded that Ca2+ channel subtypes responsible for spontaneous transmitter release are different from those mediating the transmitter release evoked by nerve stimulation.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2003

Ethanol increases GABAergic transmission at both pre- and postsynaptic sites in rat central amygdala neurons.

We examined the interaction of ethanol with the gamma-aminobutyric acid (GABA)ergic system in neurons of slices of the rat central amygdala nucleus (CeA), a brain region thought to be critical for the reinforcing effects of ethanol. Brief superfusion of 11-66 mM ethanol significantly increased GABA type A (GABA(A)) receptor-mediated inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) in most CeA neurons, with a low apparent EC(50) of 20 mM. Acute superfusion of 44 mM ethanol increased the amplitude of evoked GABA(A) IPSPs and IPSCs in 70% of CeA neurons. The ethanol enhancement of IPSPs and IPSCs occurred to a similar extent in the presence of the GABA type B (GABA(B)) receptor antagonist CGP 55845A, suggesting that this receptor is not involved in the ethanol effect on CeA neurons. Ethanol superfusion also decreased paired-pulse facilitation of evoked GABA(A) IPSPs and IPSCs and always increased the frequency and sometimes the amplitude of spontaneous miniature GABA(A) IPSCs as well as responses to local GABA application, indicating both presynaptic and postsynaptic sites of action for ethanol. Thus, the CeA is the first brain region to reveal, without conditional treatments such as GABA(B) antagonists, consistent, low-dose ethanol enhancement of GABAergic transmission at both pre- and postsynaptic sites. These findings add further support to the contention that the ethanol-GABA interaction in CeA plays an important role in the reinforcing effects of ethanol.

Amygdala↗
PubMed2001

Adenosine-mediated inhibition of striatal GABAergic synaptic transmission during in vitro ischaemia.

Several reports have shown that energy deprivation, as a result of hypoxia, hypoglycaemia or ischaemia, depresses excitatory synaptic transmission in virtually all brain areas. How this pathological condition affects inhibitory synaptic transmission is still unclear. In the present in vitro study, we coupled whole-cell patch clamp recordings from striatal neurones with focal stimulation of GABAergic nerve terminals in order to characterize the electrophysiological effects of combined oxygen and glucose deprivation (in vitro ischaemia) on inhibitory postsynaptic currents (IPSCs) in this brain area. We found that brief periods (2-5 min) of in vitro ischaemia invariably caused a marked depression of IPSC amplitude. This inhibitory effect was fully reversible on removal of the ischaemic challenge. It was coupled with an increased paired-pulse facilitation, suggesting the involvement of presynaptic mechanisms. Accordingly, the ischaemic inhibition of striatal GABAergic IPSCs was not caused by a shift in the reversal potential of GABA(A)-receptor mediated synaptic currents, and was independ- ent of postsynaptic ATP concentrations. Endogenous adenosine, acting on A1 receptors, appeared responsible for this presynaptic action as the ischaemic depression of IPSCs was prevented by CPT [8-(4-chlorophenylthio) adenosine] and DPCPX, two adenosine A1 receptor antagonists, and mimicked by the application of adenosine in the bathing solution. Conversely, ATP-sensitive potassium channels were not involved in the inhibition of IPSCs by ischaemia, as demonstrated by the fact that tolbutamide and glipizide, two blockers of these channels, were ineffective in preventing this electrophysiological effect. The early depression of GABA-mediated synaptic transmission might play a role in the development of irreversible neuronal injury in the course of brain ischaemia.

Adenosine↗
PubMed2004

Differential effects of ethanol on gamma-aminobutyric acid-A receptor-mediated synaptic currents in congenic strains of inbred long and short-sleep mice.

