Receptor-mediated presynaptic facilitation of quantal release of acetylcholine induced by pralidoxime inAplysia

1. Possible interactions of contrathion (pralidoxime sulfomethylate), a reactivator of phosphorylated acetylcholinesterase (AChE), with the regulation of cholinergic transmission were investigated on an identified synapse in the buccal ganglion of Aplysia californica. 2. Transmitter release was evoked either by a presynaptic action potential or, under voltage clamp, by a long depolarization of the presynaptic cell. At concentrations higher than 10(-5) M, bath-applied contrathion decreased the amplitude of miniature postsynaptic currents and increased their decay time. At the same time, the quantal release of ACh was transiently facilitated. The facilitatory effect of contrathion was prevented by tubocurarine but not by atropine. Because in this preparation, these drugs block, respectively, the presynaptic nicotinic-like and muscarinic-like receptors involved in positive and negative feedback of ACh release, we proposed that contrathion activates presynaptic nicotinic-like receptors. 3. Differential desensitization of the presynaptic receptors is proposed to explain the transience of the facilitatory action of contrathion on ACh release. 4. The complexity of the synaptic action of contrathion raises the possibility that its therapeutic effects in AChE poisonings are not limited to AChE reactivation.     

Differential Postmortem Delay Effect on Agonist-Mediated Phospholipase Cβ Activity in Human Cortical Crude and Synaptosomal Brain Membranes

The phosphoinositide signal transduction system, and particularly, phospholipase Cbeta isozymes, are relevant in the etiopathogeny of human neuropsychiatric pathologies such as depression. Stimulation of phospholipase Cbeta activity by muscarinic receptors and G proteins was determined in crude and synaptosomal membrane preparations from nine postmortem human frontal cortices (postmortem delay range 8 to 50 h). Thus, the phospholipase Cbeta activity was determined by measuring the hydrolysis of exogenous [3H]-phosphatidylinositol 4,5-bisphosphate. There was a postmortem delay-mediated decrease in the PIP2 hydrolysis irrespective of the membrane preparation used (P < 0.05). Moreover, there were statistically significant differences for exponential decay curve parameters (K factor and Span) of PLCbeta activity induced by agonist-mediated activation between crude and synaptosomal membrane preparations. These results show that the postsynaptic component of the PLCbeta activity is more sensible to the postmortem delay effect.     

Possible Involvement of GABAA and GABAB Receptors in the Inhibitory Action of Lindane on Transmitter Release from Cerebellar Granule Neurons

Cerebellar granule cells in culture express receptors for GABA belonging to the GABA_A and GABA_B classes. In order to characterize the ability of the insecticide lindane to interact with these receptors cells were grown in either plain culture media or media containing 150 M THIP as this is known to influence the properties of both GABA_A and GABA_B receptors. It was found that lindane regardless of the culture condition inhibited evoked (40 mM K⁺) release of neurotransmitter ([³H]D-aspartate as label for glutamate). In naive cells both GABA_A and GABA_B receptor active drugs prevented the inhibitory action of lindane but in THIP treated cultures none of the GABA_A and GABA_B receptor active drugs had any effect on the inhibitory action of lindane. This lack of effect was not due to inability of baclofen itself to inhibit transmitter release. It is concluded that lindane dependent on the state of the GABA_A and GABA_B receptors is able to indirectly interfere with both GABA_A and GABA_B receptors. In case of the latter receptors it was shown using [³H]baclofen to label the receptors that lindane could not displace the ligand confirming that lindane is likely to exert its action at a site different from the agonist binding site.     

金褐霉素(Aureofuscin)对神经末梢的递质释放和肌细胞膜的作用

利用细胞内微电极记录,在小鼠离体膈神经膈肌标本上观察了金褐霉素(20μg/ml,过饱和溶液)对运动神经末梢的递质释放和肌细胞膜电位的影响。主要作用是:(1)缓慢降低肌细胞膜的静息电位,这种作用是可逆的;(2)有一促进神经末梢的乙酰胆碱量子释放的时相:提高小终板电位的发放频率和终板电位的平均量子含量,但在持续作用下,它们又恢复至接近对照水平;(3)这种对自发量子释放的作用与胞外 Ca~(2+)有关,(4)不阻遏神经肌肉接头兴奋传递。     

NMDA and Non-NMDA Receptor-Mediated Release of [3H]GABA from Granule Cell Dendrites of Rat Olfactory Bulb

Abstract: We have studied the effect of glutamate and the glutamatergic agonists N-methyl-d -aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on [³H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [³H]GABA release and the release evoked by a high K⁺ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine-2,4-dicarboxylate (2,4-PDC) inhibited by 50% the glutamate-induced [³H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [³H]GABA release. The presence of glycine and the absence of Mg²⁺ have no potentiating effect on NMDA-stimulated release; however, when the tissue was previously depolarized with a high K⁺ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2-amino-5-phosphonoheptanoic acid (AP-7). The kainate and AMPA effects were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by AP-7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP-5); nevertheless, in the presence of glycine, [³H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP-5. Tetrodotoxin inhibited glutamate- and kainate-stimulated [³H]GABA release but not the NMDA-stimulated release. The present results show that in the external plexiform layer of the olfactory bulb, glutamate is stimulating GABA release through a presynaptic, receptor-mediated mechanism as a mixed agonist on NMDA and non-NMDA receptors; glutamate is apparently also able to induce GABA release through heteroexchange.     

