5-HT3 receptors mediate inhibition of acetylcholine release in cortical tissue

The release of cerebral acetylcholine from terminals in the cerebral cortex has been shown to be regulated by 5-hydroxytryptamine (5-HT) but it is not known which subtype of the 5-HT receptor is involved. 5-HT receptor agonists increase acetylcholine levels in vivo, indicating a reduced turnover, and reduce release of acetylcholine from striatal slices in vitro. Depleting 5-HT by inhibiting synthesis or by destroying the neurons containing 5-HT potentiates acetylcholine release, and increases acetylcholine turnover in the cerebral cortex and hippocampus. Selective antagonists for the 5-HT3 receptor subtypes which seem to have effects on mood and activity may exert their effect through the regulation of acetylcholine release in the cortex and limbic system. Radioligand binding studies show a high density of 5-HT3 receptors in the cholinergic-rich entorhinal cortex and we provide evidence that a reduction in cortical cholinergic function can be effected in vitro by 5-HT3 receptors.     

NMDA receptor agonists selectively block N-type calcium channels in hippocampal neurons.

The modulation of voltage-dependent calcium channels by various neurotransmitters has been demonstrated in many neurons. Because of the critical role of Ca2+ in transmitter release and, more generally, in transmembrane signalling, this modulation has important functional implications. Hippocampal neurons possess low-threshold (T-type) Ca2+ channels and both L- and N-type high voltage-activated Ca2+ channels. N-type Ca2+ channels are blocked selectively by omega-conotoxin and adenosine. These substances both block excitatory synaptic transmission in the hippocampus, whereas dihydropyridines, which selectively block L-type channels, are ineffective. Excitatory synaptic transmission in the hippocampus displays a number of plasticity phenomena that are initiated by Ca2+ entry through ionic channels operated by N-methyl-D-aspartate (NMDA) receptors. Here we report that NMDA receptor agonists selectively and effectively depress N-type Ca2+ channels which are involved in neurotransmitter release from presynaptic sites. The inhibitory effect is eliminated by the competitive NMDA antagonist D-2-amino-5-phosphonovalerate, does not require Ca2+ entry into the cell, and is probably receptor-mediated. This phenomenon may provide a negative feedback between the liberation of excitatory transmitter and entry of Ca2+ into the cell, and could be important in presynaptic inhibition and in the regulation of synaptic plasticity.     

家兔蓝斑复合核注射P物质对膈神经放电的影响及机制

目的 :探讨 P物质在蓝斑复合核对呼吸的作用及机制。方法 :实验在 45只麻醉、制动、人工呼吸的家兔上进行。结果 :将 P物质 (SP)微量注入蓝斑复合核区 (L c— Sc) ,使膈神经放电频率明显增加 ,吸气时程延长 ,呼气时程缩短 ,呼吸频率无明显变化。SP在 L c— Sc区使膈神经放电频率增加的效应可被延髓孤束核 (NTS)分别预注射 α或 β受体阻断剂拮抗。结论 :L c— Sc是 SP在中枢呼吸兴奋效应的有效作用部位 ,其作用机制可能是通过 NTS的 α和 β受体介导     

Muscarinic acetylcholine receptor is involved in acetylcholine regulating stomatal movement

In animal cells, action of acetylcholine depends on its binding with its two specific receptors on the plasma membrane: the nicotinic and muscarinic respectively. The present investigation has shown that agonists of muscarinic receptor (muscarine) could induce stomatal opening, while the antagonists (atropine) could block stomatal opening induced by acetylcholine. Their effects can only be realized in medium containing Ca²⁺, but not in medium containing K⁺. The results tend to reveal that the muscarinic receptor is involved in acetylcholine-induced stomatal movement.     

Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor.

A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.     

Anti-nociceptive role of neuropeptide Y in the nucleus accumbens in rats with inflammation, an effect modulated by mu- and kappa-opioid receptors

Recent study in our laboratory showed that neuropeptide Y (NPY) plays an antinociceptive role in the nucleus accumbens (NAc) in intact rats. The present study was performed to further investigate the effect of NPY in nociceptive modulation in the NAc of rats with inflammation, and the possible interaction between NPY and the opioid systems. Experimental inflammation was induced by subcutaneous injection of carrageenan into the left hindpaw of rats. Intra-NAc administration of NPY induced a dose-dependent increase of hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulations in rats with inflammation. The anti-nociceptive effect of NPY was significantly blocked by subsequent intra-NAc injection of the Y1 receptor antagonist NPY28-36, suggesting an involvement of Y1 receptor in the NPY-induced anti-nociception. Furthermore, intra-NAc administration of the opioid antagonist naloxone significantly antagonized the increased HWLs induced by preceding intra-NAc injection of NPY, suggesting an involvement of the endogenous opioid system in the NPY-induced anti-nociception in the NAc during inflammation. Moreover, the NPY-induced anti-nociception was attenuated by following intra-NAc injection of the μ-opioid antagonist β-funaltrexamine (β-FNA), and κ-opioid antagonist nor-binaltorphimine (nor-BNI), but not by δ-opioid antagonist naltrindole, indicating that μ- and κ-opioid receptors, not δ-opioid receptor, are involved in the NPY-induced anti-nociception in the NAc in rats with inflammation.     

