GABAA receptor immunoreactivity in adult and developing monkey sensory-motor cortex

Summary The areal and laminar distribution of GABA_A receptor immunoreactivity was examined in fetal, early postnatal and adult monkey sensory-motor cortex by using a monoclonal antibody to the purified GABA_A receptor complex (Vitorica et al. 1988). GABA_A receptor immunoreactivity was distributed throughout the neuropil, often outlining the unstained somata of pyramidal and non-pyramidal cells. In all areas of the adult sensory-motor cortex, layers I–IIIA exhibited the most intense immunostaining. In deeper layers of the four cytoarchitectonic fields of the first somatic sensory area (SI), layers IIIB and V were lightly stained and alternated with somewhat more intensely stained layers IV and VI. In deeper layers of area 4, the deeper half of layer IIIA through layer VA was lightly immunostained, but layers VB and VI were slightly more intensely immunoreactive. A variable number of nonpyramidal cell somata in the cortex and underlying white matter showed immunoreactive staining. GABA_A receptor immunoreactivity was present throughout the sensory-motor cortex from the youngest fetal age examined (E121), but the pattern of immunostaining differed from that in the adult. In all areas, the densest immunoreactivity was found in a diffuse band in layers III and IV and in the subplate zone. Within the subplate zone, the presence of receptor immunoreactivity and some intensely stained neuronal somata at all fetal ages suggests the presence of a synaptic neuropil. With increasing age, gradual changes in the distribution of receptor immunoreactivity occurred, resulting in an adult-like pattern of immunostaining by postnatal day 1.5. These results show that the laminar pattern of GABA_A receptor distribution closely follows the major concentrations of GABA immunoreactive neurons in adults and it is suggested that laminar changes seen in development are associated with the establishment of afferent connections and inhibitory circuits in the sensory-motor cortex.     

The distribution and cellular localization of the serotonin 1C receptor mRNA in the rodent brain examined by in situ hybridization histochemistry. Comparison with receptor binding distribution

The regional distribution and cellular localization of mRNA coding for the serotonin 1C receptor were investigated in tissue sections of mouse and rat brain by in situ hybridization histochemistry. Several 32P-labelled riboprobes derived from mouse genomic clones were used. The serotonin 1C receptor binding sites were visualized autoradiographically and quantified using [3H]mesulergine as ligand, in the presence of spiperone to block serotonin 1C receptors. Strong hybridization signal was observed in the choroid plexus of all brain ventricles. High levels of hybridization were also seen in the anterior olfactory nucleus, pyriform cortex, amygdala, some thalamic nuclei, especially the lateral habenula, the CA3 area of the hippocampal formation, the cingulate cortex, some components of the basal ganglia and associated areas, particularly the nucleus subthalamicus and the substantia nigra. The midbrain and brainstem showed moderate levels of hybridization. The distribution of the serotonin 1C receptor mRNA corresponded well to that of the serotonin 1C receptors. The highest levels of serotonin 1C receptor binding were observed in the choroid plexus. In addition, significant levels of the serotonin 1C receptor binding were seen in the anterior olfactory nucleus, pyriform cortex, nucleus accumbens, ventral aspects of the striatum, paratenial and paracentral thalamic nuclei, amygdaloid body and substantia nigra pars reticulata. The cingulate and retrosplenial cortices as well as the caudal aspects of the hippocampus (CA3) were also labelled. Binding in brainstem and medulla was low and homogeneously distributed. No significant binding was seen in the habenular and subthalamic nuclei. Similar findings were obtained in rat brain. These results demonstrate that, in addition to their enrichment in the choroid plexus, the serotonin 1C receptor mRNA and binding sites are heterogeneously distributed in the rodent brain and thus could be involved in the regulation of many different brain functions. The combination of in situ hybridization histochemistry with receptor autoradiography opens the possibility of examining the regulation of the serotonin 1C receptor synthesis after pharmacological or physiological alterations.     

