Chemically distinct rat olivocochlear neurons.

We have produced a neurochemical map of the cell bodies of origin of the cochlear efferent terminals in rat by combining glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT), or calcitonin gene-related peptide (CGRP) immunocytochemistry with retrograde transport of horseradish peroxidase. The locations of cochlear efferent cell bodies are in general agreement with the medial and lateral systems described by White and Warr (J. Comp. Neurol. 219:203-214, 1983) with some minor modifications. The lateral system consists of at least two pools of chemically distinct neurons located within the lateral superior olive (LSO) ipsilateral to the injected cochlea. One pool immunostains with an antibody to GAD while the other immunostains with antibodies to ChAT and to CGRP. The medial efferent system consists of periolivary neurons that are almost exclusively large and ChAT-positive but CGRP-negative. They are located both ipsilateral and contralateral to the cochlea they project to. There are a few GAD-positive small neurons in the medioventral and rostral periolivary regions that project ipsilaterally, but these may prove tobe ectopic neurons. The ipsilateral lateroventral periolivary region (LVPO) contains some efferent neurons, all of which are ChAT-positive but CGRP-negative. Additional cochlear efferent neurons, some of which are ChAT-positive and others GAD-positive, are present within and immediately dorsal to the fiber capsule surrounding the medial limb, and to a lesser extent the lateral limb, of the ipsilateral LSO. Not all GAD-positive or ChAT-positive olivary cells project to the cochlea. We have complemented the results in the brainstem by demonstrating two immunocytochemically distinct populations of efferent terminals in the cochlea simultaneously, one CGRP-positive and the other GAD-positive. Approximately equal numbers of boutons immunoreactive for both markers are present beneath inner hair cells throughout the entire length of the cochlea. Surprisingly high numbers of GAD-positive and CGRP-positive boutons are also present on outer hair cells, with each class having its spatially and morphologically distinct features. The lack of CGRP-positive periolivary cells that are retrogradely labeled by cochlear injections of HRP suggests that the lateral olivocochlear system sends projections to outer hair cells. Our results raise questions about species differences in the organization of targets of the lateral and medial olivocochlear systems.     

Pathophysiology of idiopathic dystonia: findings from genetic animal models

Dystonia is a common movement disorder which is thought to represent a disease of the basal ganglia. However, the pathogenesis of the idiopathic dystonias, i.e. the neuroanatomic and neurochemical basis, is still a mystery. Research in dystonia is complicated by the existence of various phenotypic and genotypic subtypes of idiopathic dystonia, probably related to heterogeneous dysfunctions.In neurological diseases in which no obvious neuronal degeneration can be found, such as in idiopathic dystonia, the identification of a primary defect is difficult, because of the large number of chemically distinct, but functionally interrelated, neurotransmitter systems in the brain.The variable response to pharmacological agents in patients with idiopathic dystonia supports the notion that the underlying biochemical dysfunctions vary in the subtypes of idiopathic dystonia. Hence, in basic research it is important to clearly define the involved type of dystonia.Animal models of dystonias were described as limited. However, over the last years, there has been considerable progress in the evaluation of animal models for different types of dystonia.Apart from animal models of symptomatic dystonia, genetic animal models with inherited dystonia which occurs in the absence of pathomorphological alterations in brain and spinal cord are described.This review will focus mainly on genetic animal models of different idiopathic dystonias and pathophysiological findings. In particular, in the case of the mutant dystonic (dt) rat, a model of generalized dystonia, and in the case of the genetically dystonic hamster (dt^sz), a model of paroxysmal dystonic choreoathetosis has been used, as these show great promise in contributing to the identification of underlying mechanisms in idiopathic dystonias, although even a proper animal model will probably never be equivalent to a human disease.Several pathophysiological findings from animal models are in line with clinical observations in dystonic patients, indicating abnormalities not only in the basal ganglia and thalamic nuclei, but also in the cerebellum and brainstem. Through clinical studies and neurochemical data several similarities were found in the genetic animal models, although the current data indicates different defects in dystonic animals which is consistent with the notion that dystonia is a heterogenous disorder.Different supraspinal dysfunctions appear to lead to manifestation of dystonic movements and postures. In addition to increasing our understanding of the pathophysiology of idiopathic dystonia, animal models may help to improve therapeutic strategies for this movement disorder.     