Glutamate agonists and [3H]GABA release from rat hippocampal slices: Involvement of metabotropic glutamate receptors in the quisqualate-evoked release

The effects of glutamate agonists and their selective antagonists on the Ca²⁺-dependent and independent releases of [³H]GABA from rat coronal hippocampal slices were studied in a superfusion system. The Ca²⁺-dependent release evoked by glutamate, kainate and N-methyl-D-aspartate (NMDA) gradually declined with time despite the continuous presence of the agonists. Quisqualate (QA) caused a sustained release which exhibited no tendency to decline within the 20-min period of stimulation. This release was enhanced in Ca²⁺-free medium. The release evoked by QA in Ca²⁺-containing medium was significantly inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing that QA activates NMDA receptors directly or indirectly through (RS)--amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. The inhibition of MK-801 was slightly diminished and that of CNQX totally abolished in Ca²⁺-free medium. Verapamil inhibited the QA-activated release in both Ca²⁺-containing and Ca²⁺-free media. The effect of QA but not that of AMPA was blocked in Ca²⁺-free medium by L(+)-2-amino-3-phosphonopropionate (L-AP3), a selective antagonist of the metabotropic glutamate receptor. It is suggested that the sustained release of GABA is also mediated partly by activation of metabotropic receptors and mobilization of Ca²⁺ from intracellular stores.     

Halothane Anesthesia Affects NMDA-Stimulated Cholinergic and GABAergic Modulation of Striatal Dopamine Efflux and Metabolism in the Rat In Vivo

Microdialysis of the striatum of halothane-anesthetized rats was used to study the participation of local cholinergic and GABAergic neurotransmission in NMDA receptor-modulated striatal dopamine release and metabolism. Reverse dialysis.of NMDA (1 mM) evoked a 10-fold increase in dopamine efflux and reduced DOPAC and HVA to > 20% of basal values. The effect of NMDA on dopamine efflux was abolished by atropine (10 microM) but unaffected by (+)-bicuculline (50 microM). NMDA-induced decrease in DOPAC (but not HVA) efflux was potentiated by atropine, whereas (+)-bicuculline attenuated the decrease in DOPAC and HVA. Compared to our previous studies in unanesthetised rats, our data suggest that halothane anesthesia alters the balance between NMDA-stimulated cholinergic and GABAergic influences on striatal dopamine release and metabolism. Differential sensitivity to halothane of NMDA receptors expressed by the neurones mediating these modulatory influences, or loss of specific NMDA receptor populations through voltage-dependent Mg2+ block under anesthesia, could underlie these observations.     

GABA Release Modified by Adenosine Receptors in Mouse Hippocampal Slices under Normal and Ischemic Conditions

The excitatory glutamatergic neurons in the hippocampus are modulated by inhibitory GABA-releasing interneurons. The neuromodulator adenosine is known to inhibit the presynaptic release of neurotransmitters and to hyperpolarize postsynaptic neurons in the hippocampus, which would imply that it is an endogenous protective agent against cerebral ischemia and excitotoxic neuronal damage. Interactions of the GABAergic and adenosinergic systems in regulating neuronal excitability in the hippocampus is of crucial importance, particularly under cell-damaging conditions. We now characterized the effects of adenosine receptor agonists and antagonists on the release of preloaded [³H]GABA from hippocampal slices prepared from adult (3-month-old) mice, using a superfusion system. The effects were tested both under normal conditions and in ischemia induced by omitting glucose and oxygen from the superfusion medium. Basal and K⁺-evoked GABA release in the hippocampus were depressed by adenosinergic compounds. Under normal conditions activation of both adenosine A₁ and A_2A receptors by the agonists R(-)N⁶-(2-phenylisopropyl)adenosine and CGS 21680 inhibited the K⁺-evoked release, which effects were blocked by their specific antagonists, 8-cyclopentyl-1,3-dipropyl-xanthine and 3,7-dimethyl-1-propargylxanthine, respectively. Under ischemic conditions the release of both GABA and adenosine is markedly enhanced. The above receptor agonists then depressed both the basal and K⁺-evoked GABA release, only the action of A_2A receptors being however receptor-mediated. The demonstrated depression of GABA release by adenosine in the hippocampus could be deleterious to neurons and contribute to excitotoxicity.     