Expression and localization of ionotropic glutamate receptor subunits in the goldfish retina--an in situ hybridization and immunocytochemical study

The expression and distribution of AMPA, kainate and NMDA glutamate receptor subunits was studied in the goldfish retina. For the immunocytochemical localization of the AMPA receptor antisera against GluR2, GluR2/3 and GluR4 were used, and for in situ hybridization rat specific probes for GluR1 and GluR2 and goldfish specific probes for GluR3 and GluR4 were used. The localization of the low affinity kainate receptor and NMDA receptor was studied using antisera against GluR5-7 and NR1. All AMPA receptor subtypes were demonstrated to be present in the goldfish retina both by immunocytochemistry and in situ hybridization. In situ hybridization revealed expression of all AMPA receptors subunit at the inner border of the INL. Only GluR3 was also strongly expressed in the outer border of the INL. Some of the ganglion cells displayed a strong signal for GluR1, GluR3 and GluR4. GluR1-immunoreactivity was present in subsets of bipolar, amacrine, and ganglion cells. GluR2 and GluR2/3-immunoreactivity was mainly localized in the outer plexiform layer. GluR2 and GluR2/3-immunoreactivity are associated with the photoreceptor synaptic terminals. GluR4-immunoreactivity is present on Müller cells in the inner retina and on dendrites of bipolar cells in the OPL, whereas GluR5-7-immunoreactivity was prominently present on horizontal cell axon terminals. Finally, NR1-immunoreactivity was confined to amacrine cells, the inner plexiform layer and ganglion cells. This study shows that there is a strong heterogeneity of glutamate receptor subunit expression in the various layers of the retina. Of the AMPA receptor subunits GluR3 seems to be expressed the most widely in all layers with strong glutamatergic synaptic interactions whereas all the other subunits seem to have a more restricted expressed pattern.     

Immunocytochemical and in situ hybridization studies of the expression and distribution of three subunits of a complex with N-methyl-D-aspartate receptor-like properties

A group of four proteins with recognition sites for L-glutamate, N-methyl-D-aspartate, glycine, and competitive and non-competitive inhibitors of N-methyl-D-aspartate receptors was previously purified from rat brain synaptic membranes. The biochemical and immunochemical characteristics of this complex, as well as the sequences of the complementary DNAs of three subunits, are distinct from those of other glutamate receptors, transporters, or enzymes. The function of this complex has not yet been defined, but it appears to be involved in glutamate-induced neuronal excitation and toxicity. It is not known whether all protein components of the complex are expressed in the same populations of brain cells. In the present study, immunohistochemical and in situ hybridization were used to map the distribution of the glutamate-binding, glycine/thienylcyclohexylpiperidine-binding, and carboxypiperazinyl-propylphosphonate-binding protein subunits of the complex. These proteins were abundantly expressed in pyramidal neurons of the hippocampus and cerebral cortex, and in granule cells of the dentate gyrus, cerebellum, and olfactory tubercle. Based on these results, it was concluded that the three subunits of the complex have similar patterns of expression in rat brain. The distribution of one subunit of the complex, glutamate-binding protein, was traced throughout the rat brain, thus providing a potential map of the expression of the complex in rodent brain. In addition, probes were developed in the present study that should be useful in future explorations of the role of these proteins in brain function and of the possible co-localization of the protein subunits in single cells or cell processes.     

No interaction of GABAA alpha-1 subunit and dopamine receptor D4 exon 3 genes in symptomatology of major psychoses

Previously, we reported on an association of the dopamine receptor D4 (DRD4) gene with delusional symptomatology of major psychoses. However, despite the strength of the association, it only accounted for 2% of the variance, indicating that contributions from other genes were probable. In the present study, we investigated the original cohort of subjects to evaluate the gene for the γ-aminobutyric acid type A (GABA_A) receptor alpha-1 subunit (GABRA1). The possible association of GABRA1 with the psychopathology of major psychoses was tested both alone and in interaction with DRD4. Four hundred and sixty-one inpatients affected by major psychoses were assessed by the operational criteria checklist for psychotic illness (OPCRIT) and were also typed for the DRD4 and GABRA1 variants using PCR techniques. Mania, depression, delusion, and disorganization were the four symptomatologic factors used as phenotype definitions. GABRA1 variants were not associated with these symptomatologic factors, and consideration of possible stratification effects such as sex and psychiatric diagnosis also did not reveal any association. GABRA1 variants did not significantly influence the association of DRD4 with delusional symptoms. No interaction was observed on the other symptom factors. The GABA_A alpha-1 subunit gene does not, therefore, interact with DRD4 in the symptomatology of major psychoses. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:44–49, 1999. © 1999 Wiley-Liss, Inc.     

Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development

Gamma‐aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABA_A and metabotropic GABA_B receptors. During the development of the nervous system, GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation, and activity‐dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABA_A and GABA_B receptors during early development of Xenopus laevis. Using in situ hybridization and qRT‐PCR, GABA_A α2 was detected as a maternal mRNA. All other α‐subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern, suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner. Developmental Dynamics 240:862–873, 2011. © 2011 Wiley‐Liss, Inc.     

Erythropoietin and erythropoietin-receptor producing cells demonstrated by in situ hybridization in mouse visceral yolk sacs

We have previously demonstrated that mRNAs for erythropoietin and the erythropoietin receptor temporarily express on the visceral yolk sacs on days 9–11 of gestation in mice. In order to investigate the sites of expression, we performed in situ hybridization on visceral yolk sacs. Visceral yolk sacs from 10-day-old mice embryos were frozen in liquid nitrogen, and processed for cryosections. Sections were hybridized with a ³⁵S-labeled RNA probe complementary to mRNA coding for erythropoietin or erythropoietin receptor. Erythropoietin mRNA was detectable in 57.6% of the endodermal epithelial cells, while erythropoietin-receptor mRNA was discerned in 90.8% of the endodermal cells and mesodermal cells, including hemocyteblasts. Moreover, erythropoietin protein was detectable in 52.8% of the endodermal epithelial cells, and on the surface of hemocyteblasts and mesothelial cells. Erythropoietin-receptor protein was discernible in 87.2% of the endodermal cells and in the corresponding mesodermal cells to those where erythropoietin protein was expressed by immunohistochemical examinations. The results indicate that erythropoietin-synthesizing cells are located in half of the endodermal epithelial cells, while the majority of cells in the visceral yolk sac are erythropoietin-receptor-producing cells, indicating that almost all cell population in the visceral yolk sac is erythropoietin-responding cells via both autocrine and paracrine routes.     

Characterization of benzodiazepine-sensitive behaviors in the A/J and C57BL/6J inbred strains of mice

Exploratory behaviors as well as pharmacological actions of -aminobutyric acid_A (GABA_A)/benzodiazepine receptor agonists and inverse agonists were characterized in C57BL/6J and A/J strains of mice. C57BL/6J mice displayed higher levels of exploratory behavior than A/J mice in the lightdark exploration model of anxiety and in an openfield test, suggesting that C57BL/6J mice are less emotional and more active than A/J mice, respectively. However, C57BL/6J mice were more sensitive than A/J mice to the anxiolytic effects of diazepam in the lightdark exploration model. In contrast, A/J mice,were more sensitive than C57BL/6J mice to the convulsant effects of methyl--carboline-3-carboxylate. C57BL/6J mice showed no evidence of acquisition of a passive avoidance task, while A/J readily acquired this memory task at low levels of footshock. C57BL/6J and A/J mice should be useful parental strains in recombinant inbred lines for investigating the genetic determinants of benzodiazepine-sensitive behaviors and sensitivity to drugs acting on the GABA_A/benzodiazepine receptor complex.     

Spatial distribution of Escherichia coli in the mouse large intestine inferred from rRNA in situ hybridization.