A -243A-->G polymorphism upstream of the gene encoding GAD65 associates with lower levels of body mass index and glycaemia in a population-based sample of 5857 middle-aged White subjects.

AIMS: The glutamate decarboxylase gene (GAD2) encodes GAD65, an enzyme catalysing the production of the gamma-aminobutyric acid (GABA) which interacts with neuropeptide Y to stimulate food intake. It has been suggested that in pancreatic islets, GABA serves as a functional regulator of pancreatic hormone release. Conflicting results have been reported concerning the potential impact of GAD2 variation on estimates of energy metabolism. The aim of this study was to elucidate potential associations between the GAD2-243A-->G polymorphism and levels of body mass index (BMI) and estimates of glycaemia. METHODS: Using high-throughput chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the GAD2-243A-->G (rs2236418) polymorphism was genotyped in a population-based sample (Inter99) of 5857 middle-aged, unrelated Danish White subjects. RESULTS: The G-allele was associated with modestly lower BMI (P = 0.01). In a case-control study of obesity, the G-allele frequency in 2582 participants with BMI < 25 kg/m2 was 19.5% (18.4-20.6) compared with 17.1% (15.5-18.8) in 968 participants having BMI > or = 30 kg/m2 (P = 0.03), odds ratio 0.9 (0.7-1.0). Of the 5857 subjects, GG carriers had lower fasting plasma glucose levels (mmol/l) [AA (n = 3859) 5.6 +/- 0.8; AG (n = 1792) 5.5 +/- 0.8; GG (n = 206) 5.5 +/- 0.8, P = 0.008] and lower 30-min oral glucose tolerance test (OGTT)-related plasma glucose levels (AA 8.7 +/- 1.9; AG 8.6 +/- 1.9; GG 8.6 +/- 2.0, P = 0.04), adjusted for sex, age and BMI. Analysing subjects who were both normoglycaemic and glucose tolerant (n = 4431) GG carriers still had lower fasting plasma glucose concentrations: AA (n = 2895) 5.3 +/- 0.4; AG (n = 1383) 5.3 +/- 0.4; GG (n = 153) 5.2 +/- 0.4 (P = 9.10(-5)). CONCLUSION: The present study suggests that the GAD2-243A-->G polymorphism in a population of middle-aged White people associates with a modest reduction in BMI and fasting and OGTT-related plasma glucose levels.     

Endogenous Immune Response to Glutamic Acid Decarboxylase (GAD67) in NOD Mice is Modulated by Adjuvant Immunotherapy

We have shown that immunization of non-obese diabetic (NOD) mice with adjuvants (CFA or BCG) prevents the onset of diabetes by induction of regulatory cells. Since autoimmune responses to glutamic acid decarboxylase (GAD) are up-regulated in insulin-dependent diabetes mellitus (IDDM), in this study GAD67-specific antibody, T cell proliferation and lymphokine production patterns were analysed in the adjuvant-treated mice to characterize the regulatory mechanisms underlying the protection. We used both spontaneous diabetes and syngeneic islet transplantation models in NOD mice. Protection against spontaneous diabetes and prevention of syngeneic islet graft rejection by CFA or BCG treatment was found to be accompanied by the production of long lasting and high titre anti-GAD67 antibody of IgG1 isotype in the sera. Uponin vitrostimulation with GAD67, draining lymph node and spleen cells from CFA-immunized NOD mice or syngeneic islet-grafted and BCG-protected NOD mice produced much more IL-4, whereas there was no significant change in IFN-γ production. The strong early T cell proliferative response to GAD67 in CFA or BCG-immunized NOD mice was followed by a low or unresponsiveness state. Taken together, these results suggest a shift in Th1/Th2 balance in the GAD67-specific endogenous immune response to a change in Th2 levels after adjuvant treatment. We postulate that the protective effect of CFA or BCG is due to the diversion of GAD-specific endogenous cellular immune response to a non-pathogenic humoral response.     

