Anglade I, Mazurais D, Douard V, Le Jossic-Corcos C, Mañanos E L, Michel D, Kah O
Endocrinologie Moléculaire de la Reproduction, UPRES-A-CNRS 6026, Institut de Biologie et d'Ecologie des Poissons, IFR 43, Rennes, France.
J Comp Neurol. 1999 Jul 26;410(2):277-89. doi: 10.1002/(sici)1096-9861(19990726)410:2<277::aid-cne9>3.0.co;2-v.
By using degenerate primers designed from glutamate decarboxylase (GAD) sequences of mammals, Xenopus and Drosophila, a 270-bp cDNA fragment was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) from cerebellum total RNA of rainbow trout. This partial cDNA shows 90% identity with mammalian GAD 65 and presents the Asn-Pro-His-Lys (NPHK) sequence corresponding to the pyridoxal-binding region of porcine DOPA decarboxylase or mammalian GAD. The distribution of GAD 65 mRNA-expressing neurons in the forebrain of the trout was studied by in situ hybridization using either digoxigenin- or 35S-labeled probes. The results demonstrate that gamma-amino butyric acid (GABA) neurons are widely distributed throughout the forebrain, with a high density in the periventricular regions. In this study, we report their precise distribution in the telencephalon and diencephalon. GAD mRNA-expressing cells were particularly abundant in the preoptic region and the mediobasal hypothalamus, two major neuroendocrine and estrogen-sensitive regions in fish. The presence of GAD mRNA-expressing neurons was observed in visually related structures such as the suprachiasmatic nucleus, the pretectal region, and the thalamus. Immunohistochemistry with antibodies directed against mouse GAD failed to demonstrate the presence of immunoreactive cell bodies, but showed a very high concentration of GAD-immunoreactive fibers in many brain regions, notably in the preoptic area, hypothalamus, and neurohypophyseal digitations of the pituitary, in particular in the proximal pars distalis. These results indicate that GABA neurons are ideally placed to modulate neuroendocrine activities at the hypothalamic and pituitary levels and to participate in the processing of sensorial information.
通过使用根据哺乳动物、非洲爪蟾和果蝇的谷氨酸脱羧酶（GAD）序列设计的简并引物，利用逆转录聚合酶链反应（RT-PCR）从小虹鳟鱼小脑总RNA中克隆出一个270 bp的cDNA片段。这个部分cDNA与哺乳动物GAD 65具有90%的同一性，并呈现出与猪多巴脱羧酶或哺乳动物GAD的吡哆醛结合区域相对应的天冬酰胺-脯氨酸-组氨酸-赖氨酸（NPHK）序列。使用地高辛或35S标记的探针，通过原位杂交研究了鳟鱼前脑表达GAD 65 mRNA的神经元分布。结果表明，γ-氨基丁酸（GABA）能神经元广泛分布于整个前脑，在脑室周围区域密度较高。在本研究中，我们报告了它们在端脑和间脑中的精确分布。表达GAD mRNA的细胞在视前区和中基底部下丘脑特别丰富，这是鱼类两个主要的神经内分泌和雌激素敏感区域。在与视觉相关的结构如视交叉上核、顶盖前区和丘脑中观察到了表达GAD mRNA的神经元。用针对小鼠GAD的抗体进行免疫组织化学未能证明存在免疫反应性细胞体，但在许多脑区显示出GAD免疫反应性纤维的高浓度，特别是在视前区、下丘脑和垂体的神经垂体指状部，尤其是在远侧部近端。这些结果表明，GABA能神经元处于理想位置，可在下丘脑和垂体水平调节神经内分泌活动，并参与感觉信息的处理。