A neurochemical approach for studying response to acetylcholine in Alzheimer's disease

The proposal of cholinomimetic treatment as a rational basis for the therapy of Alzheimer's disease has been prematurely dismissed by some workers on the hypothesis of impaired coupling/signal transduction of postsynaptic cholinergic receptors. Disparity of reports studying such impairment may be due to inappropriate extrapolation of experimental systems to the physiological stituation, as well as inadequate consideration of disease epiphenoma. In the present study we have used samples with short duration of terminal coma, collected using techniques to minimise postmortem autolysis, and samples obtained during neurosurgery to examine carbachol stimulated hydrolysis of [³H]phosphatidylinositol (PI) as a marker for receptor/signal transduction integrity. The influence of postmortem delay was also studied using another series of samples and a rat model. While a significant correlation of postmortem delay and carbachol stimulated [³H]PI hydrolysis was found, comparison of pooled neurosurgical and postmortem controls with AD samples revealed no significant reduction. Thus this study concurs with a similar one previously reported here, using [³H]phosphatidylinositol 4,5-bisphosphate (1). They provide evidence for competent receptor-signal transduction events in AD, supporting the use of cholinomimetic therapy for disease treatment.     

Muscarinic M1 acetylcholine receptors regulate the non-quantal release of acetylcholine in the rat neuromuscular junction via NO-dependent mechanism

Nitric oxide (NO), previously demonstrated to participate in the regulation of the resting membrane potential in skeletal muscles via muscarinic receptors, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh release was estimated by the amplitude of endplate hyperpolarization (H-effect) following a blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine. The muscarinic agonists oxotremorine and muscarine lowered the H-effect and the M1 antagonist pirenzepine prevented this effect occurring at all. Another muscarinic agonist arecaidine but-2-ynyl ester tosylate (ABET), which is more selective for M2 receptors than for M1 receptors and 1,1-dimethyl-4-diphenylacetoxypiperidinium (DAMP), a specific antagonist of M3 cholinergic receptors had no significant effect on the H-effect. The oxotremorine-induced decrease in the H-effect was calcium and calmodulin-dependent. The decrease was negated when either NO synthase was inhibited by N(G)-nitro-L-arginine methyl ester or soluble guanylyl cyclase was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. The target of muscle-derived NO is apparently nerve terminal guanylyl cyclase, because exogenous hemoglobin, acting as an NO scavenger, prevented the oxotremorine-induced drop in the H-effect. These results suggest that oxotremorine (and probably also non-quantal ACh) selectively inhibit the non-quantal secretion of ACh from motor nerve terminals acting on post-synaptic M1 receptors coupled to Ca(2+) channels in the sarcolemma to induce sarcoplasmic Ca(2+)-dependent synthesis and the release of NO. It seems that a substantial part of the H-effect can be physiologically regulated by this negative feedback loop, i.e., by NO from muscle fiber; there is apparently also Ca(2+)- and calmodulin-dependent regulation of ACh non-quantal release in the nerve terminal itself, as calmidazolium inhibition of the calmodulin led to a doubling of the resting H-effect.     

DGKι regulates presynaptic release during mGluR-dependent LTD

Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (～2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι-/- synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.     

Metabotropic glutamate receptors modulate GABA release from mouse hippocampal slices

The effects of metabotropic glutamate receptor agonists on the basal and potassium (50 mM K⁺)-stimulated release of [³H]GABA from mouse hippocampal slices were investigated using a superfusion system. The group I agonist (1±)-1-aminocyclopentane-trans-1,3-dicarboxylate enhanced the basal GABA release and reduced the K⁺-evoked release by a mechanism antagonized by (RS)-1-aminoindan-1,5-dicarboxylate in both cases. The group II agonist (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine failed to have any effect on the basal release, but inhibited the stimulated release. This inhibition was not affected by the antagonist (2S)-2-ethylglutamate. The group III agonists L(+)-amino-4-phosphonobutyrate and O-phospho-L-serine inhibited the basal GABA release, which effects were blocked by the antagonist (RS)-2-cyclopropyl-4-phosphonophenylglycine. Moreover, the suppression of the K⁺-evoked release by L(+)2-amino-4-phosphonobutyrate was apparently receptor-mediated, being blocked by (RS)-2-cyclopropyl-4-phosphonophenylglycine. The results show that activation of metabotropic glutamate receptors of group I is able to potentiate the basal release of GABA, whereas activation of groups I and III receptors reduce K⁺-stimulated release in mouse hippocampal slices.     