Analysis of children with type 1 diabetes in Korea: high prevalence of specific anti-islet autoantibodies, immunogenetic similarities to Western populations with "unique" haplotypes, and lack of discrimination by aspartic acid at position 57 of DQB

This study analyzed the expression of anti-islet autoantibodies and HLA-DR and -DQ genotypes in Korean children with type 1 diabetes mellitus (T1DM). The positivity of the anti-ICA512, anti-GAD65, and anti-insulin autoantibodies in the newly onset T1DM patients (n = 15) was 66.7%, 86.7%, and 46.7%, respectively, and all of them had one or more of the autoantibodies. HLA analysis showed higher frequencies of HLA-DRB1*0301, *0405, *09012 and -DQB1*0201, *0401, *03032 alleles in T1DM patients compared to controls (P(c) < 0.05). Because HLA-DQB1*0401, *03032 alleles carry aspartic acid at position 57 of DQB, susceptibility to T1DM in Korean children was not related to the presence of aspartic acid at position 57 of DQB1 locus. We suggest this unique HLA-DR, -DQ allele distribution might be an important factor for the low incidence of T1DM in Korea, and the combined anti-islet autoantibody assays could be valuable screening markers for the early detection of T1DM in Korea.     

谷氨酸脱羧酶67-绿色荧光蛋白基因敲入小鼠三叉神经尾侧亚核内GFP阳性神经元的分布及与小白蛋白的共存

目的观察谷氨酸脱羧酶67-绿色荧光蛋白（GAD67-GFP）基因敲入小鼠三叉神经尾侧亚核（Vc）浅层内，表达GFP的GABA能神经元的分布及其与小白蛋白（PV）的共存。方法分别运用原位分子杂交与免疫组织化学相结合；GFP与神经元标记物——神经元核蛋白（NeuN）或PV免疫荧光染色相结合的双重标记方法，在光学显微镜和激光共聚焦显微镜下进行观察。结果1．Vc浅层内90％以上的GFP阳性神经元同时表达GAD67 mRNA，而几乎所有表达GAD67 mRNA的阳性神经元都呈GFP阳性；2．GFP阳性神经元主要分布于Ｖc的Ⅰ－Ⅱ层内，细胞较小，尤其在Ⅱ层内可见大量密集分布的GFP阳性细胞和突起。GFP阳性神经元分别占Ⅰ、Ⅱ层内NeuN阳性神经元总数的19．4％和24．3％；3．GFP／PV双标神经元主要分布于Ｖc的Ⅰ－Ⅱ层，这些双标神经元大约占PV阳性神经元的62．4％，占GFP阳性神经元的12．8％。结论在Ｖc表达GFP的GABA能神经元主要密集分布于与外周伤害性信息传递关系密切的板层内，且大部分PV样阳性神经元属于GABA能神经元。     

The nucleus basalis magnocellularis: The origin of a cholinergic projection to the neocortex of the rat