Fluorescent oligonucleotide probes targeting rRNA were used to develop an in situ hybridization technique by which the spatial distribution of Escherichia coli in the large intestines of streptomycin-treated mice was determined. Single E. coli cells were identified in thin frozen sections from the large intestines by the use of a probe specific for E. coli 23S rRNA. Furthermore, the total bacterial population was visualized with an rRNA probe targeting the domain Bacteria. By this technique, all E. coli cells were seen embedded in the mucosal material overlying the epithelial cells of the large intestine, and no direct attachment to the epithelium was observed.     

Cajal's nucleus of the stria medullaris: Characterization by in situ hybridization and immunohistochemistry for enkephalin

While analyzing the distribution of enkephalinergic neurons by in situ hybridization and immunohistochemistry in the septal region of untreated or colchicine-injected rats, a densely packed enkephalinergic group of neurons was identified that corresponds to a small nucleus first described by Cajal as the nucleus of the stria medullaris. It contains mostly irregular or fusiform small neurons differing from those in adjacent structures by their size, and by their intensity of staining with in situ hybridization and immunohistochemistry. The connections of this nucleus (which is called here the bed nucleus of the stria medullaris, BSM) are unclear, but evidence in the literature suggests that it may receive inputs from the fornix and project through the stria medullaris to the medial habenula.     

Distribution of neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding radioautography and in situ hybridization histochemistry.

The neuronal distribution of cannabinoid receptor in the adult rat brain is reported, combining receptor binding radioautography using the synthetic psychoactive cannabinoid ligand CP55,940 with in situ hybridization histochemistry using oligonucleotide probes complementary to rat cannabinoid receptor cDNA. In the cerebral cortex, especially in the frontal and cingulate cortex, dense binding was found in layers I and VI together with slight mRNA levels in a majority of both pyramidal and non-pyramidal-shaped neurons and of high mRNA levels in a moderate number of non-pyramidal-shaped neurons especially in layers II-III and V-VI. In the hippocampal dentate gyrus, very dense staining was found in the molecular layer together with high mRNA levels in a moderate number of hilar neurons close to the granular layer. In Ammon's horn, especially in the CA3 sector, very dense binding was found in the dendritic layers together with slight mRNA levels in the majority of the pyramidal cells and high mRNA levels in a moderate number of interneurons. In the basal ganglia, binding was very dense in the lateral putamen, substantia nigra pars reticulata, globus pallidus and entopeduncular nucleus, moderate in the medial putamen and caudate; and slight in the accumbens, together with slight to moderate mRNA levels in the striatal medium-sized neurons. Together with slight binding, slight to moderate mRNA levels were found in the majority of the neurons in the subthalamic nucleus. No binding and mRNA were found in the substantia nigra pars compacta and ventral tegmental area. Slight to moderate binding was found together with slight to moderate mRNA levels in the majority of neurons in the anterior olfactory nucleus; septum, especially medial septum and diagonal band of Broca; amygdala, especially basolateral amygdala; lateral habenula; ventromedial hypothalamic nucleus; lateral interpeduncular nucleus; central gray, dorsal cochlear nucleus; parabrachial nucleus; dorsal pontine tegmentum; pontine nuclei; commissural part of the nucleus tractus solitarius; inferior olive and dorsal horn of the spinal cord. In the cerebellum, very dense binding was found in the molecular layer together with slight mRNA levels in the majority of the granule cells and moderate mRNA levels in the basket and stellate cells. In conclusion, this study provides, for the first time, indirect assessment of the neurons containing cannabinoid receptor in the entire adult rat brain and will serve as a basis for future direct morphological confirmation using receptor immunohistochemistry and for functional studies.     