BACKGROUND: Ethanol enhances gamma-aminobutyric acid (GABA)A receptor-mediated responses in the brain, and this enhancement is greater in a mouse line behaviorally more sensitive to ethanol (long sleep) than in a line (short sleep) behaviorally less ethanol sensitive (assayed by loss of righting; sleep time). Quantitative trait locus (QTL) analysis of inbred long sleep (ILS) and inbred short sleep (ISS) phenotypes revealed four chromosomal regions (Lore1, Lore2, Lore4, and Lore5) that together account for approximately 50% of ethanol-induced sleep-time variance. Congenic strains were generated, each of which is homozygous for one of four ISS Lore QTLs on the ILS background. These congenic mouse strains are ideally suited for asking which QTL regions might correlate with other phenotypes that differ between ILS and ISS mice. Here we used the congenics to investigate altered GABAA responses to ethanol. METHODS: Evoked GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were measured by whole-cell voltage-clamp recording procedures in CA1 pyramidal neurons in hippocampal brain slices. RESULTS: GABAA IPSC responses in hippocampal brain slices from ILS mice were significantly enhanced by 80 mM ethanol, whereas those from ISS mice were not affected. ILS.Lore2 and ILS.Lore5 congenic strains were significantly enhanced by 80 mM ethanol, similar to the background (control) ILS mice. However, ethanol had no significant effect on GABAA responses in ILS.Lore1 and ILS.Lore4 congenic mice, similar to the ISS mice, thus reflecting the influence of ISS alleles on the ILS phenotype. CONCLUSIONS: Our results suggest that alleles located in the Lore1 and Lore4 QTL regions confer ethanol sensitivity of GABAA receptor-mediated IPSCs. Thus, for these QTLs, GABAA IPSCs may represent an endophenotype of sedative/hypnotic sensitivity to ethanol. Although the Lore2 and Lore5 QTL regions have a significant effect on sleep time, they do not play a significant role in the differential ethanol enhancement of GABAA IPSCs between ILS and ISS mice.

Animals↗
PubMed2006

Characterization of the glycinergic input to bipolar cells of the mouse retina.

Glycine and gamma-aminobutyric acid (GABA) are the major inhibitory transmitters of the mammalian retina, and bipolar cells receive GABAergic and glycinergic inhibition from multiple amacrine cell types. Here we evaluated the functional properties and subunit composition of glycine receptors (GlyRs) in bipolar cells. Patch-clamp recordings were performed from retinal slices of wild-type, GlyRalpha1-deficient (Glra1(spd-ot)) and GlyRalpha3-deficient (Glra3(-/-)) mice. Whole-cell currents following glycine application and spontaneous inhibitory postsynaptic currents (IPSCs) were analysed. During the recordings the cells were filled with Alexa 488 and, thus, unequivocally identified. Glycine-induced currents of bipolar cells were picrotoxinin-insensitive and thus represent heteromeric channels composed of alpha and beta subunits. Glycine-induced currents and IPSCs were absent from all bipolar cells of Glra1(spd-ot) mice, indicating that GlyRalpha1 is an essential subunit of bipolar cell GlyRs. By comparing IPSCs of bipolar cells in wild-type and Glra3(-/-) mice, no statistically significant differences were found. OFF-cone bipolar (CB) cells receive a strong glycinergic input from AII amacrine cells, that is preferentially based on the fast alpha1beta-containing channels (mean decay time constant tau = 5.9 +/- 1.4 ms). We did not observe glycinergic IPSCs in ON-CB cells and could elicit only small, if any, glycinergic currents. Rod bipolar cells receive a prominent glycinergic input that is mainly mediated by alpha1beta-containing channels (tau = 5.5 +/- 1.6 ms). Slow IPSCs, the characteristic of GlyRs containing the alpha2 subunit, were not observed in bipolar cells. Thus, different bipolar cell types receive kinetically fast glycinergic inputs, preferentially mediated by GlyRs composed of alpha1 and beta subunits.

Animals↗
PubMed1997

Presynaptic GABAB and adenosine A1 receptors regulate synaptic transmission to rat substantia nigra reticulata neurones.

