Heterogeneity in expression of functional ionotropic glutamate and GABA receptors in astrocytes across brain regions: insights from the thalamus

Astrocytes may express ionotropic glutamate and gamma-aminobutyric acid (GABA) receptors, which allow them to sense and to respond to neuronal activity. However, so far the properties of astrocytes have been studied only in a few brain regions. Here, we provide the first detailed receptor analysis of astrocytes in the murine ventrobasal thalamus and compare the properties with those in other regions. To improve voltage-clamp control and avoid indirect effects during drug applications, freshly isolated astrocytes were employed. Two sub-populations of astrocytes were found, expressing or lacking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. AMPA receptor-bearing astrocytes displayed a lower Kir current density than cells lacking the receptors. In contrast, all cells expressed GABA_A receptors. Single-cell RT-PCR was employed to identify the receptor subunits in thalamic astrocytes. Our findings add to the emerging evidence of functional heterogeneity of astrocytes, the impact of which still remains to be defined.     

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K_a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABA_A receptors in neuronal populations within the olfactory bulb.     

Nonneuronal expression of the GABA(A) beta3 subunit gene is required for normal palate development in mice

Cleft palate is one of the most common birth defects in humans, in which both genetic and environmental factors are involved. In mice, loss of the GABA(A) receptor beta3 subunit gene (Gabrb3) or the targeted mutagenesis of the GABA synthetic enzyme (Gad1) leads to cleft palate. These observations indicate that a GABAergic system is important in normal palate development. To determine what cell types, neuronal or nonneuronal, are critical for GABA signaling in palate development, we used the neuron-specific enolase promoter to express the beta3 subunit in Gabrb3 mutant mice. Expression of this construct was able to rescue the neurological phenotype, but not the cleft palate phenotype. Combined with the previous observation demonstrating that ubiquitous expression of the beta3 subunit rescued the cleft palate phenotype, a nonneuronal GABAergic system is implicated in palate development. Using immunohistochemistry, we detected GABA in the developing palate, initially in the nasal aspect of palatal epithelium of the vertical shelves; later in the medial edge epithelium of the horizontally oriented palatal shelves and in the epithelial seam during fusion. Based on these observations, we propose that GABA, synthesized by the palatal epithelium, acts as a signaling molecule during orientation and fusion of the palate shelves.     

Synthesis and evaluation of highly potent GABA(A) receptor antagonists based on gabazine (SR-95531)

A selection of highly potent analogues based on the gabazine structure is described. Their syntheses are carried out in just four steps, and their potencies for antagonism at the GABA_A receptor were measured. All antagonists showed significantly higher potencies compared to the parent competitive antagonist, gabazine.     

Cerebellar GABA(A) receptor alterations in hypoxic neonatal rats: Role of glucose, oxygen and epinephrine supplementation

Hypoxia in neonates causes dysfunction of excitatory and inhibitory neurotransmission resulting in permanent brain damage. The present study is to understand the cerebellar GABA(A) receptor alterations and neuroprotective effect of glucose supplementation prior to current sequence of resuscitation - oxygen and epinephrine supplementation in hypoxic neonatal rats. Hypoxic insult caused a significant decrease in GABA(A) receptor number along with down regulated expression of GABA(Aα1,) GABA(Aα5), GABA(Aδ) and GABA(Aγ3) receptor subunits in the cerebellum which accounts for the respiratory inhibition. Hypoxic rats supplemented with glucose alone and with oxygen showed a reversal of the receptor alterations and changes in GABA(A) receptor subunits expression to near control. Glucose can reduce ATP-depletion-induced alterations in GABA receptors, thereby assisting in overcoming the neuronal damage caused by hypoxia. Resuscitation with oxygen alone and epinephrine was less effective in reversing the receptor alterations. The reduction in the GABA(A) receptors functional regulation during hypoxia plays an important role in cerebellar damage. Resuscitation with glucose alone and glucose with oxygenation to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.     

Effects of Bisphenol A and its Derivatives on the Response of GABAA Receptors Expressed in Xenopus Oocytes

To study the effects of bisphenol-A (BPA) known to have estrogenic actions, and its derivatives, 3,5-dimethylphenol (DMP) and p-t-butylphenol (TBP), on ionotropic γ-aminobutyric acid (GABA) receptors, GABA_A receptors were expressed in Xenopus oocytes by injecting both poly(A)⁺RNA prepared from rat whole brain and cRNAs synthesized from cloned cDNAs of α₁ and β₁ subunit of the bovine receptors, and their electrical responses were measured by the voltage clamping method. BPA caused the potentiation and inhibition of the former receptor-responses, while it caused only inhibition of the latter ones. In the presence of low concentrations of GABA, DMP and TBP potentiated the responses of both receptors. DMP and TBP also increased the rate of decay of the response, possibly by desensitization of the receptors when GABA solution was continuously bath-applied. Diethyl terephthalate (DTP), which is also known to have estrogenic actions, had little effect on both the responses and the decay of both receptors.     