GABA Induces Functionally Active Low-Affinity GABA Receptors on Cultured Cerebellar Granule Cells

The effect of γ-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo[5-4-c]pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 μM GABA in the culture media led to the induction of low-affinity GABA receptors (K_D 546 ± 117 nM) in addition to the high-affinity receptors (K_D 7 ± 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED₅₀ 10 ± 3 (μM) only when the cells had been cultured in the presence of 50 νM GABA, 50 μM muscimol, or 150 μM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 μM bicuculline and mimicked by 50 μM muscimol or 150 μM THIP whereas 150 μM (-)-baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABA_A receptors.     

A Search for Receptors Modulating the Release of γ-[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices

Various putative striatal transmitters and related compounds were studied for their effects on the release of gamma-aminobutyric acid (GABA) from slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused and stimulated electrically at 5 or 20 Hz. Aminooxyacetic acid was present throughout. The main changes observed were the following. The basal and, less consistently, the electrically evoked overflow of [3H]GABA were enhanced by 3,4-dihydroxyphenylethylamine (dopamine), an effect not blocked by cis-flupentixol or domperidone and not mimicked by apomorphine and D1-selective agonists. The electrically evoked overflow was diminished by 5-hydroxytryptamine (serotonin); the inhibition was prevented by methiothepin. The basal but not the electrically evoked overflow was enhanced by carbachol; acetylcholine and nicotine also accelerated the basal outflow whereas oxotremorine caused no consistent change; the effect of carbachol and acetylcholine were blocked by hexamethonium but not by atropine or by tetrodotoxin. These findings indicate that the GABA neurons in the caudate nucleus may be stimulated by dopamine, although the receptor type involved remains unclear; inhibited by serotonin; and stimulated by acetylcholine acting via a nicotine receptor. However, all drug effects observed were relatively small. No evidence was obtained for autoreceptors, alpha 2-adrenoceptors or receptors for opioids, adenosine or substance P at the GABA neurons.     

Single GABAergic synaptic terminals from rat midbrain exhibit functional P2X and dinucleotide receptors, able to induce GABA secretion

GABAergic terminals from rat midbrain characterized by immunolocalization of glutamic acid decarboxylase and/or the vesicular inhibitory amino acid transporter respond to ATP or P(1),P(5)-di(adenosine-5') pentaphosphate (Ap(5)A) with an increase in the intrasynaptosomal calcium concentration measured by a microfluorimetric technique in single synaptic terminals. The ATP response is mediated through the activation of P2X receptors with an abundant presence of P2X(3) subunits. Ap(5)A, however, exerts its effects by acting through a different receptor termed the dinucleotide receptor. Both receptors, once activated in the presence of extrasynaptosomal calcium, induce a concentration-dependent GABA release from synaptosomal populations with EC(50) values of 16 and 20 microM for ATP and Ap(5)A, respectively. Specific inhibition of GABA release is obtained with pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (80 microM) on the ATP effect and with P(1),P(5)-di(inosine-5') pentaphosphate (100 nM) on the dinucleotide receptor.     

Increased GABA Release in the Human Brain Cortex as a Potential Pathogenetic Basis of Hyperosmolar Diabetic Coma

Human cerebral cortical slices preincubated with [³H]GABA, [³H]noradrenaline, or 5-[³H]hydroxytryptamine and superfused with Krebs solution or Mg²⁺-free Krebs solution were used to investigate the influence of increased D-glucose concentrations on the release of these [³H]-neurotransmitters evoked by high K⁺ content or NMDA receptor activation, respectively. An increase in level of D-glucose (normal content, 11.1 mM) by 32, 60, and/or 100 mM (a range characteristic for hyperosmolar diabetic coma) increased the [³H]GABA release and inhibited the [³H]noradrenaline release evoked by both methods of stimulation. The K⁺-induced 5-[³H]hydroxytryptamine release was also inhibited by high D-glucose content. Blockade of GABA_B receptors by p-(3-aminopropyl)-p-diethoxymethylphosphinic acid (CGP 35348) attenuated the inhibitory effect of high D-glucose content on the K⁺-evoked release of [³H]noradrenaline and 5-[³H]hydroxy-tryptamine, suggesting that the effect on monoamine release is, at least to a major part, the result of the increased GABA release and, as a consequence, of an increased GABA concentration at inhibitory GABA_B receptors. The membrane-impermeable sorbitol mimicked the increasing effect of D-glucose on [³H]GABA release and its inhibitory effect on 5-[³H]hydroxytryptamine release. However, dimethyl sulfoxide, which is known to permeate rapidly through biological membranes, had no effect at concentrations equiosmolar to D-glucose. It is concluded that a reduction in brain cell volume caused by increased extracellular, compared with cytoplasmic, osmolarity is crucial for the changes in neuronal function observed at high D- glucose and sorbitol content, In view of the fact that GABA is the main inhibitory neurotransmitter in the brain, the increased GABA release may be assumed to contribute to the pathogenesis of hyperosmolar diabetic coma.