Presynaptic GABA(B) receptors inhibit synaptic inputs to rat subthalamic neurons.

Effects of baclofen on synaptic transmission were studied in rat subthalamic neurons using whole-cell patch clamp recording from brain slices. Focal electrical stimulation of the brain slice evoked GABAergic inhibitory postsynaptic currents and glutamatergic excitatory postsynaptic currents. Baclofen reduced the amplitude of evoked inhibitory postsynaptic currents in a concentration-dependent manner with an IC(50) of 0.6+/-0.2 microM. Evoked excitatory postsynaptic currents were also reduced by baclofen concentration-dependently (IC(50) of 1.6+/-0.2 microM), but baclofen was more potent at reducing the GABA(A) receptor inhibitory postsynaptic currents. The GABA(B) receptor antagonist CGP 35348 blocked these inhibitory effects of baclofen on evoked inhibitory and excitatory postsynaptic currents. Baclofen increased the paired-pulse ratios of evoked inhibitory and excitatory postsynaptic currents. Furthermore, baclofen reduced the frequency of spontaneous miniature excitatory postsynaptic currents, but had no effect on their amplitude.These results provide evidence for presence of presynaptic GABA(B) receptors that modulate both GABA and glutamate release from afferent terminals in the subthalamus.     

Retrograde signaling changes the venue of postsynaptic inhibition in rat substantia nigra

Both endocannabinoids through cannabinoid receptor type I (CB1) receptors and dopamine through dopamine receptor type D1 receptors modulate postsynaptic inhibition in substantia nigra by changing GABA release from striatonigral terminals. By recording from visually identified pars compacta and pars reticulata neurons we searched for a possible co-release and interaction of endocannabinoids and dopamine. Depolarization of a neuron in pars reticulata or in pars compacta transiently suppressed evoked synaptic currents which were blocked by GABA(A) receptor antagonists (inhibitory postsynaptic currents [IPSCs]). This depolarization-induced suppression of inhibition (DSI) was abrogated by the cannabinoid CB1 receptor antagonist AM251 (1 microM). A correlation existed between the degree of DSI and the degree of reduction of evoked IPSCs by the CB1 receptor agonist WIN55,212-2 (1 microM). The cholinergic receptor agonist carbachol (0.5-5 microM) enhanced DSI, but suppression of spontaneous IPSCs was barely detectable pointing to the existence of GABA release sites without CB1 receptors. In dopamine, but not in GABAergic neurons DSI was enhanced by the dopamine D1 receptor antagonist SCH23390 (3-10 microM). Both the antagonist for CB1 receptors and the antagonist for dopamine D1 receptors enhanced or reduced, respectively, the amplitudes of evoked IPSCs. This tonic influence persisted if the receptor for the other ligand was blocked. We conclude that endocannabinoids and dopamine can be co-released. Retrograde signaling through endocannabinoids and dopamine changes inhibition independently from each other. Activation of dopamine D1 receptors emphasizes extrinsic inhibition and activation of CB1 receptors promotes intrinsic inhibition.     

Muscarinic receptors mediate enhancement of spontaneous GABA release in the chick brain

The functional role of muscarinic acetylcholine receptors in the lateral spiriform nucleus was studied in chick brain slices. Whole-cell patch-clamp recordings of neurons in the lateral spiriform nucleus revealed that carbachol enhanced GABAergic spontaneous inhibitory postsynaptic currents. The duration of the response to carbachol was significantly reduced after blockade of muscarinic receptors with atropine. In the presence of the nicotinic receptor antagonist dihydro-beta-erythroidine, carbachol produced a delayed but prolonged enhancement of spontaneous GABAergic inhibitory postsynaptic currents that was completely blocked by atropine. Muscarine also enhanced the frequency of spontaneous GABAergic inhibitory postsynaptic currents in a dose-dependent manner, but had no effect on inhibitory postsynaptic current amplitude. While 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride, a M3 antagonist, completely blocked muscarine's effect, telenzepine, a M1 antagonist, and tropicamide, a M4 antagonist, only partially decreased the response to muscarine. Pirenzepine, a M1 antagonist, and methoctramine, a M2 antagonist, potentiated muscarine's enhancement of spontaneous GABAergic inhibitory postsynaptic currents. Muscarine's action was blocked by tetrodotoxin, cadmium chloride and omega-conotoxin GVIA, but was not affected by dihydro-beta-erythroidine, 6-cyano-7-nitroquinoxaline-2,3-dione, D(-)-2-amino-5-phosphonopentanoic acid, naloxone or fluphenazine. These results demonstrate that activation of both muscarinic and nicotinic acetylcholine receptors can enhance GABAergic inhibitory postsynaptic currents in the lateral spiriform nucleus. The muscarinic response has a slower onset but lasts longer than the nicotinic effect. The M3 receptor subtype is predominantly involved in enhancing spontaneous GABAergic inhibitory postsynaptic currents. These M3 receptors must be located some distance from GABA release sites, since activation of voltage-dependent sodium channels, and consequent activation of N-type voltage-dependent calcium channels, is required to trigger enhanced GABA release following activation of muscarinic receptors.     