C57BL/6小鼠胚胎成纤维细胞生物学特性及饲养层制备

背景：昆明小鼠胚胎成纤维细胞是目前最常用的饲养层细胞,C57BL/6小鼠胚胎成纤维细胞作为饲养层的研究鲜有报道。目的：体外分离和培养C57BL/6小鼠胚胎成纤维细胞,制备饲养层,力求扩大小鼠胚胎成纤维细胞的来源。方法：用不同浓度胰蛋白酶分步消化法体外分离和培养C57BL/6小鼠胚胎成纤维细胞,观察其生物学特性,研究其生长规律,并制备小鼠胚胎成纤维细胞饲养层,检测干细胞在所制备饲养层上的生长状态。结果与结论：不同浓度胰蛋白酶分步消化法制备的C57BL/6小鼠胚胎成纤维细胞生长状态好,获得的成纤维细胞数量多,增殖活跃。在细胞冻存后1,2周、1,3,6个月内复苏的细胞存活率差异无显著性意义。C57BL/6小鼠胚胎成纤维细胞在第2-5代增殖旺盛,第6代以后细胞增殖出现明显下降。种植到培养皿上的C57BL/6小鼠饲养层细胞在种植后3 d内活力高,种植4 d以后细胞活力急剧下降。所以C57BL/6小鼠胚胎成纤维细胞来源的饲养层的最佳使用时间为灭活后3 d内,C57BL/6小鼠胚胎成纤维细胞饲养层和昆明小鼠胚胎成纤维细胞饲养层一样,能很好地支持胚胎干细胞及诱导多能干细胞生长。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Long-term fate and distribution of newborn cells in the adult mouse olfactory bulb: Influences of olfactory deprivation

The adult subventricular zone produces neuroblasts that migrate to the main olfactory bulb, where they differentiate into interneurons in the glomerular and granular layers. Using bromodeoxyuridine labeling, the survival of newborn cells was assessed in these two layers of the MOB in control mice and in mice unilaterally deprived from sensory input by naris occlusion. In control main olfactory bulbs, bromodeoxyuridine-positive cell density decreased about 70% between 15 and 180 days post-bromodeoxyuridine administration but earlier in the glomerular layer than in the granular layer. At all time points examined, newborn cell density was higher in the deep granular layer than in the superficial granular layer. Occlusion started at the age of 2 months and lasted for 15, 30, 45, 60 or 180 days. The newborn cell survival was similarly reduced in both layers by occlusion, during a critical period 15 and 45 days post-occlusion. Interestingly, olfactory deprivation decreased bromodeoxyuridine-positive cell density in the deep granular layer only, indicating a greater dependence of cell fate on sensory input in this sub-layer. Neuronal differentiation was assessed in the granular layer and glomerular layer by multiple double-labeling 45 days post-bromodeoxyuridine-injections, the time point at which the proportion of bromodeoxyuridine-positive cells expressing a neuronal marker reached approximately 85% in the granular layer and approximately 50% in the glomerular layer. Naris occlusion did not significantly affect these proportions. Taken together, our results reveal that the survival of newborn cells has a different time course in the glomerular layer and in the granular layer, but is similarly decreased in each layer by olfactory deprivation. In addition, our data suggest a functional heterogeneity of neurogenesis within the granular layer.     

Altered CB1 receptor and endocannabinoid levels precede motor symptom onset in a transgenic mouse model of Huntington's disease

Huntington's disease (HD) is an inherited neurodegenerative disease characterised by cell dysfunction and death in the basal ganglia and cortex. Currently there are no effective pharmacological treatments available. Loss of cannabinoid CB1 receptor ligand binding in key brain regions is detected early in HD in human postmortem tissue [Glass M, Dragunow M, Faull RL (2000) The pattern of neurodegeneration in Huntington's disease: a comparative study of cannabinoid, dopamine, adenosine and GABA(A) receptor alterations in the human basal ganglia in Huntington's disease. Neuroscience 97:505–519]. In HD transgenic mice environmental enrichment upregulates the CB1 receptors and slows disease progression [Glass M, van Dellen A, Blakemore C, Hannan AJ, Faull RL (2004) Delayed onset of Huntington's disease in mice in an enriched environment correlates with delayed loss of cannabinoid CB1 receptors. Neuroscience 123:207–212]. These findings, combined with data from lesion studies have led to the suggestion that activation of cannabinoid receptors may be protective. However, studies suggest that CB1 mRNA may be decreased early in the disease progression in HD mice, making this a poor drug target. We have therefore performed a detailed analysis of CB1 receptor ligand binding, protein, gene expression and levels of endocannabinoids just prior to motor symptom onset (12 weeks of age) in R6/1 transgenic mice. We demonstrate that R6/1 mice exhibit a 27% decrease in CB1 mRNA in the striatum compared to wild type (WT). Total protein levels, determined by immunohistochemistry, are not significantly different to WT in the striatum or globus pallidus, but are significantly decreased by 19% in the substantia nigra. CB1 receptor ligand binding demonstrates significant but small decreases (<20%) in all basal ganglia regions evaluated. The levels of the endocannabinoid 2-arachidonoyl glycerol are significantly increased in the cortex (147%) while anandamide is significantly decreased in the hippocampus to 67% of WT. Decreases are also apparent in the ligand binding of neuronal D1 and D2 dopamine receptors co-located with CB1, while there is no change in GABA_A receptor ligand binding. These results suggest that in this R6/1 mouse colony at 12 weeks there are only very small changes in CB1 protein and receptors and thus this would be an appropriate time point to evaluate therapeutic interventions.     

