The instructive role of binocular vision in the Xenopus tectum

This review presents the fascinating neurobiology underlying the development of the frog optic tectum, the brain structure where the two separate inputs from the two eye are combined into a single, integrated map. In the species Xenopus laevis, binocular visual information has a dramatic impact on axon growth and connectivity, and the formation of binocular connections in this system provides a rich basis for both theoretical and experimental investigations.     

The effect of experimental diabetes on the twenty-four-hour pattern of the vasodilator responses to acetylcholine and isoprenaline in the rat aorta

The aim of this study was to investigate whether time-dependent variations in the relaxant effect of acetylcholine, an endothelium-dependent vasorelaxant via muscarinic receptors, and isoprenaline, a nonselective β-adrenoceptor agonist in rat aorta, are influenced by streptozotocin (STZ)-induced experimental diabetes. Adult male rats were divided randomly into two groups: control and STZ-induced (STZ, 55 mg/kg, intraperitoneal) diabetes. The animals were synchronized to a 12:12 h light-dark cycle (lights on 08:00 h) and sacrificed at six different times of day (1, 5, 9, 13, 17, and 21 hours after lights on; HALO) eight weeks after STZ injection. The in vitro responsiveness of thoracic aorta rings obtained from control and diabetic rats to acetylcholine (10^-9-10^-5 M) and isoprenaline (10^-10-10^-3 M) was determined in six different times. EC₅₀ (the concentration inducing half of the maximum response) values and maximum responses were calculated from cumulative concentration-response curves of the agonists and were analyzed with respect to time and STZ treatment. Treatment, time, and interactions between treatment and time were tested by two-way analysis of variance (ANOVA). To analyze differences due to biological time, one-way ANOVA was used. STZ treatment did not significantly change EC₅₀ values or maximum responses for both agonists. There were statistically significant time-dependent variations in the EC₅₀ values for isoprenaline and maximum responses for both acetylcholine and isoprenaline in control groups by one-way ANOVA, but significant time-dependent variations disappeared in the aortas isolated from STZ-induced diabetic rats. The vasodilator responses to acetylcholine and isoprenaline failed to show any significant interaction (treatment×time of study) between STZ treatment and time of sacrifice in both EC₅₀ values and maximum responses by two-way ANOVA. These results indicate there is a basic temporal pattern in the responses to acetylcholine and isoprenaline in rat aorta which continues in diabetes. It is shown for the first time that experimental diabetes does not change the 24 h pattern of responses to acetylcholine and isoprenaline, and that time-dependent variations in the responses to these agonists disappear in diabetic animals. Although further studies are required to define the underlying mechanism(s) of these findings, results suggest that experimental diabetes can modify the time-dependent vasorelaxant responses of rat aorta. This may help to understand the circadian rhythms in cardiovascular physiology and pathology or in drug effects in diabetes.     

Neuromodulation by Glutamate and Acetylcholine can Change Circuit Dynamics by Regulating the Relative Influence of Afferent Input and Excitatory Feedback

Substances such as acetylcholine and glutamate act as both neurotransmitters and neuromodulators. As neuromodulators, they change neural information processing by regulating synaptic transmitter release, altering baseline membrane potential and spiking activity, and modifying long-term synaptic plasticity. Slice physiology research has demonstrated that many neuromodulators differentially modulate afferent, incoming information compared to intrinsic and recurrent processing in cortical structures such as piriform cortex, neocortex, and the hippocampus. The enhancement of afferent (external) pathways versus the suppression at recurrent (internal) pathways could cause cortical dynamics to switch between a predominant influence of external stimulation to a predominant influence of internal recall. Modulation of afferent versus intrinsic processing could contribute to the role of neuromodulators in regulating attention, learning, and memory effects in behavior.     

Single-channel properties of N- and L-subtypes of acetylcholine receptor in Ascaris suum

We are interested in the properties of the target site of cholinergic anti-nematodal drugs for therapeutic reasons. The target receptors are ligand-gated ion channels that have different subtypes, and each subtype may have a different pharmacology. In a contraction assay using the parasitic nematode Ascaris suum, our laboratory has identified several subtypes, including an N-subtype, preferentially activated by nicotine, and an L-subtype, preferentially activated by levamisole. Here we use patch-clamp recordings to test the hypothesis that the single-channel selectivities of nicotine and levamisole are different. Unitary currents evoked by nicotine in this preparation were characterised for the first time. In some patches, both nicotine and levamisole activated small- and large-conductance channels. In other patches, the agonists activated just one channel amplitude. Discriminant analysis allowed classification of the one-conductance patch channels into the small or large categories, based on sets defined by the two-conductance patch data. The small channels had a conductance of 26.1+/-1.5 pS, n=18 (mean+/-SEM); the large conductance channels had a conductance of 38.8+/-1.2 pS, n=23 (mean+/-SEM). Analysis of amplitude histograms of the two-conductance patches showed that nicotine preferentially activated the small-conductance channels and levamisole preferentially activated the large-conductance channels. Our observations suggest that the N-subtype receptor channel has a conductance of 26 pS channel and the L-subtype receptor channel has a conductance of 39 pS.     