Dopamine depresses glutamatergic synaptic transmission in the rat parabrachial nucleus in vitro

Nystatin-perforated patch recordings were made from rat parabrachial neurons in an in vitro slice preparation to examine the effect of dopamine on parabrachial cells and on excitatory synaptic transmission in this nucleus. In current clamp mode, dopamine reduced the amplitude of the evoked excitatory postsynaptic potential without significant change in membrane potential. In cells voltage-clamped at -65 mV, dopamine dose dependently and reversibly decreased evoked, pharmacologically isolated, excitatory postsynaptic currents with an EC50 of 31 microM. The reduction in excitatory postsynaptic current was accompanied by an increase in paired pulse ratio (a protocol used to detect presynaptic site of action) with no change in the holding current or in the decay of the evoked excitatory postsynaptic currents. In addition, dopamine altered neither postsynaptic (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate-induced currents, nor steady-state current voltage curves. Miniature excitatory postsynaptic current analysis revealed that dopamine caused a rightward shift of the frequency-distribution curve with no change in the amplitude-distribution curve, which is consistent with a presynaptic mechanism. The dopamine-induced attenuation of the excitatory postsynaptic current was almost completely blocked by the D1-like receptor antagonist SCH23390 (10 microM), although the D2-like antagonist sulpiride (10 microM) also partially blocked it. Combined application of both antagonists blocked all dopamine-induced synaptic effects. The synaptic effect of dopamine was mimicked by the D1-like agonist SKF38393 (50 microM), but the D2-1ike agonist quinpirole (50 microM) also had a small effect. Combined application of both agonists did not produce potentiated responses. Dopamine's effect on the excitatory postsynaptic current was independent of serotonin, GABA and adenosine receptors, but may have some interactions with adrenergic receptors. These results suggest that dopamine directly modulates excitatory synaptic events in the parabrachial nucleus predominantly via presynaptic D1-like receptors.     

Regulation of synaptic input to hypothalamic presympathetic neurons by GABA(B) receptors

The hypothalamic paraventricular (PVN) neurons projecting to the spinal cord and brainstem play an important role in the control of homeostasis and the sympathetic nervous system. Although GABA(B) receptors are present in the PVN, their function in the control of synaptic inputs to PVN presympathetic neurons is not clear. Using retrograde tracing and whole-cell patch-clamp recordings in rat brain slices, we determined the role of presynaptic GABA(B) receptors in regulation of glutamatergic and GABAergic inputs to spinally projecting PVN neurons. The GABA(B) receptor agonist baclofen (1-50 microM) dose-dependently decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs). The effect of baclofen on sEPSCs and sIPSCs was completely blocked by 10 microM CGP52432, a selective GABA(B) receptor antagonist. Baclofen also significantly reduced the frequency of both miniature excitatory and miniature inhibitory postsynaptic currents (mEPSCs and mIPSCs). Furthermore, uncoupling pertussis toxin-sensitive G(i/o) proteins with N-ethylmaleimide abolished baclofen-induced inhibition of mEPSCs and mIPSCs. However, the inhibitory effect of baclofen on the frequency of mIPSCs and mEPSCs persisted in the presence of either Cd2+, a voltage-gated Ca2+ channel blocker, or 4-aminopyridine, a blocker of voltage-gated K+ channels. Our results suggest that activation of presynaptic GABA(B) receptors inhibits synaptic GABA and glutamate release to PVN presympathetic neurons. This presynaptic action of GABA(B) receptors is mediated by the N-ethylmaleimide-sensitive G(i/o) proteins, but independent of voltage-gated Ca2+ and K+ channels.     