Modulation of glycine receptor chloride channels by cAMP-dependent protein kinase in spinal trigeminal neurons

Glycine is an important inhibitory transmitter in the brainstem and spinal cord. In the trigeminal subnucleus caudalis (medullary dorsal horn) and in the spinal dorsal horn (the relaying centres for processing pain and sensory information), glycine inhibits the glutamate-evoked depolarization and depresses firing of neurons. The binding of glycine to its receptor produces a large increase in Cl- conductance, which causes membrane hyperpolarization. The selectivity and gating properties of glycine receptor channels have been well characterized; the glycine receptor molecules have also been purified. The amino-acid sequence, deduced from complementary DNA clones encoding one of the peptides (the 48K subunit), shows significant homology with gamma-aminobutyric acid A (GABAA) and nicotinic acetylcholine receptor subunits, suggesting that glycine receptors may belong to a superfamily of chemically gated channel proteins. However, very little is known about the modulation of glycine receptor channels. We have investigated the regulation of strychnine-sensitive glycine receptor channels by cyclic AMP-dependent protein kinase in neurons isolated from spinal trigeminal nucleus of rat and report here that the protein kinase A dramatically increased the glycine-induced Cl- currents by increasing the probability of the channel openings. GS protein, which is sensitive to cholera toxin, was involved in the modulation.     

Reduced antinociception in mice lacking neuronal nicotinic receptor subunits

Nicotine exerts antinociceptive effects by interacting with one or more of the subtypes of nicotinic acetylcholine receptors (nAChRs) that are present throughout the neuronal pathways that respond to pain. To identify the particular subunits involved in this process, we generated mice lacking the alpha4 subunit of the neuronal nAChR by homologous recombination techniques and studied these together with previously generated mutant mice lacking the beta2 nAChR subunit. Here we show that the homozygous alpha4-/- mice no longer express high-affinity [3H]nicotine and [3H]epibatidine binding sites throughout the brain. In addition, both types of mutant mice display a reduced antinociceptive effect of nicotine on the hot-plate test and diminished sensitivity to nicotine in the tail-flick test. Patch-clamp recordings further reveal that raphe magnus and thalamic neurons no longer respond to nicotine. The alpha4 nAChR subunit, possibly associated with the beta2 nAChR subunit, is therefore crucial for nicotine-elicited antinociception.     

Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors

Worldwide, 100 million people are expected to die this century from the consequences of nicotine addiction, but nicotine is also known to enhance cognitive performance. Identifying the molecular mechanisms involved in nicotine reinforcement and cognition is a priority and requires the development of new in vivo experimental paradigms. The ventral tegmental area (VTA) of the midbrain is thought to mediate the reinforcement properties of many drugs of abuse. Here we specifically re-expressed the beta2-subunit of the nicotinic acetylcholine receptor (nAChR) by stereotaxically injecting a lentiviral vector into the VTA of mice carrying beta2-subunit deletions. We demonstrate the efficient re-expression of electrophysiologically responsive, ligand-binding nicotinic acetylcholine receptors in dopamine-containing neurons of the VTA, together with the recovery of nicotine-elicited dopamine release and nicotine self-administration. We also quantified exploratory behaviours of the mice, and showed that beta2-subunit re-expression restored slow exploratory behaviour (a measure of cognitive function) to wild-type levels, but did not affect fast navigation behaviour. We thus demonstrate the sufficient role of the VTA in both nicotine reinforcement and endogenous cholinergic regulation of cognitive functions.     

