Ke Zhong Shen

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

1 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. 2 Dopamine reversibly reduced the amplitude of GABAA IPSCs by up to 48% with an IC50 value of 3.4 ± 0.8μm. 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. 3 Muscarine reversibly reduced the amplitude of GABAA IPSCs by up to 70 % with an IC50 value of 0.6 ± 0.1μm. Inhibition of IPSCs by muscarine was completely blocked by scopolamine (10 μm), a muscarinic receptor antagonist. The M3 muscarinic receptor antagonist 4‐DAMP effectively reversed muscarine‐induced inhibition of IPSCs with an IC50 of 0.11 ± 0.03μm. 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 ± 9.4μm). 4 Dopamine and muscarine both increased the paired‐pulse ratio of GABAA IPSCs. Neither agent produced sustained changes in postsynaptic holding current. 5 Glutamate EPSCs were also inhibited reversibly by dopamine (by up to 29 %; IC50= 16 ± 3 μm) and muscarine (by up to 41 %; IC50=1.0 ± 0.4μm). However, both agents were more potent and efficacious for reducing GABA IPSCs compared with glutamate EPSCs. 6 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.

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 mm. The GABAB antagonist CGP 35348 blocked this effect with a Kd value estimated by Schild analysis of 5 mm. 3 Adenosine reduced IPSCs by 48% at its IC50 value of 56 mm. 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‐l,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 mm) and adenosine (IC50= 57 mm) Schild analysis yielded a Kd value of 11 mm for antagonism of baclofen‐induced inhibition of EPSCs by CGP 35348. DPCPX (1 mm) completely blocked the inhibitory effects of adenosine (100 mm) 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.

Muscarine increases cation conductance and decreases potassium conductance in rat locus coeruleus neurones

1. Whole‐cell patch‐clamp recordings were made from rat locus coeruleus neurones in slices of brain tissue in vitro. Muscarine (30 microM) caused an inward current of about 100 pA in neurones voltage clamped at ‐60 mV. 2. In about 75% of cells, the current elicited by muscarine was independent of potential in the range ‐60 to ‐120 mV and had no associated conductance change. 3. In about 25% of cells, the current became smaller with hyperpolarization, was associated with a decreased conductance, and reversed polarity between ‐100 and ‐140 mV. The reversal potential changed with the logarithm of the extracellular potassium concentration. Barium and caesium blocked inward rectification and also prevented reversal of the muscarine current. 4. When potassium ions of the intracellular and extracellular solutions were replaced by caesium, the current evoked by muscarine became smaller with depolarization at reversed polarity at +9 mV. This current was associated with an increase in conductance, and was greatly reduced when the extracellular sodium concentration was reduced to 20 mM. 5. The results could be quantitatively accounted for by a model in which muscarine both increases a voltage‐independent cation conductance and decreases the inward rectifier potassium conductance.

Vasodilatation of arterioles by acetylcholine released from single neurones in the guinea‐pig submucosal plexus.