Presynaptic inhibition of synaptic transmission by adenosine in rat subthalamic nucleus in vitro

Whole-cell patch clamp recordings were made from the subthalamic nucleus in rat brain slice preparations to examine the effect of adenosine on inhibitory and excitatory synaptic transmission. Adenosine reversibly inhibited both GABA-mediated inhibitory and glutamate-mediated excitatory postsynaptic currents. Adenosine at 100 microM reduced the amplitude of inhibitory and excitatory postsynaptic currents by 42+/-5% and 34+/-6%, respectively. Reductions in the amplitude of both inhibitory and excitatory postsynaptic currents were accompanied by increases in paired-pulse ratios. In addition, adenosine decreased the frequency of spontaneous miniature excitatory postsynaptic currents but had no effect on their amplitude. These results are consistent with a presynaptic site of action. The adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine completely reversed the adenosine-induced attenuation of inhibitory and excitatory postsynaptic currents, but 8-cyclopentyl-1,3-dipropylxanthine alone had no effect on synaptic currents evoked at 0.1 Hz. However, 8-cyclopentyl-1,3-dipropylxanthine inhibited a time-dependent depression of excitatory postsynaptic currents that was normally observed in response to a 5 Hz train of stimuli, suggesting that endogenous adenosine could be released during higher frequencies of stimulation. These results suggest that adenosine inhibits synaptic release of GABA and glutamate by stimulation of presynaptic A(1) receptors in the subthalamic nucleus.     

Metabotropic glutamate and muscarinic cholinergic receptor-mediated preferential inhibition of N-methyl-D-aspartate component of transmissions in rat ventral tegmental area

Presynaptic inhibition is one of the major control mechanisms in the CNS. Our laboratory recently reported that presynaptic GABA(B) and adenosine A(1) receptors mediate a preferential inhibition on N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents recorded in rat midbrain dopamine neurons. Here we extended these findings to metabotropic glutamate and muscarinic cholinergic receptors. Intracellular voltage clamp recordings were made from dopamine neurons in rat ventral tegmental area in slice preparations. (+/-)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (agonist for groups I and II metabotropic glutamate receptors) and L(+)-2-amino-4-phosphonobutyric acid (L-AP4; agonist for group III metabotropic glutamate receptors) were significantly more potent for inhibiting N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents, as compared with inhibition of excitatory postsynaptic currents mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Such preferential inhibition of the N-methyl-D-aspartate component was also observed for muscarine (agonist for muscarinic cholinergic receptors). Inhibitory effects of (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid, L-AP4, and muscarine were blocked reversibly by their respective antagonists [(RS)-alpha-methyl-4-carboxyphenylglycine, (RS)-alpha-methyl-4-phosphonophenylglycine, and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide]. In addition, all three agonists increased the ratio of excitatory postsynaptic currents in paired-pulse studies and did not reduce currents induced by exogenous N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. Interestingly, the glutamate release stimulator 4-aminopyridine (30 microM) and the glutamate uptake inhibitor L-anti-endo-3,4-methanopyrrolidine dicarboxylate (300 microM) preferentially increased the amplitude of N-methyl-D-aspartate excitatory postsynaptic currents.Thus, agonists for metabotropic glutamate and muscarinic cholinergic receptors act presynaptically to cause a preferential reduction in the N-methyl-D-aspartate component of excitatory synaptic transmissions. Together with the evidence for GABA(B) and adenosine A(1) receptor-mediated preferential inhibition of the N-methyl-D-aspartate component, the present results suggest that limiting glutamate spillover onto postsynaptic N-methyl-D-aspartate receptors may be a general rule for presynaptic modulation in midbrain dopamine neurons.     