A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits

We show that the five subunits of a gamma-aminobutyric acid type A receptor (GABA(A) receptor) can be concatenated to yield a functional receptor. This concatenated receptor alpha(1)-beta(2)-alpha(1)-gamma(2)-beta(2) has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non-concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non-concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABA(A) receptor subtype selective compounds.     

Chorein deficiency leads to upregulation of gephyrin and GABA(A) receptor

Chorea-acanthocytosis (ChAc) is a hereditary neurodegenerative disorder caused by loss of function mutations in the VPS13A gene encoding chorein. Recently, using a gene-targeting technique to delete exons 60-61, we produced a ChAc-model mouse that corresponds to a human disease mutation. In this study, a comparative microarray analysis of gene expression in the striatum revealed an increased level of gephyrin gene expression in the ChAc-model mice compared with wild type mice. Since gephyrin is known as a GABA(A) receptor-anchoring protein, we compared the protein-level expression and localization of gephyrin and the GABA(A) receptor alpha1 (GABRA1) and gamma2 (GABRG2) subunits. Gephyrin and GABRG2 immunoreactivities in the striatum and hippocampus of the ChAc-model mice were significantly higher than those in the wild types. Our results suggest that chorein functional loss may lead to a compensatory upregulation of gephyrin and GABRG2 in the pathologic condition in ChAc.     

Isolation and characterization of endogenous modulators for GABA system

Pig brain extracts from both soluble and membrane fractions were found to contain potent inhibitors for GABA synthesizing enzyme, GAD, referred to as endogenous GAD inhibitors (EGIs) and for the binding of GABA agonist, muscimol, referred to as muscimol binding inhibitors (MBIs). EGIs and MBIs were first purified through gel-filtration Bio-Gel P-2 columns, in which multiple activity peaks were observed. One of them appears to be co-eluted with eitherl-glutamate or GABA. However, others are clearly separated froml-glutamate or GABA. EGIs were found to be low MW (<1,800 dalton), heat and acid-base stable, negatively charged, non hydrophobic substances. MBIs were found to be low MW (<1,800 dalton) neutral or positively charged substances. MBIs had no effect on [³H]flunitrazepam (FNZP) binding, indicating that they are not endogenous benzodiazepine receptor ligands and they may act specifically on GABA binding site.Special issue dedicated to Dr. Frederick E. Samson     

Spatial patterns of gene expression in preimplantation mouse embryos

The distribution of total polyadenylated RNA and mRNAs from the beta-actin, fibronectin, and cytokeratin Endo A genes was examined in preimplantation mouse embryos using in situ hybridization of riboprobes to RNA in sections of embryos. Polyadenylated RNA was found in the cytoplasm of all cells of blastocyst-stage embryos, whereas the specific mRNAs displayed three distinct patterns of expression: uniform throughout the embryo (beta-actin), enriched in the inner cell mass (fibronectin), and enriched in the trophectoderm (Endo A). In eight-cell embryos, the polyadenylated RNA was more concentrated in nuclei than in the cytoplasm (as noted previously), although this was not the case in blastocysts, nor was it true for the specific mRNAs that were examined. These experiments demonstrate that there is localized gene expression in the early mouse embryo, which correlates with the formation of the trophectoderm and the inner cell mass.     

海洋酸化对海洋鱼类行为的影响及机制研究进展

自工业革命以来,在人类活动的影响下,大气CO_2浓度持续增加,其中有大约1/3被海洋吸收,造成海水pH值降低和碳酸盐平衡体系的波动,即"海洋酸化"现象(Ocean Acidification)。据联合国政府间气候变化专门委员会预测,如果以当前速率排放CO_2,到21世纪末表层海水的pH值将降低至7.7—7.8,而到2300年将降低至7.3—7.4。作为鱼类对外界刺激最直接的反应,行为在鱼类的繁衍、捕食、避敌等过程中发挥着关键作用。基于此,海洋酸化对海洋鱼类行为的影响受到了越来越多关注。现有研究结果显示海洋酸化不仅会显著干扰包括嗅觉、听觉、视觉在内的感官功能,还将对神经生理功能和细胞信号传导等过程产生不利影响,从而影响海洋鱼类的捕食、逃避捕食、行为侧向化、栖息地识别与选择和集群等行为。行为异常将直接损害鱼类种群的生存与繁衍,继而威胁海洋生态系统的稳定和功能。我国海岸线漫长,海域辽阔,鱼类资源丰富,鱼类捕捞和养殖业发达。但与国外相比,国内此类研究十分匮乏,仅见零星报道。这种现状极大的制约了我国相关应对策略的制定,对我国海洋生态保育和渔业发展非常不利。此外,当前的研究也存在研究范围窄、研究手段不合理、行为效应、潜在机制及生态风险考察不足、研究结果难以整合等问题亟待改进。为此,研究对国内外相关研究进展进行了梳理和总结,并对未来的研究进行展望,以期弥补上述缺憾,促进国内相关研究的广泛开展。     