1. Patch pipettes were used to record whole-cell currents under voltage clamp in substantia nigra zona reticulata (SNR) neurones in the rat midbrain slice. Bipolar electrodes evoked synaptic currents mediated by glutamate (EPSCs) and GABAA receptors (IPSCs). 2. Baclofen reduced the amplitude of IPSCs by 48% at its IC50 value of 0.60 microM. The GABAB antagonist CGP 35348 blocked this effect with a Kd value estimated by Schild analysis of 5 microM. 3. Adenosine reduced IPSCs by 48% at its IC50 value of 56 microM. Adenosine agonists reduced IPSCs with the following rank order of potency: CPA (N6-cyclopentyladenosine) > R-PIA (R(-)N6-(2-phenylisopropyl)adenosine) > CHA (N6-cyclohexyladenosine) = NECA (5'-N-ethylcarboxamidoadenosine) > 2-CADO (2-chloroadenosine) > adenosine. Schild analysis yielded a Kd value of 0.4 nM for antagonism of CPA by the adenosine A1 receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine). 4. Both baclofen and adenosine reduced the magnitude of paired-pulse depression of IPSCs, and neither blocked currents evoked by GABA, which was pressure-ejected from micropipettes. 5. Glutamate EPSCs were reduced by baclofen (IC50 = 0.78 microM) and adenosine (IC50 = 57 microM). Schild analysis yielded a Kd value of 11 microM for antagonism of baclofen-induced inhibition of EPSCs by CGP 35348. DPCPX (1 microM) completely blocked the inhibitory effects of adenosine (100 microM) and CPA (100 nM) on EPSCs. Neither adenosine nor baclofen reduced inward currents evoked by glutamate which was pressure-ejected from micropipettes. 6. These results show that presynaptic GABAB and A1 receptors reduce glutamate and GABA release from nerve terminals in the SNR.

Adenosine↗
PubMed1998

Muscarinic facilitation of GABA release in substantia gelatinosa of the rat spinal dorsal horn.

1. Blind patch clamp recordings were made from substantia gelatinosa (SG) neurones in the adult rat spinal cord slice to study the mechanisms of cholinergic modulation of GABAergic inhibition. 2. In the majority of SG neurones tested, carbachol (10 microM) increased the frequency (677 % of control) of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs). A portion of these events appeared to result from the generation of spikes by GABAergic interneurones, since large amplitude IPSCs were eliminated by tetrodotoxin (1 microM). 3. The effect of carbachol on spontaneous IPSCs was mimicked by neostigmine, suggesting that GABAergic interneurones are under tonic regulation by cholinergic systems. 4. The frequency of GABAergic miniature IPSCs in the presence of tetrodotoxin (1 microM) was also increased by carbachol without affecting amplitude distribution, indicating that acetylcholine facilitates quantal release of GABA through presynaptic mechanisms. 5. Neither the M1 receptor agonist McN-A-343 (10-300 microM) nor the M2 receptor agonist, arecaidine (10-100 microM), mimicked the effects of carbachol. All effects of carbachol and neostigmine were antagonized by atropine (1 muM), while pirenzepine (100 nM), methoctramine (1 microM) and hexahydrosiladifenidol hydrochloride, p-fluoro-analog (100 nM) had no effect. 6. Focal stimulation of deep dorsal horn, but not dorsolateral funiculus, evoked a similar increase in IPSC frequency to that evoked by carbachol and neostigmine. The stimulation-induced facilitation of GABAergic transmission lasted for 2-3 min post stimulation, and the effect was antagonized by atropine (100 nM). 7. Our observations suggest that GABAergic interneurones possess muscarinic receptors on both axon terminals and somatodendritic sites, that the activation of these receptors increases the excitability of inhibitory interneurones and enhances GABA release in SG and that the GABAergic inhibitory system is further controlled by cholinergic neurones located in the deep dorsal horn. Those effects may be responsible for the antinociceptive action produced by the intrathecal administration of muscarinic agonists and acetylcholinesterase inhibitors.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed1998

Vagally evoked synaptic currents in the immature rat nucleus tractus solitarii in an intact in vitro preparation.