The nervous control of arterioles in the guinea‐pig submucosal plexus was studied. Outside diameters of arterioles were recorded using a video‐monitoring system. Changes in arteriolar diameter in response to electrical stimulation of single neurones or ganglia in the plexus were measured. 2. When the arteriole was pre‐constricted with the prostaglandin analogue U46619 or with phenylephrine, electrical stimulation (2‐20 Hz, 10 s) of a ganglion dilated the blood vessel. This vasodilatation was abolished by tetrodotoxin or by cutting the fine nerve strands running between the ganglion and the arteriole. 3. The vasodilatations caused by ganglionic stimulation were blocked by the muscarinic antagonists atropine, pirenzepine, (11[[2‐[(diethylamino)methyl]‐1‐piperidinyl]acetyl]‐5,11‐dihydro‐6H‐ pyrido[2,3‐b][1,4]benzodiazepine‐6‐)‐one (AFDX‐116), 4‐diphenylacetoxy‐N‐methyl‐piperidine methiodide (4‐DAMP) and hexahydrosilodifenidol (HSDF). IC50 values for the inhibition of nerve‐evoked vasodilatation by pirenzepine, AFDX‐116 and HSDF were 500 nM, 4 microM and 25 nM respectively. Physostigmine (1 microM) increased the dilatation by 90%. 4. Muscarine dilated all submucosal arterioles; the concentration causing half‐maximum effects was 200 nM. Muscarinic vasodilatations were inhibited by pirenzepine, AFDX‐116, and HSDF in a competitive manner; dissociation equilibrium constants determined by Schild analyses were 125 nM, 1.3 microM and 4 nM respectively. 5. Gossypol, an irreversible inhibitor of the production of endothelium‐derived relaxing factor (EDRF), did not reduce the vasodilatation produced by either ganglionic stimulation or muscarine in submucosal arterioles. 6. Intracellular recordings were made from submucosal neurones and action potentials were elicited by depolarizing current pulses (10 ms duration, 10 Hz/10 s). In seven neurones vasodilatation was associated with intracellularly evoked action potentials; this vasodilatation was blocked by pirenzepine. Cell bodies of reidentified vasodilator neurones were subsequently shown to contain immunoreactive choline acetyltransferase. 7. These results show that cholinergic neurones in the submucosal plexus project to submucosal arterioles and that they release acetylcholine onto muscarinic receptors to produce vasodilatation. The muscarinic receptor activated by nerve‐released acetylcholine is the M3 subtype and its location appears to be on the vascular smooth muscle rather than the endothelium.

Dopamine depletion alters responses to glutamate and GABA in the rat subthalamic nucleus.

We used whole-cell recordings to compare currents evoked by glutamate and GABA receptor agonists in subthalamic nucleus neurons located ipsilateral and contralateral to unilateral 6-hydroxydopamine (6-OHDA) injections into the substantia nigra zona compacta. The ratio of currents evoked by AMPA (0.6 μM) and NMDA (20 μM) was significantly greater in neurons recorded ipsilateral to 6-OHDA lesions compared with the ratio of currents recorded in control (contralateral) neurons. Both the GABAA agonist isoguvacine (20 μM) and the GABAB agonist baclofen (10 μM) evoked significantly greater outward currents in subthalamic nucleus neurons ipsilateral to the lesion compared to contralateral neurons. We conclude that chronic dopamine depletion up-regulates expression of GABA receptors and shifts the functional expression of ionotropic glutamate receptor subtype from NMDA to AMPA receptors in subthalamic nucleus.

Calcium‐dependent subthreshold oscillations determine bursting activity induced by N‐methyl‐d‐aspartate in rat subthalamic neurons in vitro

We used whole‐cell patch recordings in current clamp to investigate the ionic dependence of burst firing induced by N‐methyl‐d‐aspartate (NMDA) in neurons of the subthalamic nucleus (STN) in slices of rat brain. NMDA (20 µm) converted single‐spike firing to burst firing in 87% of STN neurons tested. NMDA‐induced bursting was blocked by AP5 (50 µm), and was not mimicked by the non‐NMDA receptor agonist AMPA (0.6 µm). Tetrodotoxin (1 µm) converted bursts to oscillations of membrane potential, which were most robust when oscillations ranged between −50 and −70 mV. The NMDA bursts were blocked by an elevated extracellular concentration of Mg2+, but superfusate containing no added Mg2+ either reduced or increased burst firing, depending upon the amount of intracellular current injection. Block of K+ conductances by apamin and tetraethylammonium prolonged burst duration, but iberiotoxin had no effect. NMDA‐induced burst firing and membrane oscillations were completely blocked by superfusate containing no added Ca2+, and they were significantly reduced when patch pipettes contained BAPTA. Selective antagonists for T‐type (mibefradil, 10 µm), L‐type (nifedipine, 3 µm), and N‐type (ω‐conotoxin GVIA, 1 µm) Ca2+ channels had no effect on NMDA burst firing. Superfusate containing a low concentration of Na+ (20 mm) completely abolished NMDA‐induced burst firing. Flufenamic acid (10 µm), which blocks current mediated by Ca2+‐activated nonselective cation channels (ICAN), reversibly abolished NMDA‐depended bursting. These results are consistent with the hypothesis that NMDA‐induced burst firing in STN neurons requires activation of either an ICAN or a Na+–Ca2+ exchanger.