Dual modulation of gabaergic transmission by metabotropic glutamate receptors in rat ventral tegmental area

The effects of metabotropic glutamate receptor (mGluR) activation on non-dopamine (putative GABAergic) neurons and inhibitory synaptic transmission in the ventral tegmental area were examined using intracellular recordings from rat midbrain slices. Perfusion of (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD; agonist for group I and II mGluRs), but not L-amino-4-phosphonobutyric acid (L-AP4; agonist for group III mGluRs), produced membrane depolarization (current clamp) and inward current (voltage clamp) in non-dopamine neurons. The t-ACPD-induced depolarization was concentration-dependent (concentration producing 50% maximal depolarization [EC(50)]=6.1+/-2.5 microM), and was blocked by the antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine, but not by tetrodotoxin and ionotropic glutamate-receptor antagonists. The t-ACPD-evoked responses were mimicked comparably by selective group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG). Furthermore, the DHPG-induced depolarization in non-dopamine neurons was greatly reduced by mGluR1-specific antagonist 7(hydroxyimino)cyclopropachromen-1a-carboxylate ethyl ester. When recorded in dopamine neurons, the frequency of spontaneous GABA(A) receptor-mediated inhibitory postsynaptic potentials was increased by t-ACPD but not L-AP4. However, the amplitude of evoked inhibitory postsynaptic currents in dopamine neurons was reduced by all three group mGluR agonists.These results reveal a dual modulation of mGLuR activation on inhibitory transmission in midbrain ventral tegmental area: enhancing putative GABAergic neuronal excitability and thus potentiating tonic inhibitory synaptic transmission while reducing evoked synaptic transmission at inhibitory terminals.     

Distinct muscarinic receptors enhance spontaneous GABA release and inhibit electrically evoked GABAergic synaptic transmission in the chick lateral spiriform nucleus.

The effects of muscarinic agonists on GABAergic synaptic transmission were examined using whole-cell patch-clamp recording in chick brain slices containing the lateral spiriform nucleus. Bath application of muscarine (10 microM) both increased the frequency of spontaneous GABAergic postsynaptic currents and reduced the amplitude of evoked GABAergic polysynaptic postsynaptic currents elicited by focal afferent fiber electrical stimulation. Both of these muscarinic actions were reversible and dose-dependent. Two M(1) antagonists, telenzepine and pirenzipine, and to a lesser extent the M(2) antagonist methoctramine, protected against muscarine's inhibition of the evoked polysynaptic currents. Other M(2) antagonists (tripitramine and gallamine) as well as the M(3) antagonist 4-DAMP mustard (4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride) and an M(4) antagonist (tropicamide) provided little or no protection against muscarine in this assay. In contrast, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride, tropicamide and telenzepine, but not pirenzepine, methoctramine, tripitramine and gallamine, blocked muscarine's enhancement of spontaneous GABAergic currents. McN-A-343 [(4-hydroxy-2-butynyl)-1-trimethylammonium-m-chlorocarbanilate chloride] and CDD-0097 (5-propargyloxycarbonyl-1,4,5,6-tetrahydropyrimidine hydrochloride), two M(1) agonists, mimicked muscarine's inhibition of the evoked polysynaptic GABAergic currents but did not mimic muscarine's enhancement of spontaneous GABAergic currents. Both actions of muscarine persisted when slices were pretreated with pertussis toxin or N-ethylmaleimide, which inactivate G-proteins coupled to M(2) and M(4) receptors while leaving G-proteins coupled to M(1), M(3) and M(5) receptors intact. Muscarine had no significant effect on the amplitude of the direct postsynaptic current elicited by exogenous GABA in the presence of tetrodotoxin.The results demonstrate that distinct muscarinic receptors oppositely modulate GABAergic transmission in the lateral spiriform nucleus. The receptor mediating the inhibition of evoked GABAergic polysynaptic currents is pharmacologically similar to an M(1) receptor, while the enhancement of spontaneous GABAergic currents appears to be mediated by an M(3) receptor.     