The cells of origin of a neocortical cholinergic afferent projection have been identified by anterograde and retrograde methods in the rat. Horseradish peroxidase injected into neocortex labelled large, acetylcholinesterase-rich neurons in the ventromedial extremity of the globus pallidus. This same group of neurons underwent retrograde degeneration following cortical ablations. The region in which cell depletion occurred also showed significant decreases in the activities of choline acetyltransferase and acetylcholinesterase. Discrete electrolytic and kainic acid lesions restricted to the medial part of the globus pallidus each resulted in significant depletions of neocortical choline acetyltransferase and acetylcholinesterase. Hemitransections caudal to this cell group did not result in such depletions. Taken together these observations suggest that the acetylcholinesterase-rich neurons lying in the ventromedial extremity of the globus pallidus, as mapped in this study, constitute the origin of a major subcortical cholinergic projection to the neocortex. The utility of acetylcholinesterase histochemistry in animals pretreated with di-isopropylphosphorofluoridate in identifying cholinergic neurons is discussed in the light of this example; specifically, it is proposed that high acetylcholinesterase activity 4–8 h after this pretreatment is a necessary, but not sufficient, criterion for the identification of cholinergic perikarya.The neurons in question appear to be homologous to the nucleus basalis of the substantia innominata of primates, and are thus termed ‘nucleus basalis magnocellularis’ in the rat. No evidence was obtained to support the hypothesis that nucleus of the diagonal band projects to neocortex. However, striking similarities in size and acetylcholinesterase activity were observed among the putative cholinergic perikarya of the nucleus basalis magnocellularis, the nucleus of the diagonal band, and the medial septal nucleus.Kainic acid lesions of the neocortex produced uniform and complete destruction of neuronal perikarya. These lesions decreased neocortical glutamic acid decar?ylase activity, suggesting that there are GABAergic perikarya in the neocortex. However, the same lesions did not affect neocortical choline acetyltransferase. This observation suggests that there are no cholinergic perikarya in the neocortex, a conclusion that is consistent with the absence of intensely acetylcholinesterase-reactive neurons in the neocortex.     

乌灵胶囊和左洛复治疗广泛性焦虑临床观察

目的:观察左洛复和乌灵胶囊对广泛性焦虑(GAD)的临床疗效.方法:入组对象均符合<中国精神疾病分类与诊断标准-3>(CCMD-3)GAD的诊断标准,随机分左洛复合用乌灵胶囊组(观察组)和左洛复组(对照组).结果:观察组在第1周末SAS、HAMA量表评分与疗前有高度显著性差异(P＜0.01),2周末、4周末、6周末观察组及对照组SAS、HAMA量表评分与疗前均有高度显著性差异(P＜0.01).阿普唑仑使用率对照组高于观察组,有高度显著性差异(P＜0.01);对照组左洛复平均剂量高于观察组,有显著性差异(P＜0.05).在第4周末对照组副反应高于观察组,有高度显著性差异(P＜0.01).第1周末观察组有效率高于观察组,有高度显著性差异(P＜0.01);第2周末观察组有效率高于对照组,有显著性差异(P＜0.05).结论:左洛复合用乌灵胶囊,较单用左洛复治疗,起效快,是广泛性焦虑有效安全的治疗方法.     

GAD67-GFP精神分裂症小鼠行为学改变及海马齿状回颗粒细胞层GABA能神经元的表达

目的:探讨谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠制备精神分裂症模型后学习与记忆功能的改变及海马齿状回颗粒细胞层GABA能神经元的表达。方法:利用聚合酶链式反应(PCR)鉴定GAD67-GFP基因敲入小鼠,MK-801连续腹腔注射2周制备精神分裂症动物模型,通过悬尾实验、Morris水迷宫实验、免疫荧光标记技术等,观察GAD67-GFP基因敲入小鼠的学习与记忆功能的改变及GABA能神经元在海马齿状回颗粒细胞层的表达。结果:停药后实验组与对照组比较:(1)实验组体重增加明显低于对照组(P0.05);(2)行为学改变:1悬尾实验:实验组不动时间明显小于对照组(P0.05);2Morris水迷宫实验:定位航行实验中实验组逃避潜伏期,游泳总路程明显长于对照组(P0.05),而其平均游泳速度与对照组没有明显差异(P0.05);空间探查实验中实验组经过平台所在点的次数和在平台所在象限的时间明显小于对照组(P0.05);(3)在海马齿状回颗粒细胞层中实验组的GFP阳性细胞明显多于对照组(P0.05)。结论:通过对GAD67-GFP基因敲入小鼠进行腹腔注射MK-801制备精神分裂症模型后,其学习与记忆功能显著下降,且海马齿状回颗粒细胞层GABA能神经元明显增加。提示精神分裂症后学习记忆功能减退可能与GABA能神经元的表达有关。