1. Whole-cell voltage-clamp recordings in an in vitro brainstem-cranial nerve explant preparation were used to assess the local circuitry activated by vagal input to nucleus tractus solitarii (NTS) neurones in immature rats. 2. All neurones that responded to vagal stimulation displayed EPSCs of relatively constant latency. Approximately 50 % of these also demonstrated variable-latency IPSCs, and approximately 31 % also displayed variable-latency EPSCs to vagal stimulation. All neurones also had spontaneous EPSCs and IPSCs. 3. Evoked and spontaneous EPSCs reversed near 0 mV and were blocked by the glutamate AMPA/kainate receptor antagonists 6,7-nitroquinoxaline-2,3-dione (DNQX) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) at rest. Evoked EPSCs had rapid rise times (< 1 s) and decayed monoexponentially (tau = 2. 04 +/- 0.03 ms) at potentials near rest. 4. At holding potentials positive to approximately -50 mV, a slow EPSC could be evoked in the presence of DNQX or CNQX. This current peaked at holding potentials near -25 mV and was blocked by the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5). It was therefore probably due to activation of NMDA receptors by vagal afferent fibres. 5. Fast IPSCs reversed near -70 mV and were blocked by the GABAA receptor antagonist bicuculline. In addition, bicuculline enhanced excitatory responses to vagal stimulation and increased spontaneous EPSC frequency. Antagonists to AMPA/kainate receptors reversibly blocked stimulus-associated IPSCs and also decreased the frequency of spontaneous IPSCs. 6. These findings suggest that glutamate mediates synaptic transmission from the vagus nerve to neurones in the immature NTS by acting at non-NMDA and NMDA receptors. NTS neurones may also receive glutamatergic and GABAergic synaptic input from local neurones that can be activated by vagal input and/or regulated by amino acid inputs from other brainstem neurones.1. Whole-cell voltage-clamp recordings in an in vitro brainstem-cranial nerve explant preparation were used to assess the local circuitry activated by vagal input to nucleus tractus solitarii (NTS) neurones in immature rats.

2-Amino-5-phosphonovalerate↗
PubMed1998

Laminar difference in GABA uptake and GAT-1 expression in rat CA1.

1. The axonal plexus of most hippocampal interneurons is restricted to certain strata within the target region. This lamination suggests a possible functional heterogeneity of inhibitory synapses between different interneurons and CA1 pyramidal cells. 2. We therefore compared inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) in CA1 pyramidal cells, which were evoked from two stimulation sites (stratum oriens and stratum radiatum). Stimulation in stratum oriens yielded faster decaying IPSPs and IPSCs than stimulation in stratum radiatum. 3. IPSP and IPSC kinetics were regulated by GABA uptake in both layers as indicated by the prolongation of the signals under tiagabine, a GAT-1 (neuronal GABA plasma membrane transporter)-specific GABA-uptake blocker. However, the effect of tiagabine was significantly more pronounced following stimulation in stratum radiatum than in stratum oriens (prolongation of IPSC half-decay time by 167 vs. 115 %, respectively). 4. In situ hybridization with antisense mRNA for the GABA-synthesizing enzyme glutamate decarboxylase (GAD65/67) and the GABA transporter GAT-1 showed that the proportion of interneurons expressing GAT-1 was lower in stratum oriens than in stratum radiatum/lacunosum-moleculare. 5. From these functional and molecular data we conclude that the regulation of IPSP and IPSC kinetics in CA1 pyramidal cells by neuronal GABA uptake differs between layers. Our findings suggest that this laminar difference is caused by a lower expression of GAT-1 in interneurons in stratum oriens than in stratum radiatum/lacunosum-moleculare.

Animals↗
PubMed2001

Actions of nociceptin/orphanin FQ and other prepronociceptin products on rat rostral ventromedial medulla neurons in vitro.