Presynaptic GABAA receptors facilitate spontaneous glutamate release from presynaptic terminals on mechanically dissociated rat CA3 pyramidal neurons

Mossy fiber-derived giant spontaneous miniature excitatory postsynaptic currents have been suggested to be large enough to generate action potentials in postsynaptic CA3 pyramidal neurons. Here we report on the functional roles of presynaptic GABA(A) receptors on excitatory terminals in contributing to spontaneous glutamatergic transmission to CA3 neurons. In mechanically dissociated rat hippocampal CA3 neurons with adherent presynaptic nerve terminals, spontaneous excitatory postsynaptic currents were recorded using conventional whole-cell patch clamp recordings. In most recordings, unusually large spontaneous excitatory postsynaptic currents up to 500 pA were observed. These large spontaneous excitatory postsynaptic currents were highly sensitive to group II metabotropic glutamate receptor activation, and were still observed even after the blockade of voltage-dependent Na(+) or Ca(2+) channels. Exogenously applied muscimol (0.1-3 microM) significantly increased the frequency of spontaneous excitatory postsynaptic currents including the large ones. This facilitatory effect of muscimol was completely inhibited in the presence of 10 microM 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid HBr, a specific GABA(A) receptor antagonist. Pharmacological data suggest that activation of presynaptic GABA(A) receptors directly depolarizes glutamatergic terminals resulting in the facilitation of spontaneous glutamate release. In the current-clamp condition, a subset of large spontaneous excitatory postsynaptic potentials triggered action potentials, and muscimol greatly increased the frequency of spontaneous excitatory postsynaptic potential-triggered action potentials in postsynaptic CA3 pyramidal neurons. The results suggest that presynaptic GABA(A) receptors on glutamatergic terminals play an important role in the excitability of CA3 neurons as well as in the presynaptic modulation of glutamatergic transmission onto hippocampal CA3 neurons.     

Synaptically released GABA activates both pre- and postsynaptic GABA(B) receptors in the rat globus pallidus

The globus pallidus (GP) contains abundant GABAergic synapses and GABA(B) receptors. To investigate whether synaptically released GABA can activate pre- and postsynaptic GABA(B) receptors in the GP, physiological recordings were performed using rat brain slice preparations. Cell-attached recordings from GABA(A) antagonist-treated preparations revealed that repetitive local stimulation induced a GABA(B) antagonist-sensitive pause in spontaneous firings of GP neurons. Whole cell recordings revealed that the repetitive stimulation evoked fast excitatory postsynaptic potentials followed by a slow inhibitory postsynaptic potential (IPSP) in GP neurons. The slow IPSP was insensitive to a GABA(A) receptor antagonist, increased in amplitude with the application of ionotropic glutamate receptor antagonists, and was suppressed by the GABA(B) antagonist CGP55845. The reversal potential of the slow IPSP was close to the potassium equilibrium potential. These results suggest that synaptically released GABA activated postsynaptic GABA(B) receptors and induced the pause and the slow IPSP. On the other hand, in the neurons that were treated to block postsynaptic GABA(B) responses, CGP55845 increased the amplitudes of repetitive local stimulation-induced GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) but not the ionotropic glutamate-mediated excitatory postsynaptic currents. Moreover, the GABA(B) receptor specific agonist baclofen reduced the frequency of miniature IPSCs without altering their amplitude distributions. These results suggest that synaptically released GABA also activated presynaptic GABA(B) autoreceptors, resulting in decreased GABA release in the GP. Together, we infer that both pre- and postsynaptic GABA(B) receptors may play crucial roles in the control of GP neuronal activity.     

Serotonin modulates synaptic transmission in immature rat ventrolateral medulla neurons in vitro.

Patch-clamp recordings in whole-cell configuration were made from ventrolateral medulla neurons of brainstem slices from 8-12-day-old rats. 5-Hydroxytryptamine (3-30 microM) concentration-dependently suppressed excitatory and inhibitory postsynaptic currents evoked by focal stimulation. An augmentation of inhibitory synaptic currents by 5-hydroxytryptamine was noted in a small number of neurons. 5-Hydroxytryptamine depressed synaptic currents with or without causing a significant change in holding currents and membrane conductances; the inward or outward currents induced by exogenously applied glutamate or GABA/glycine were also not significantly changed by 5-hydroxytryptamine. In paired-pulse paradigms designed to evaluate a presynaptic site of action, 5-hydroxytryptamine suppressed synaptic currents but enhanced the paired-pulse facilitation. 5-Hydroxytryptamine reduced the frequency of miniature excitatory postsynaptic currents without significantly affecting the amplitude. 5-Carboxamidotryptamine, 8-hydroxy-2(di-n-propylamino)tetralin, sumatriptan and N-(3-trifluoromethylphenyl)piperazine which exhibit 5-hydroxytryptamine1 receptor agonist activity, depressed synaptic currents with different potencies, with 5-carboxamidotryptamine being the most potent. The non-selective 5-hydroxytryptamine1 receptor antagonist pindolol attenuated the presynaptic effect of 5-hydroxytryptamine, whereas the 5-hydroxytryptamine1A antagonist pindobind-5-hydroxytryptamine1A and 5-hydroxytryptamine2 receptor antagonist ketanserin were ineffective. Our results indicate that 5-hydroxytryptamine suppressed synaptic transmission in ventrolateral medulla neurons by activating presynaptic 5-hydroxytryptamine1 receptors, probably the 5-hydroxytryptamine1B/5-hydroxytryptamine1D subtype. In addition, 5-hydroxytryptamine augmented inhibitory synaptic currents in a small number of neurons the site and mechanism of this potentiating action are not known.     