Pharmacological identification of inward current evoked by dopamine in rat subthalamic neurons in vitro.

Dopaminergic mechanisms in the subthalamic nucleus (STN) are implicated in the pathophysiology of Parkinsons disease. Here, electrophysiological responses of STN neurons to dopamine (DA) were investigated by using whole-cell patch-clamp recordings in the rat brain slice preparation. Under current-clamp, DA depolarized membrane potential and increased the frequency of spontaneous action potentials of STN neurons. Under voltage-clamp, DA (3-300 microM) produced a reversible concentration-dependent inward current (I(DA); 6-40 pA) with an EC(50) of 13 microM. This DA-induced current had a negative slope conductance which reversed at -102 mV. It was partially reduced by barium and by superfusion with an elevated concentration of extracellular K(+). Moreover, TTX and glutamate receptor antagonists (CNQX and AP5) did not significantly affect the DA responses, indicating that I(DA) is not dependent upon afferent synaptic activity in the STN. Quinpirole, a D(2) receptor agonist, mimicked the DA action more effectively than did the D(1) agonist SKF-38393. The D(2) antagonist sulpiride, but not the D(1) antagonist SCH-23390, blocked responses induced by DA. Intracellular application of G-protein inhibitor GDP-beta-S also suppressed I(DA). GTP-gamma-S, added to the pipette solution, evoked a sustained inward shift in the absence of DA. These results suggest that DA increases the activity of STN neurons via activation of G-protein-coupled D(2)-like receptors which reduce a K(+) conductance.

Substance P mediates neurogenic vasodilatation in extrinsically denervated guinea-pig submucosal arterioles.

1. Arteriolar diameter was measured using an optical method in preparations of guinea‐pig submucosal plexus in vitro. Electrical stimulation of one or more neurones in ganglia of the submucosal plexus causes a cholinergic vasodilatation in normal animals. The vasomotor innervation to the arterioles was studied in guinea‐pigs in which the extrinsic nerves to the intestine had been removed. Tissues were processed for immunohistochemistry after the in vitro experiments. 2. Extrinsic denervation resulted in complete loss of catecholamine fluorescence, NPY (neuropeptide Y) and CGRP (calcitonin gene‐related peptide) immunofluorescence around the blood vessels and no neurogenic vasoconstriction was observed up to 60 days post‐denervation. Vasodilatation in response to ganglionic stimulation was increased; smaller arterioles (outside diameter less than 40 microns) showed a greater enhancement of neurogenic vasodilatation than larger arterioles. 3. Nerve‐evoked vasodilatations were only partially inhibited by muscarinic antagonists at 30‐60 days after extrinsic denervations. 4. The non‐cholinergic neurogenic vasodilatation was abolished by the substance P antagonists, spantide, [D‐Arg1, D‐Pro2, D‐Trp7.9, Leu11]substance P and [D‐Arg1, D‐Phe5, D‐Trp7.9, Leu11]substance P. These antagonists did not alter the cholinergic vasodilatation in normal or extrinsically denervated arterioles. 5. Exogenous substance P dilated all submucosal arterioles; the concentration which produced half‐maximal vasodilatations was 2.5 mM in both normal and extrinsically denervated arterioles. Substance P antagonists inhibited the vasodilatation caused by substance P at concentrations similar to those needed to block nerve‐mediated vasodilatation. 6. There was a strong correlation between the finding of non‐cholinergic vasodilatation in response to ganglionic stimulation, and the presence of substance P‐immunoreactive fibres running from ganglion to arteriole. This correlation did not exist for VIP (vasoactive intestinal peptide). 7. These results suggest that intrinsic intestinal substance P‐containing nerve fibres supply submucosal arterioles after sympathetic efferents and sensory afferents are removed. Stimulation of these nerves releases substance P to produce arteriolar dilatation.