1. Whole-cell patch clamp recordings were made from rat rostral ventromedial medulla (RVM) neurons in vitro to investigate the cellular actions of the opioid-like receptor ORL1 (NOP), ligand nociceptin/orphanin FQ and other putative prepronociceptin products. 2. Primary and secondary RVM neurons were identified as responding to the kappa-opioid receptor agonist U-69593 (300 nM to 1 microM) and the mu- and delta-opioid receptor agonist met-enkephalin (10 microM), respectively. Both primary and secondary RVM neurons responded to nociceptin (3 nM to 1 microM) with an outward current that reversed polarity at -115 mV in brain slices and with inhibition of Ca(2+) channel currents in acutely isolated cells. 3. The putative ORL1 antagonist J-113397 (1 microM) produced no change in membrane current and abolished the outward current produced by nociceptin (100 nM). In contrast, Phe(1)psi(CH(2)-NH)Gly(2)]-nociceptin-(1-13)NH(2) (300 nM to 1 microM) alone produced an outward current and partially reduced the outward current produced by nociceptin (300 nM) when co-applied. 4. In brain slices nociceptin (300 nM) reduced the amplitude of evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) but not non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs). 5. Met-enkephalin (10 microM), but not nociceptin (300 nM), reduced the rate of spontaneous miniature IPSCs in normal external potassium solution (K(+) 2.5 mM). In high external potassium (K(+) 17.5 mM), nociceptin reduced the rate of miniature IPSCs in the presence (Ca(2+) 2.4 mM, Mg(2+) 1.2 mM) but not in the absence of external calcium (Ca(2+) 0 mM, Mg(2+) 10 mM, Cd(2+) 10 microM). Nociceptin and met-enkephalin had no effect on the amplitude of miniature IPSCs. 6. The putative nociceptin precursor products nocistatin (rat prepronociceptin(125-132)) and rat prepronociceptin(154-181) had no effect on membrane currents, evoked IPSCs and evoked EPSCs. 7. These results indicate that nociceptin acts via the ORL1 receptor to directly inhibit both primary and secondary RVM neurons by activating a potassium conductance and by inhibiting calcium conductances. In addition, nociceptin inhibits GABA release within the RVM via a presynaptic Ca(2+)-dependent mechanism. Thus, nociceptin has the potential to exert both disinhibitory and inhibitory effects on neuronal action potential firing within the RVM.

Animals↗
PubMed2001

The peptide TRH uncovers the presence of presynaptic 5-HT1A receptors via activation of a second messenger pathway in the rat dorsal vagal complex.

It is well recognized that brainstem microinjections of 5-hydroxytryptamine (serotonin, 5-HT) and thyrotropin-releasing hormone (TRH) act synergistically to stimulate gastric function in vivo. Previous in vitro experiments have shown that this synergism does not occur at the level of the dorsal motor nucleus of the vagus (DMV) motoneurone. In order to determine the mechanism of this action, whole cell patch clamp recordings were made from identified gastric-projecting rat DMV neurones to investigate the effects of 5-HT and TRH on GABAergic inhibitory postsynaptic currents (IPSCs) evoked by stimulation of the nucleus of the tractus solitarius (NTS). 5-HT (30 microM) decreased IPSC amplitude by 26 +/- 2.5% in approximately 43% of DMV neurones. In the remaining neurones in which 5-HT had no effect on IPSC amplitude, exposure to TRH (1 microM) uncovered the ability of subsequent applications of 5-HT to decrease IPSC amplitude by 28 +/- 3%. Such TRH-induced 5-HT responses were prevented by the 5-HT1A antagonist NAN-190 (1 microM) and mimicked by the 5-HT1A agonist 8-OH-DPAT (1 microM). Increasing cAMP levels using the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 10 microM), the non-hydrolysable cAMP analogue 8-bromo-cAMP (1 mM), or the adenylate cyclase activator forskolin (10 microM), like TRH, uncovered the ability of 5-HT to decrease evoked IPSC amplitude (17 +/- 2.2 %, 28.5 +/- 5.3 % and 30 +/- 4.8%, respectively), in neurones previously unresponsive to 5-HT. Conversely, the adenylate cyclase inhibitor, dideoxyadenosine (10 microM) and the protein kinase A inhibitor, Rp-cAMP (10 microM), blocked the ability of TRH to uncover the presynaptic inhibitory actions of 5-HT. These results suggest that activation of presynaptic TRH receptors initiates an intracellular signalling cascade that raises the levels of cAMP sufficient to uncover previously silent 5-HT1A receptors on presynaptic nerve terminals within the dorsal vagal complex.

Action Potentials↗
PubMed1990

Voltage dependence of Ia reciprocal inhibitory currents in cat spinal motoneurones.