GABA(B) modulation of GABA(A) and glycine receptor-mediated synaptic currents in hypoglossal motoneurons

We studied the effects of GABA(B) receptor activation on either glycine or GABA(A) receptor-mediated synaptic transmission to hypoglossal motoneurons (HMs, P8-13) using a rat brainstem slice preparation. Activation of GABA(B) receptors with baclofen, a GABA(B) receptor agonist, inhibited the amplitude of evoked glycine and GABA(A) receptor-mediated inhibitory postsynaptic currents. Additionally, with blockade of postsynaptic GABA(B) receptors baclofen decreased the frequency of both glycine and GABA(A) receptor-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs), indicating a presynaptic site of action. Conversely, the GABA(B) receptor antagonist CGP 35348 increased the frequency of glycine receptor-mediated mIPSCs. Application of the GABA transport blocker SKF 89976A decreased the frequency of glycinergic mIPSCs. Lastly, we compared the effects of baclofen on the frequency of glycine and GABA(A) receptor-mediated mIPSC during HM development. At increased postnatal ages (P8-13 versus P1-3) mIPSC frequency was more strongly reduced by baclofen. These results show that presynaptic GABA(B) receptors inhibits glycinergic and GABAergic synaptic transmission to HMs, and the presynaptic sensitivity to baclofen is increased in P8-13 versus P1-3 HMs. Further, endogenous GABA is capable of modulating inhibitory synaptic transmission to HMs.     

Nicotine modulates evoked GABAergic transmission in the brain

The effects of nicotine on evoked GABAergic synaptic transmission were examined using whole cell recordings from neurons of the lateral spiriform nucleus in embryonic chick brain slices. All synaptic activities were abolished by the GABA(A) receptor antagonist, bicuculline (20 microM). Under voltage-clamp with KCl-filled pipettes (holding potential -70 mV), nicotine (0.1-1.0 microM) increased the frequency of spontaneous GABAergic currents in a dose-dependent manner. Nicotine enhanced electrically evoked GABAergic transmission only at relatively low concentrations of 50-100 nM (but not 25 nM), which approximate the concentrations of nicotine in the blood produced by cigarette smoking. At higher concentrations nicotine had either no effect (0.25 microM) or diminished (0.5-1.0 microM) evoked GABAergic neurotransmission. Nicotine had no significant effect on the postsynaptic current induced by exogenous GABA (30-50 microM). These data imply that nicotine levels attained in smokers are sufficient to enhance evoked GABAergic transmission in the brain, and that this effect is most likely mediated through activation of presynaptic nicotinic receptors.     

Muscarinic receptors mediate enhancement of spontaneous GABA release in the chick brain