Musciomol and flunitrazepam binding sites in a subcellular fraction enriched in rat cerebellar glomeruli

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of GABA_A and of benzodiazepine binding sites between membranes derived from this fraction (fraction G) and from a total cerebellar homogenate (fraction T) was studied. The benzodiazepine and GABA binding sites were measured by the binding of agonists [³H]flunitrazepam and [³H]muscimol, respectively. The results indicate that both binding sites are present, but only slightly enriched, in the glomerular synapses. We found a muscimol/flunitrazepam binding site ratio of two, which is consistent with the enrichement of muscimol binding sites in the granular layer shown by both autoradiographic with radioactive glutamatergic ligands and in situ hybridization experiments respectively.     

Effects of GABA antagonists,SR 95531 and bicuculline,on GABAA receptor-regulated chloride flux in rat cortical synaptoneurosomes

Synaptoneurosomes isolated from cerebral cortices of male Sprague-Dawley rats were used for studying GABA_A receptor-regulated chloride influx. The in vitro effects of GABA antagonists, SR 95531 (a pyridazinyl GABA derivative) and bicuculline, on pentobarbital-stimulated, muscimol-stimulated or flunitrazepam-enhanced, muscimol-stimulated chloride uptake were studied. The chloride uptake was determined at 30°C, for 5 sec. Pentobarbital and muscimol produced a maximal stimulation of chloride uptake in cortical synaptoneurosomes at 500 M and 50M, respectively. SR 95531 as well as bicuculline had no effect on the basal uptake of chloride. Whereas, SR 95531 (0.3–30 M) and bicuculline (0.1–100 M), when added 5 min before muscimol (50 M), produced a significant concentration-dependent inhibition of muscimol (50 M)-stimulated chloride uptake (IC₅₀ s of 0.89±0.11 M and 13.45±2.10M, respectively). In studies of the inhibitory effects of SR 95531 and bicuculline on pentobarbital (500 M)-stimulated chloride uptake, the IC₅₀ s were 0.81±0.12 M and 3.86±1.14 M, respectively. SR 95531 exhibited a more potent inhibitory effect than bicuculline on flunitrazepam-enhanced, muscimol-stimulated chloride uptake. The results revealed that SR 95531 has a more potent antagonistic effect than bicuculline on GABA_A-regulated chloride flux.     

Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors

Based on a pharmacophore model of the benzodiazepine-binding site of GABA_A receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K_i values of around 0.20 nM were determined. They show a structural resemblance with the previously described 2-phenyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones (II) and 2-phenyl-[1,2,4]triazolo[1,5-a]quinoxalin-4(5H)-one (III). The 9-bromo substituted compounds 8a-d were prepared in an 8-step synthesis in an overall yield of approximately 40%, and a library of 9-substituted analogues was prepared by cross-coupling reactions. Compound 8e, 21, 22, and 24 were tested on recombinant rat ??₁??₃??₂, ??₂??₃??₂, ??₃??₃??₂, and ??₅??₃??₂ subtypes, and displayed selectivity for the ??₁??₃??₂ isoform.     

EphB3 receptor and ligand expression in the adult rat brain

Summary Eph receptors and ligands are two families of proteins that control axonal guidance during development. Their expression was originally thought to be developmentally regulated but recent work has shown that several EphA receptors are expressed postnatally. The EphB3 receptors are expressed during embryonic development in multiple regions of the central nervous system but their potential expression and functional role in the adult brain is unknown. We used in situ hybridization, immunohistochemistry, and receptor affinity probe in situ staining to investigate EphB3 receptors mRNA, protein, and ligand (ephrin-B) expression, respectively, in the adult rat brain. Our results indicate that EphB3 receptor mRNA and protein are constitutively expressed in discrete regions of the adult rat brain including the cerebellum, raphe pallidus, hippocampus, entorhinal cortex, and both motor and sensory cortices. The spatial profile of EphB3 receptors was co-localized to regions of the brain that had a high level of EphB3 receptor binding ligands. Its expression pattern suggests that EphB3 may play a role in the maintenance of mature neuronal connections or re-arrangement of synaptic connections during late stages of development.