Regulation of zymogen granule exocytosis by Ca2+, cAMP, and PKC in pancreatic acinar cells

The effect of cAMP and PKC on zymogen granule exocytosis was investigated by simultaneously measuring cytosolic Ca2+ concentration ([Ca2+]c) and individual zymogen granule exocytosis in isolated mouse pancreatic acini. When acinar cells were stimulated with acetylcholine (ACh, 10 microM), exocytic events were detected through granule-attached apical membranes with [Ca2+]c rise. Application of secretin, forskolin (an adenylate cyclase activator), or PMA (a PKC activator) alone did not elicit any [Ca2+]c rise or zymogen granule exocytosis, but co-stimulation with ACh led to exocytosis in that the total number of secreted granules increased markedly without a significant difference in [Ca2+]c rises. When we evoked exocytosis by [Ca2+]c ramps, pretreatment with forskolin or PMA elicited exocytosis at lower [Ca2+]c levels. These results indicate that PKC or cAMP alone could not directly elicit zymogen granule exocytosis, but that they increase the total releasable pool by rendering zymogen granules more sensitive to Ca2+.     

Acetylcholine-induced calcium signalling in adrenaline- and noradrenaline-containing adrenal chromaffin cells

Adrenal chromaffin cells secrete catecholamines in response to cholinergic receptor activation by acetylcholine (ACh). Characteristics of Ca(2+) transients induced by activation of nicotinic (nAChRs) and muscarinic (mAChRs) receptors were analyzed using Fura-2 fluorescent measurements on rat chromaffin cells. We first found two populations of chromaffin cells, which differently responded on AChR stimulation. In the first group (n-cells), consecutive ACh applications evoked persistent Ca(2+) transients, whereas desensitizing transients were observed in the other group (m-cells). The AChR agonists and antagonists precisely imitated or abolished the ACh action on n- and m-type cells, respectively. Cytochemical staining showed that n-cells contained adrenaline, whereas m-cells-noradrenaline. Thus, for the first time we found that nAChRs and mAChRs are differentially expressed in adrenergic and noradrenergic chromaffin cells, respectively. Our data suppose that chromaffin cells can be differentially regulated by incoming ACh signals and in such way release different substances-adrenaline and noradrenaline.     

重症肌无力小鼠模型的建立

目的建立重症肌无力小鼠模型。方法电鳗乙酰胆碱受体(TnAChR)和完全弗氏佐剂(CFA)混合物免疫C57BL/6J小鼠,经二次加强免疫后,检测肌力、腓肠肌肌电图、膈肌终板电镜、血清抗体水平等指标。结果与对照小鼠相比,发病小鼠表现出肌力减弱的症状,肌电图显示小鼠腓肠肌复合动作电位振幅幅度显著下降,电镜证实神经肌接头处突触后膜变平、皱褶减少、空泡样变性,发病小鼠血清抗体水平明显升高。结论成功建立了重症肌无力小鼠模型,有助于探讨其发病机理及探索治疗自身免疫病新的途径和方法。     

Asparagine of z8 Insert Is Critical for the Affinity,Conformation, and Acetylcholine Receptor-clustering Activity of Neural Agrin

Agrin isoforms with different bioactivities are synthesized by the nerve and the muscle. Neural agrin containing an 8-amino acid insert (z8) introduced by alternative splicing is the active form that induces synaptic differentiation at the neuromuscular junction. In addition to alternative splicing, extracellular calcium is also required for the activity of neural agrin. To understand better how the activity of agrin is regulated by alternative splicing, we have applied alanine substitution mutagenesis to the z8 insert and the calcium binding site in the minimally functional AgG3z8 fragment. Single alanine substitutions in the 4th through the 7th amino acid of the z8 splice insert significantly reduced the function of agrin, in terms of acetylcholine receptor clustering activity and the affinity for binding to the muscle surface. Mutation of the asparagine at the 4th position drastically reduces bioactivity such that it is equivalent to that of muscle form AgG3z0. These reduced activity mutants also show reduced magnitudes of the calcium-induced CD spectrum change from that observed in AgG3z8 fragments, indicating that cross-talk between calcium and the z8 insert is critical for the normal activity of agrin. However, removal of Ca²⁺ binding via mutation of both aspartic acids in the calcium binding site did not totally eliminate the activity of AgG3z8. These results suggest a model wherein the z8 insert is a Ca²⁺-responsive allosteric element that is essential in forming an active conformation in neuronal agrin.     

Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.     

Choline Promotes Nicotinic Receptor ��4 + ��2 Up-regulation

Neuronal nicotinic acetylcholine receptors (nAChR) composed of α4 + β2 subunits, the high affinity nicotine-binding site in the mammalian brain, up-regulate in response to chronic nicotine exposure. The identities of endogenous mediators of this process are unknown. We find that choline also up-regulates α4 + β2 nAChRs stably expressed by HEK293 cells as measured by increased [³H]epibatidine density. Choline-mediated up-regulation is dose-dependent and corresponds with an increase in β2 subunit protein expression. The choline kinase inhibitor hemicholinium-3 inhibits ∼60% of choline-mediated up-regulation revealing both an HC3-dependent and -independent pathway. Furthermore, choline-mediated up-regulation is not additive with up-regulation agents such as nicotine, but it is additive with weaker promoters of the up-regulation process. When co-applied with the pro-inflammatory cytokine tumor necrosis factor α, choline-mediated up-regulation is increased further through a mechanism that includes an increase in both α4 and β2 protein expression, and this is inhibited by the p38 MAPK inhibitor SB202190. These findings extend the view that up-regulation of α4 + β2 nAChRs is a normal physiological response to altered metabolic and inflammatory conditions.