The functional role of muscarinic acetylcholine receptors in the lateral spiriform nucleus was studied in chick brain slices. Whole-cell patch-clamp recordings of neurons in the lateral spiriform nucleus revealed that carbachol enhanced GABAergic spontaneous inhibitory postsynaptic currents. The duration of the response to carbachol was significantly reduced after blockade of muscarinic receptors with atropine. In the presence of the nicotinic receptor antagonist dihydro-β-erythroidine, carbachol produced a delayed but prolonged enhancement of spontaneous GABAergic inhibitory postsynaptic currents that was completely blocked by atropine. Muscarine also enhanced the frequency of spontaneous GABAergic inhibitory postsynaptic currents in a dose-dependent manner, but had no effect on inhibitory postsynaptic current amplitude. While 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride, a M₃ antagonist, completely blocked muscarine's effect, telenzepine, a M₁ antagonist, and tropicamide, a M₄ antagonist, only partially decreased the response to muscarine. Pirenzepine, a M₁ antagonist, and methoctramine, a M₂ antagonist, potentiated muscarine's enhancement of spontaneous GABAergic inhibitory postsynaptic currents. Muscarine's action was blocked by tetrodotoxin, cadmium chloride and ω-conotoxin GVIA, but was not affected by dihydro-β-erythroidine, 6-cyano-7-nitroquinoxaline-2,3-dione, d(−)-2-amino-5-phosphonopentanoic acid, naloxone or fluphenazine.These results demonstrate that activation of both muscarinic and nicotinic acetylcholine receptors can enhance GABAergic inhibitory postsynaptic currents in the lateral spiriform nucleus. The muscarinic response has a slower onset but lasts longer than the nicotinic effect. The M₃ receptor subtype is predominantly involved in enhancing spontaneous GABAergic inhibitory postsynaptic currents. These M₃ receptors must be located some distance from GABA release sites, since activation of voltage-dependent sodium channels, and consequent activation of N-type voltage-dependent calcium channels, is required to trigger enhanced GABA release following activation of muscarinic receptors.     

Muscarinic M2 and M1 receptors reduce GABA release by Ca2+ channel modulation through activation of PI3K/Ca2+ -independent and PLC/Ca2+ -dependent PKC

We measured pharmacologically isolated GABAergic currents from layer II/III neurons of the rat auditory cortex using patch-clamp recording. Activation of muscarinic receptors by muscarine (1 microM) or oxotremorine (10 microM) decreased the amplitude of electrically evoked inhibitory postsynaptic currents to about one third of their control value. Neither miniature nor exogenously evoked GABAergic currents were altered by the presence of muscarinic agonists, indicating that the effect was spike-dependent and not mediated postsynaptically. The presence of the N- or P/Q-type Ca(2+) channel blockers omega-conotoxin GVIA (1 microM) or omega-AgaTx TK (200 nM) greatly blocked the muscarinic effect, suggesting that Ca(2+)-channels were target of the muscarinic modulation. The presence of the muscarinic M(2) receptor (M(2)R) antagonists methoctramine (5 muM) or AF-DX 116 (1 microM) blocked most of the muscarinic evoked inhibitory postsynaptic current (eIPSC) reduction, indicating that M(2)Rs were responsible for the effect, whereas the remaining component of the depression displayed M(1)R-like sensitivity. Tissue preincubation with the specific blockers of phosphatidyl-inositol-3-kinase (PI(3)K) wortmannin (200 nM), LY294002 (1 microM), or with the Ca(2+)-dependent PKC inhibitor G? 6976 (200 nM) greatly impaired the muscarinic decrease of the eIPSC amplitude, whereas the remaining component was sensitive to preincubation in the phospholipase C blocker U73122 (10 microM). We conclude that acetylcholine release enhances the excitability of the auditory cortex by decreasing the release of GABA by inhibiting axonal V-dependent Ca(2+) channels, mostly through activation of presynaptic M(2)Rs/PI(3)K/Ca(2+)-independent PKC pathway and-to a smaller extent-by the activation of M(1)/PLC/Ca(2+)-dependent PKC.     

Presynaptic muscarinic (M3) receptors reduce excitatory transmission in dopamine neurons of the rat mesencephalon.