1. Inhibitory postsynaptic currents (IPSCs) were recorded in voltage clamped posterior biceps or semitendinosus motoneurones of the cat during reciprocal inhibition. 2. Population IPSCs, recorded following stimulation of the whole quadriceps muscle nerve, had an average time-to-peak of 0.51 +/- 0.02 ms (+/- S.E.M., n = 22) and decayed exponentially, with an average time constant of 0.99 +/- 0.04 ms (at 37 degrees C) at resting membrane potentials. 3. Unitary IPSCs, recorded following spike-triggered averaging from an identified reciprocal inhibitory interneurone, had amplitudes of 120-220 pA with an average time-to-peak of 0.40 +/- 0.06 ms (n = 5). The decay of these unitary currents was exponential, with an average time constant of 0.82 +/- 0.07 ms (at 37 degrees C) at resting membrane potentials. 4. The time course of IPSCs was unaffected by either alpha-chloralose or pentobarbitone at concentrations necessary for deep anaesthesia. 5. The peak synaptic current varied linearly with the membrane potential over the range -90 to -30 mV, and had an average reversal potential of -80.7 +/- 1.5 mV (+/- S.E.M., n = 6) when measured using KCH3SO4-filled electrodes. 6. The reversal potential for the IPSC was used to calculate [Cl-]i. This was estimated to be 6.5 mM assuming that the inhibitory synaptic current was mediated purely by Cl- ions. 7. The rate at which synaptic currents decayed was exponentially dependent on the postsynaptic membrane potential, the decay time constant increasing e-fold for a 91 mV depolarization. This result was independent of [Cl-]i or of the magnitude of the synaptic conductance and was interpreted as a voltage dependence of the glycine channel open time. 8. The average unitary peak conductance was 9.1 +/- 1.7 nS (+/- S.E.M., n = 5), corresponding to the opening of approximately 200 glycine-activated postsynaptic channels following neurotransmitter release from a single Ia reciprocal interneurone.

Action Potentials↗
PubMed1995

Dual modulation of synaptic inhibition by distinct metabotropic glutamate receptors in the rat hippocampus.

1. The effects of metabotropic glutamate receptor (mGluR) activation on synaptic inhibition were examined using whole-cell recordings of spontaneous and miniature inhibitory synaptic currents from CA3 pyramidal cells in rat hippocampal slices. 2. The mGluR agonist (1S,3R)trans-1-aminocyclopentane-1,3-dicarboxylic acid (tACPD) increased spontaneous IPSC (spIPSC) frequency by up to 5-fold. At doses above 5 microM the increase was transient (15-45 s) and was followed by a decline to control frequency. In these conditions, elevating external K+ from 2 to 8 mM could still increase spIPSC frequency. 3. Miniature IPSCs (mIPSCs) were recorded in the presence of 1 microM TTX, 5 mM Mg2+ and nominally zero Ca2+. At concentrations above 50 microM, tACPD induced a sustained, reversible reduction in mIPSC frequency by up to 43%. 4. Quisqualate, at doses as low as 50 nM, increased spIPSC frequency, but did not affect mIPSC frequency at concentrations up to 10 microM. 5. The specific mGluR2 and 3 agonist (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV, 3 microM) reduced mIPSC frequency by 40 +/- 4% but did not increase spIPSC frequency. 6. The putative mGluR antagonist L-2-amino-3-phosphonopropionate (L-AP3, 1 mM) blocked the effect of tACPD on mIPSC but not spIPSC frequency. The broad-spectrum antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM) blocked both responses. 7. mGluR activation also had dual effects on IPSCs evoked by focal extracellular stimulation. Application of 5 microM tACPD increased the mean amplitude of evoked IPSCs by 112 +/- 9%, largely by reducing the proportion of response failures. In contrast, IPSC amplitude was reduced to 44 +/- 1% of control values by 3 microM DCG-IV. 8. These results suggest hippocampal inhibitory cells express two distinct mGluR subtypes. One receptor (possibly mGluR1 or 5) is located on somato-dendritic membrane and enhances cell excitability. Another (mGluR2 or 3) is present at inhibitory terminals and reduces the probability of GABA release.

2-Amino-5-phosphonovalerate↗
PubMed1995

Erosion of inhibition contributes to the progression of low magnesium bursts in rat hippocampal slices.