Additivity and independence of neuroprotective effects of GABAA and GABAB receptor agonists in complete global cerebral ischemia

Neuroprotective effects of GABA_B agonist baclofen and GABA_A agonists THIP and muscimol are completely additive. GABA_A and GABA_B receptor antagonists block the neuroprotective effects of the corresponding agonists without cross-blocking. In fact, the blockade of combined effect of two agonists with selective GABA_A and GABA_B antagonists separately and in combination is not below the estimated value. Therefore, GABA_A and GABA_B receptor mechanisms of the neuroprotective effects independently and separately contribute to the improvement of brain resistance to global ischemia. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 130, No. 8, pp. 187–189, August, 2000     

The fate of interneurons,GABAA receptor sub-types and perineuronal nets in Alzheimer's disease

Alzheimer's disease (AD) is the most common neurological disease, which is associated with gradual memory loss and correlated with synaptic hyperactivity and abnormal oscillatory rhythmic brain activity that precedes phenotypic alterations and is partly responsible for the spread of the disease pathology. Synaptic hyperactivity is thought to be because of alteration in the homeostasis of phasic and tonic synaptic inhibition, which is orchestrated by the GABA_A inhibitory system, encompassing subclasses of interneurons and GABA_A receptors, which play a vital role in cognitive functions, including learning and memory. Furthermore, the extracellular matrix, the perineuronal nets (PNNs) which often go unnoticed in considerations of AD pathology, encapsulate the inhibitory cells and neurites in critical brain regions and have recently come under the light for their crucial role in synaptic stabilisation and excitatory-inhibitory balance and when disrupted, serve as a potential trigger for AD-associated synaptic imbalance. Therefore, in this review, we summarise the current understanding of the selective vulnerability of distinct interneuron subtypes, their synaptic and extrasynaptic GABA_AR subtypes as well as the changes in PNNs in AD, detailing their contribution to the mechanisms of disease development. We aim to highlight how seemingly unique malfunction in each component of the interneuronal GABA inhibitory system can be tied together to result in critical circuit dysfunction, leading to the irreversible symptomatic damage observed in AD.     

Cerebellar benzodiazepine receptor distribution: an autoradiographic study of the normal C57BL/6J and Purkinje cell degeneration mutant mouse

The distribution of [3H]flunitrazepam binding sites in the cerebella of normal mice and Purkinje cell degeneration mutant mice was studied by light microscopic autoradiography. In the cerebellar cortex of normal mice, a high density of [3H]flunitrazepam binding was observed over the molecular layer, an intermediate density over the Purkinje cell layer and a low density over the granule cell layer; the white matter was devoid of labeling. The deep cerebellar nuclei were labeled to an intermediate density. In the 54-day-old Purkinje cell degeneration mutant cerebellum, which is depleted of Purkinje cells, a 36% reduction in labeling density of the cerebellar cortex was observed. The density was reduced by approximately equal amounts in both the molecular and granule cell layers; labeling in the deep cerebellar nuclei was, however, substantially increased.