Mechanisms underlying presynaptic inhibition through alpha 2‐adrenoceptors in guinea‐pig submucosal neurones.

1. Intracellular recordings were made from submucosal neurones of the guinea‐pig ileum. The actions of noradrenaline, somatostatin and [Met5]enkephalin on nicotinic synaptic potentials (EPSPs) were studied. 2. In one series of experiments, agonists were applied by superfusion; noradrenaline (0.1‐20 microM) decreased EPSP amplitude by 95‐100% in all neurones. Similar application of somatostatin (1‐100 nM) inhibited EPSPs in about half the neurones by a maximum of 40%. [Met5]enkephalin (0.1‐10 microM) did not alter EPSPs. Idazoxan and yohimbine competitively antagonized the action of noradrenaline with dissociation equilibrium constants of 20 and 30 nM respectively. 3. In another series of experiments, noradrenaline and somatostatin were applied locally from a pipette so that they reached presynaptic terminals but not the cell bodies or axons of the presynaptic cell: noradrenaline inhibited EPSPs by 90% in all neurones but somatostatin had no effect. When applied locally to the cell bodies giving rise to the presynaptic fibres, both agonists inhibited EPSPs in half the neurones by 40%. 4. When noradrenaline was applied locally to presynaptic terminals, the latency to onset of noradrenaline to inhibit EPSPs was 45‐160 ms; cadmium applied similarly depressed EPSPs in 5‐50 ms. 5. Pertussis toxin pre‐treatment only partially blocked presynaptic inhibition caused by noradrenaline but abolished the reduction of EPSP amplitude by somatostatin. 6. It is concluded that noradrenaline and somatostatin reduce the amplitude of the fast EPSP because they hyperpolarize cell bodies and prevent action potential initiation. Noradrenaline, but not somatostatin, has an additional action to inhibit acetylcholine release by acting at nerve terminal receptors. 7. The presynaptic inhibitory action of noradrenaline results from activation of alpha 2‐adrenoceptors at nerve terminals but the mechanism(s) by which these presynaptic receptors act cannot be explained adequately by either activation of a potassium conductance and/or inhibition of a calcium conductance.

Functional characterization of neuronal pre and postsynaptic α2-adrenoceptor subtypes in guinea-pig submucosal plexus

1 The α2‐adrenoceptors on cell bodies of submucosal neurones, on presynaptic cholinergic nerve terminals innervating submucosal neurones, and on presynaptic sympathetic fibres innervating submucosal arterioles were characterized in functional studies by use of subtype selective ligands. 2 Both membrane hyperpolarization and presynaptic inhibition of nicotinic excitatory synaptic potentials (e.p.s.ps) produced by UK 14304 were similarly antagonized by idazoxan, yohimbine, SKF 104078, WB 4101 and ARC‐239. Antagonism was competitive and dissociation equilibrium constants were the same for both effects. 3 Vasoconstriction of submucosal arterioles in response to stimulation of the sympathetic nerves (20 Hz for 2 s) was inhibited by UK 14304 and clonidine; concentrations producing half‐maximum responses were 6 nm and 10 nm respectively. Idazoxan, yohimbine, WB 4101 and SKF 104078 antagonized this action, with dissociation constants similar to those for antagonism of the postsynaptic membrane hyperpolarization and presynaptic inhibition of nicotinic e.p.s.ps. 4 Oxymetazoline was a partial agonist when membrane hyperpolarization or presynaptic inhibition of nicotinic e.p.s.ps were measured but a full agonist when presynaptic inhibition of sympathetically‐mediated arteriolar vasoconstriction was measured. As an agonist, oxymetazoline produced half maximum responses at 80–120 nm; the dissociation constant for oxymetazoline as an antagonist was 130 nm. 5 Neither prazosin nor chlorpromazine (up to 30 μm) altered any of the three responses to α2‐adrenoceptor agonists. 6 It is concluded that α2‐adrenoceptors present on submucosal neuronal cell bodies, on presynaptic cholinergic nerve terminals and on presynaptic sympathetic nerve terminals are the α2A subtype. However, functional characterization of this subtype differs from that provided by ligand binding studies.