1. Bathing slices of rat hippocampus in media containing no magnesium ions results in epileptic discharges that originate in hippocampal area CA3. These discharges increase in severity gradually over periods of hours. 2. The progression of epileptic activity was much slower than the equilibration of extracellular magnesium activity and the resulting increase in strength of monosynaptic NMDA receptor-mediated excitation. Its time course matched that of a progressive decrease in pharmacologically isolated, evoked GABAA receptor-mediated inhibitory postsynaptic current (IPSC) in the CA3 pyramidal cells. Conductance decreased to 37 +/- 6% of control values after 4 h. Responses to exogenous GABA application decreased to 52 +/- 12%. 3. Maximal IPSC conductance in 0 mM extracellular Mg2+ ([Mg2+]o) also decreased gradually when epileptic activity was abolished by bath application of 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 microM D-2-amino-5-phosphonovaleric acid (D-APV) throughout the 4 h incubation period. It reached 61 +/- 8% of control values, a significantly smaller decline than that seen after 4 h of epileptic activity. 4. The decrease in mean IPSC conductance only partially reversed when the recording electrode contained 100 mM Mg2+. Complete recovery of IPSC strength occurred when electrodes also contained either 50 mM MgATP or 20 mM BAPTA. Reintroduction of 1 mM [Mg2+]o rapidly abolished epileptic activity and caused a slow, partial increase in IPSC conductance. 5. In the presence of 1 mM [Mg2+]o, GABAA receptor-mediated inhibition had to decrease to 17 +/- 11% of control values, in the presence of 4-7 microM bicuculline, to reach threshold for epileptic activity. 6. These data demonstrate a postsynaptic decrease in GABAA receptor-mediated inhibition in the in vitro low magnesium model of epilepsy. We propose that the apparent leaching of intracellular Mg2+ ([Mg2+]i) from cells leads to loss of ATP and consequent partial dephosphorylation of the GABAA receptor and that this is exacerbated by epileptic activity.

Animals↗
PubMed2002

Neonatal development of the rat visual cortex: synaptic function of GABAA receptor alpha subunits.

Each GABA(A) receptor consists of two alpha and three other subunits. The spatial and temporal distribution of different alpha subunit isomeres expressed by the CNS is highly regulated. Here we study changes in functional contribution of different alpha subunits during neonatal development in rat visual cortex. First, we characterized postsynaptic alpha subunit expression in layer II-III neurons, using subunit-specific pharmacology combined with electrophysiological recordings in acutely prepared brain slices. This showed clear developmental downregulation of the effects of bretazenil (1 microm) and marked upregulation of the effect of 100 nM of zolpidem on the decay of spontaneous inhibitory postsynaptic currents (sIPSCs). Given the concentrations used we interpret this as downregulation of the synaptic alpha3 and upregulation of alpha1 subunit. Furthermore, the effect of furosemide, being indicative of the functional contribution of alpha4, was increased between postnatal days 6 and 21. Our second aim was to study the effects of plasticity in alpha subunit expression on decay properties of GABAergic IPSCs. We found that bretazenil-sensitive IPSCs have the longest decay time constant in juvenile neurons. In mature neurons, zolpidem- and furosemide-sensitive IPSCs have relatively fast decay kinetics, whereas bretazenil-sensitive IPSCs decay relatively slowly. Analysis of alpha1 deficient mice and alpha1 antisense oligonucleotide deletion in rat explants showed similar results to those obtained by zolpidem application. Thus, distinct alpha subunit contributions create heterogeneity in developmental acceleration of IPSC decay in neocortex.

Animals↗
PubMed2003

Parallel decrease in omega-conotoxin-sensitive transmission and dopamine-induced inhibition at the striatal synapse of developing rats.

Whole-cell patch-clamp recordings of GABAergic IPSCs were made from cholinergic interneurones in slices of striatum from developing rats aged 21-60 days postnatal. In addition, the Ca(2+) channel subtypes involved in synaptic transmission, as well as dopamine (DA)-induced presynaptic inhibition, were investigated pharmacologically with development by bath application of Ca(2+) channel blockers and DA receptor agonists. The IPSC amplitude was reduced by omega-conotoxin GVIA (omega-CgTX) or omega-agatoxin TK (omega-Aga-TK) across the whole age range, suggesting that multiple types of Ca(2+) channels mediate transmission of the synapse. The IPSC fraction reduced by omega-CgTX significantly decreased, whereas that reduced by omega-Aga-TK remained unchanged with development. DA or quinpirole, a D(2)-like receptor agonist, presynaptically reduced the IPSC amplitude throughout development. The DA-induced inhibition decreased with age in parallel with the decrease in N-type Ca(2+) channels. DA showed no further inhibition of IPSCs after the inhibitory effect of omega-CgTX had reached steady state throughout development. These results demonstrate that there is a functional link between presynaptic N-type Ca(2+) channels and D(2)-like DA receptors at inhibitory synapses in the striatum. They also demonstrate that the suppression of GABAergic transmission by D(2)-like receptors is mediated by modulation of N-type Ca(2+) channels and decreases in parallel with the developmental decline in the contribution of N-type Ca(2+) channels to exocytosis.