乙酰胆碱对人胃粘膜上皮细胞和胃腺癌细胞内Ca2+、Mg2+浓度的影响

目的:探讨乙酰胆碱对人胃粘膜上皮细胞和胃腺癌细胞内Ca2 、Mg2 浓度的影响。方法:利用大型生化分析仪,检测了乙酰胆碱作用后培养的人胃粘膜上皮细胞和胃腺癌细胞内Ca2 、Mg2 的浓度。结果:与正常胃粘膜上皮细胞比较,胃腺癌细胞内Ca2 、Mg2 浓度偏低(P>0 01)。在乙酰胆碱作用下,胃粘膜上皮细胞内Ca2 浓度升高(P<0 01),Mg2 浓度降低(P<0 01);胃腺癌细胞仅有Ca2 浓度升高(P<0 05),Mg2 浓度无变化。阿托品能阻断乙酰胆碱的作用(P<0 05)。结论:乙酰胆碱对胃粘膜上皮细胞和胃腺癌细胞的作用是通过毒蕈碱受体实现的。     

Postsynaptic NMDA receptor-mediated calcium accumulation in hippocampal CA1 pyramidal cell dendrites

In the CA1 hippocampal region, intracellular calcium is a putative second messenger for the induction of long-term potentiation (LTP), a persistent increase of synaptic transmission produced by high frequency afferent fibre stimulation. Because LTP in this region is blocked by the NMDA (N-methyl-D-aspartate) receptor antagonist AP5 (DL-2-amino-5-phosphonovaleric acid) and the calcium permeability of NMDA receptors is controlled by a voltage-dependent magnesium block, a model has emerged that suggests that the calcium permeability of NMDA receptor-coupled ion channels is the biophysical basis for LTP induction. We have performed microfluorometric measurements in individual CA1 pyramidal cells during stimulus trains that induce LTP. In addition to a widespread component of postsynaptic calcium accumulation previously described, we now report that brief high frequency stimulus trains produce a transient component spatially localized to dendritic areas near activated afferents. This localized component is blocked by the NMDA receptor antagonist AP5. The results directly confirm the calcium rise predicted by NMDA receptor models of LTP induction.     

Regulation of AMPA receptor lateral movements

An essential feature in the modulation of the efficacy of synaptic transmission is rapid changes in the number of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors at post-synaptic sites on neurons. Regulation of receptor endo- and exocytosis has been shown to be involved in this process. Whether regulated lateral diffusion of receptors in the plasma membrane also participates in receptor exchange to and from post-synaptic sites remains unknown. We analysed the lateral mobility of native AMPA receptors containing the glutamate receptor subunit GluR2 in rat cultured hippocampal neurons, using single-particle tracking and video microscopy. Here we show that AMPA receptors alternate within seconds between rapid diffusive and stationary behaviour. During maturation of neurons, stationary periods increase in frequency and length, often in spatial correlation with synaptic sites. Raising intracellular calcium, a central element in synaptic plasticity, triggers rapid receptor immobilization and local accumulation on the neuronal surface. We suggest that calcium influx prevents AMPA receptors from diffusing, and that lateral receptor diffusion to and from synaptic sites acts in the rapid and controlled regulation of receptor numbers at synapses.     

Long-lasting self-inhibition of neocortical interneurons mediated by endocannabinoids

Neocortical GABA-containing interneurons form complex functional networks responsible for feedforward and feedback inhibition and for the generation of cortical oscillations associated with several behavioural functions. We previously reported that fast-spiking (FS), but not low-threshold-spiking (LTS), neocortical interneurons from rats generate a fast and precise self-inhibition mediated by inhibitory autaptic transmission. Here we show that LTS cells possess a different form of self-inhibition. LTS, but not FS, interneurons undergo a prominent hyperpolarization mediated by an increased K+-channel conductance. This self-induced inhibition lasts for many minutes, is dependent on an increase in intracellular [Ca2+] and is blocked by the cannabinoid receptor antagonist AM251, indicating that it is mediated by the autocrine release of endogenous cannabinoids. Endocannabinoid-mediated slow self-inhibition represents a powerful and long-lasting mechanism that alters the intrinsic excitability of LTS neurons, which selectively target the major site of excitatory connections onto pyramidal neurons; that is, their dendrites. Thus, modulation of LTS networks after their sustained firing will lead to long-lasting changes of glutamate-mediated synaptic strength in pyramidal neurons, with consequences during normal and pathophysiological cortical network activities.     

Ca^2＋ is involved in muscarine-acetylcholine-receptor-mediated acetylcholine signal transduction in guard cells of Vicia faba L.

Acetyicholine (ACh) is an important neuro-chemical transmitter in animals; it also exists in plants and plays a significant role in various kinds of physiological functions in plants. ACh has been known to induce the stomatal opening. By monitoring the changes of cytusolic Ca^2 with fluorescent probe Fiuo-3 AM under the confocal microscopy, we found that exogenous ACh increased cytosolic Ca^2 concentration of guard cells of Vicia faba L. Muscarlne, an agonist of muscarine acetyicholine receptor (mAChR), could do so as well. In contrast, atropine, the antagonist of mAChR abolished the ability of ACh to increase Ca^2 in guard cells. This mechanism is similar to mAChR in animals. When EGTA was used to chelate Ca^2 or ruthenium red to block Ca^2 released from vacuole respectively, the results showed that the increased cytosolic Ca^2 mainly come from intracellular Ca^2 store. The evidence supports that Ca^2 is involved in guard-cell response to ACh and that Ca^2 sigual is coupled to mAChRs in ACh signal transduction in guard cells.     