The effects of carbachol (0.01-30 microM) and muscarine (10-30 microM) on the excitatory synaptic potentials were studied using conventional intracellular recordings from dopaminergic neurons in rat mesencephalic slices. Both muscarinic agonists reversibly reduced the excitatory synaptic potentials, evoked by local electrical stimulation. The EC50 for carbachol was determined to be 4.5 microM. The maximal degree of the excitatory synaptic potentials suppression caused by carbachol and muscarine was around 40% of control. This suppression was completely blocked by the non-specific muscarinic antagonist atropine (1 microM) and the selective M3 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (1 microM). Other antagonists, preferentially acting at M1, M2 and M4 receptors, were not effective. Furthermore, the acetylcholinesterase inhibitor, physostigmine (50 microM), decreased the amplitude of the excitatory synaptic potentials, indicating that ambient acetylcholine can depress this potential. Direct depolarizing responses to glutamate were not changed by muscarine. In addition, muscarine facilitated the second excitatory synaptic potentials during a paired-pulse protocol. Thus, the effect of the muscarinic agonists is attributable to a presynaptic locus of action. The action of muscarine was not mediated by an N-ethylmaleimide-sensitive G-protein since it was not modified by a treatment of the slices with this agent. The calcium channels blockers, omega-conotoxin GIVA, omega-agatoxin IVA and omega-conotoxin MVIIC did not affect the action of muscarine on the excitatory synaptic potentials. When the potassium currents were reduced by extracellular barium and 4-aminopyridine, the muscarinic agonists still depressed the excitatory synaptic potentials. Our data indicate that presynaptically located M3 receptors modulate the excitatory transmission to midbrain dopaminergic neurons via a N-ethylmaleimide-insensitive G-protein which activates mechanisms neither linked to N-, P-, Q-type calcium channels nor to barium- and 4-aminopyridine-sensitive potassium channels.     

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.     

Presynaptic GABA(B) receptors modulate organum vasculosum lamina terminalis-evoked postsynaptic currents in rat hypothalamic supraoptic neurons

Whole-cell patch-clamp recordings obtained from 36 hypothalamic supraoptic nucleus neurons in explant preparations evaluated a role for GABA(B) receptors in modulating postsynaptic inhibitory and excitatory currents evoked by electrical stimulation in the organum vasculosum of the lamina terminalis. At a holding current of -65 mV, application of baclofen (1-10 microM) induced a dose-dependent reduction in the amplitude of pharmacologically isolated inhibitory and excitatory postsynaptic currents, converted paired-pulse depression in inhibitory postsynaptic currents to paired-pulse facilitation, and enhanced paired-pulse ratios for excitatory postsynaptic currents. In media containing 2-hydroxysaclofen (200-400 microM), baclofen-associated events were blocked and paired-pulse depression in evoked inhibitory postsynaptic currents was abolished. In addition, a progressive increase in the amplitude of inhibitory postsynaptic currents implied that GABA was endogenously active at presynaptic GABA(B) receptors. In contrast, no paired-pulse depression was observed for inhibitory postsynaptic currents evoked in six non-magnocellular neurons. Neither baclofen nor 2-hydroxysaclofen altered holding currents or input resistances in supraoptic neurons, or altered the kinetics of the evoked responses.These observations imply that the terminals of both inhibitory (GABAergic) and excitatory (glutamatergic) afferents to supraoptic nucleus neurons from organum vasculosum lamina terminalis neurons are subject to modulation by presynaptic GABA(B) receptors, and that this modulation is preferentially directed to the inhibitory inputs.     

Opioid inhibition of GABAergic neurotransmission in mechanically isolated rat periaqueductal gray neurons

The descending pain control system is activated by opioid peptides mainly at the midbrain periaqueductal gray (PAG). Although activation of presynaptic opioid receptors has been reported to inhibit gamma-aminobutyric acid (GABA) release, the exact electrophysiological mechanisms are controversial. To elucidate the mechanisms involved in the opioid modulation of presynaptic GABA release, we isolated single PAG neurons with functionally intact synaptic terminals by a mechanical dissociation in the absence of enzyme. With the conventional whole-cell recording mode under the voltage-clamp conditions, the spontaneous miniature inhibitory postsynaptic currents (mIPSCs) were recorded. Bicuculline completely and reversibly blocked mIPSCs. A specific mu-opioid agonist, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), reversibly reduced the frequency of mIPSCs without any alteration of amplitude. The inhibitory effect of DAMGO was blocked by N-ethylmaleimide. Blockade of presynaptic Ca(2+) influx by cadmium or depletion of extracellular Ca(2+) did not alter the DAMGO inhibition. In addition, K(+) channels blockers, Ba(2+) or 4-aminopyridine, did not affect the DAMGO effect. The present study indicates that activation of presynaptic mu-opioid receptors coupled to G-proteins inhibits GABA release through unknown intracellular mechanisms downstream of Ca(2+) influx.