Agatoxins↗
PubMed2003

GABA B receptor modulation of excitatory and inhibitory synaptic transmission onto rat CA3 hippocampal interneurons.

Hippocampal stratum radiatum inhibitory interneurons receive glutamatergic excitatory innervation via the recurrent collateral fibers of CA3 pyramidal neurons and GABAergic inhibition from other interneurons. We examined both presynaptic- and postsynaptic-GABA(B) receptor-mediated responses at both synapse types. Postsynaptic GABA(B) receptor-mediated responses were absent in recordings from young (P16-18) but present in recordings from older animals (> or =P30) suggesting developmental regulation. In young animals, the GABA(B) receptor agonist, baclofen, inhibited the amplitude of evoked EPSCs and IPSCs, an effect blocked by prior application of the selective antagonist CGP55845. Baclofen enhanced the paired-pulse ratio and coefficient of variation of evoked EPSCs and IPSCs, consistent with a presynaptic mechanism of regulation. In addition, baclofen reduced the frequency of miniature IPSCs but not mEPSCs. However, baclofen reduced the frequency of KCl-induced mEPSCs; an effect blocked by Cd(2+), implicating presynaptic voltage-gated Ca(2+) channels as a target for baclofen modulation. In contrast, although Cd(2+) prevented the KCl-induced increase in mIPSC frequency, it failed to block baclofen's reduction of mIPSC frequency. Whereas N- and P/Q-types of Ca(2+) channels contributed equally to GABA(B) receptor-mediated inhibition of EPSCs, more P/Q-type Ca(2+) channels were involved in GABA(B) receptor-mediated inhibition of IPSCs. Finally, baclofen blocked the frequency-dependent depression of EPSCs and IPSCs, but was less effective at blocking frequency-dependent facilitation of EPSCs. Our results demonstrate that presynaptic GABA(B) receptors are expressed on the terminals of both excitatory and inhibitory synapses onto CA3 interneurons and that their activation modulates essential components of the release process underlying transmission at these two synapse types.

Animals↗
PubMed2002

Effects of the anticonvulsant retigabine on cultured cortical neurons: changes in electroresponsive properties and synaptic transmission.

The whole-cell patch-clamp technique was used to examine the effects of retigabine, a novel anticonvulsant drug, on the electroresponsive properties of individual neurons as well as on neurotransmission between monosynaptically connected pairs of cultured mouse cortical neurons. Consistent with its known action on potassium channels, retigabine significantly hyperpolarized the resting membrane potentials of the neurons, decreased input resistance, and decreased the number of action potentials generated by direct current injection. In addition, retigabine potentiated inhibitory postsynaptic currents (IPSCs) mediated by activation of gamma-aminobutyric acid(A) (GABA(A)) receptors. IPSC peak amplitude, 90-to-10% decay time, weighted decay time constant, slow decay time constant, and, consequently, the total charge transfer were all significantly enhanced by retigabine in a dose-dependent manner. This effect was limited to IPSCs; retigabine had no significant effect on excitatory postsynaptic currents (EPSCs) mediated by activation of non-N-methyl-D-aspartate ionotropic glutamate receptors. A form of short-term presynaptic plasticity, paired-pulse depression, was not altered by retigabine, suggesting that its effect on IPSCs is primarily postsynaptic. Consistent with the hypothesis that retigabine increases inhibitory neurotransmission via a direct action on the GABA(A) receptor, the peak amplitudes, 90-to-10% decay times, and total charge transfer of spontaneous miniature IPSCs were also significantly increased. Therefore, retigabine potently reduces excitability in neural circuits via a synergistic combination of mechanisms.

Animals↗