Comparative and statistical analysis of nAChR sequences: An <Emphasis Type="Italic">ab initio</Emphasis> approach to the origin of molecular discrimination

Compared with traditional structure-based approaches for the identification of species-specific ligands, the ab initio approach, based on large-scale protein sequences from different species, has been used to locate specific sites that may be important to the molecular selectivity of species. Statistically significant differences in the distribution of residues in different species and differences in the physicochemical properties of residue-specific sites may largely account for species selectivity. The nicotinic acetylcholine receptor (nAChR), an important neuro-receptor with significantly different ligand selectivity in different species, was used to test our method. Because of the lack of nAChR structural information, the mechanism of ligand discrimination is unclear which hinders attempts at novel molecular design. In this study, the specific site residues 186 and 189 in the principal subunits and residues 34, 55, 56, 57, 106 and 112 in complementary subunits of nAChR were identified by applying our method with stringent statistical cutoffs. These sites were predicted to contribute to ligand selectivity and this result coincides well with the known experimental data.     

Effects of calcineurin on LTP of rats <Emphasis Type="Italic">in vivo</Emphasis>

Calcineurin (CN) is thought to play a role in the synaptic plastivity and long-term potentiation (LTP) in the hippocampus. Based on two LTP models in vivo, a specific inhibitor cyclosporin A (CsA) of CN was observed, which affected LTP in the hippocampal dentate gyrus of the rats. The results indicated that CsA blocked LTP induced by high frequency stimulation (HFS) partly, but it had no effect on the decrease of the onset and peak latency of population spikes (PS) except that it reduced the increase of the amplitude after HFS. On the other hand, CsA blocked LTP induced by ginsenosides (GSS) completely. It suppressed the GSS-enhanced amplitude of PS reversibly and blocked the decrease of the peak latency of PS induced by the GSS. These results suggest that the postsynaptic CN plays a role in the induction of LTP in the hippocampus of the rats.     

大鼠耳蜗血管纹胆碱能M受体的表达及意义

目的:研究胆碱能M受体在耳蜗血管纹的表达.方法:采用RT-PCR技术检测大鼠耳蜗血管纹上胆碱能M受体的基因表达.结果:扩增出标志M2、M3、M4和M5受体亚型基因表达的PCR产物,未扩增出标志M1受体亚型的产物.结论:胆碱能M2、M3、M4和M5受体亚型在耳蜗血管纹有表达,提示胆碱能M受体可能参与了血管纹功能的调节,为研究胆碱能M受体在血管纹上的功能提供分子生物学基础.     

Excitatory effect of histamine on neuronal activity of rat cerebellar fastigial nucleus in vitro

The cerebellar fastigial nucleus (FN) holds an important role in motor control and body balance. Previous studies have revealed that the nucleus is innervated by direct hypothalamocerebellar histaminergic fibers. However, the functional role of histaminergic projection in cerebellar FN has never been established. In this study, we investigated the effect of histamine on neuronal firing of cerebellar FN by using slice preparations. Sixty-five FN cells were recorded from 47 cerebellar slices, and a vast majority of the cells responded to histamine stimulation with an excitatory response (58/65, 89.2%). Perfusing slices with low-Ca2 /high-Mg2 medium did not block the histamine-induced excitation (n=10), supporting a direct postsynaptic action of histamine on the cells. Furthermore, the excitatory effect of histamine on FN neurons was not blocked by selective histamine H1 receptor antagonist triprolidine (n=15) or chlorpheniramine (n=10), but was effectively suppressed by ranitidine (n=15), a highly selective histamine H2 receptor antagonist. On the other hand, highly selective histamine H2 receptor agonist dimaprit (n=20) instead of histamine H1 receptor agonist 2-pyridylethylamine (n=16) mimicked the ex- citatory effect of histamine on FN neurons. The dimaprit-induced FN neuronal excitation was effectively antagonized by selective histamine H2 receptor antagonist ranitidine (n=13) but not influenced by se- lective histamine H1 receptor antagonist triprolidine (n=15). These results demonstrate that histamine excites cerebellar FN cells via the histamine H2 receptor mechanism and suggest that the hypotha- lamocerebellar histaminergic fibers may modulate cerebellar FN-mediated sensorimotor integration through their excitatory innervations on FN neurons.