Molecular Brain Research, 11 (1991) 239-247 ~) Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$03.50 ADONIS 0169328X92703347
239
BRESM 70334
Distribution of GABAA-receptor subunit gene expression in the rat forebrain Jian-Hua Zhang*, Toshiyuki Araki, Makoto Sato and Masaya Tohyama Department of Anatomy ar¢~'Neuroscience, Osaka University Medical School, Osaka (Japan) (Accepted 14 May 1991) Key words: y-Aminobutyric acid-A receptor: a ! Subunit variant mRNA; Cellular localization; Forebrain: Rat; In situ hybridization histochemistry
The localization of neurons containing mRNA of ~the a i subunit of the y-aminobutyric acid-A (GABAA) receptor was examined in the rat forebrain by in situ hybridization histochemistry using an oligonucleotide probe for the a , subunit. Moderately to strongly labeled neurons were numerous in the mitral cell layer of the olfactory bulb, the anterior olfactory nucleus, the diagonal band of Broca, the globus pallidus, the tenia tecta, the hippocampal formation, the thalamic and subthalamic nuclei, the zona incerta, and the amygdaloid complex. A few positive neurons were found in the caudate-putamen, the lateral and medial septal areas, the nucleus accumbens, the bed nucleus of the stria terminalis, the ventral pallidum, and the hypothalamus. The distribution of neurons containing a i subunit mRNA in the forebrain was very similar to that of neurons expressing t2 subunit mRNA, suggesting that these two subunits frequently coexist in the same neurons in the forebrain.
strated t2"3°'34'35. Cells containing the a~ subunit variant
INTRODUCTION
m R N A have been demonstrated in the cortex, the hippocampus, and the olfactory bulb 12'3°'34'35, but no infor-
9-Aminobutyric acid ( G A B A ) is the major inhibitory neurotransmitter in the central nervous system 5"27. Its inhibitory action is mediated via the activation of its spc ~ cific receptor, G A B A A and G A B A e receptors. U p to now, five subunits of the G A B A A receptor with some varzants have been found and their primary structures have been cloned. These are at_ 5, subunitsS,9.tl,2t,22,25,28,29,31,33,36.37.
ill-3,
91, 92,
6,
mation is yet available about other forebrain areas. In the present study, we used in situ hybridization histochemistry with an oligonudeotide probe unique to the sequence of the a~ subunit to investigate the detailed localization of cells expressing G A B A A receptor a 1 subunit m R N A in the rat forebrain.
and e
To analyze the functions of the variants of these subunits, their cellular localization needs to be explored. Recently, in situ hybridization histochemistry was used to determine the details of the region-specific expression of m R N A s encoding the fl subunit (~1, f12, and f13) and the 92 subunit of the G A B A A receptor in the rat brain~°" 40.4~. In the case of the a subunit variants, several sites have been shown to contain each of these vaI~anLs 6"12"30" 34,35, but the overall distribution of the cells expressing these a subunits is still not established. F o r example, although detailed mapping of a~ subunit m R N A expression in the lower brainstem has been reported 6, its localization in the forebrain has not been fully demon-
MATERIALS AND METHODS Animals and tissue preparation Male Wistar rats weighing approximately 100 g were decapitated under sodium pentoi~arbita! anesthesia (50 mg/kg, i.p.). The fresh brains were quickly remov¢~4 and immediately frozen on powdered dry ice. Serial sections 10-15 pm thick were cut on a cryostat and thaw-mounted onto gelatin-coated sl,,'des, which were stored at -80 °C until use (up to 1 month). Microscopic analysis revealed no appreciable change in the hybridization signals with storage. In situ hybridization histochemistry The procedure for in situ hybridization histochemistry was essentially the same as that described previously 14.41. After being warmed to room temperature, the slide-mounted sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 5 min. (All the following steps were performed at room temperature unless otherwise indicated.) The sections were then rinsed three times
* J.-H.Z. is a visiting researcher from the Department of Anatomy of the Fourth Military Medical College, Shaan Xi, Xi'an, People's Republic of China. Correspondence: M. Tohyama, Department of Anatomy and Neuroscience, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka, 565, Japan.
240
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239
BRESM 70334
Distribution of GABAA-receptor subunit gene expression in the rat forebrain Jian-Hua Zhang*, Toshiyuki Araki, Makoto Sato and Masaya Tohyama Department of Anatomy ar¢~'Neuroscience, Osaka University Medical School, Osaka (Japan) (Accepted 14 May 1991) Key words: y-Aminobutyric acid-A receptor: a ! Subunit variant mRNA; Cellular localization; Forebrain: Rat; In situ hybridization histochemistry
The localization of neurons containing mRNA of ~the a i subunit of the y-aminobutyric acid-A (GABAA) receptor was examined in the rat forebrain by in situ hybridization histochemistry using an oligonucleotide probe for the a , subunit. Moderately to strongly labeled neurons were numerous in the mitral cell layer of the olfactory bulb, the anterior olfactory nucleus, the diagonal band of Broca, the globus pallidus, the tenia tecta, the hippocampal formation, the thalamic and subthalamic nuclei, the zona incerta, and the amygdaloid complex. A few positive neurons were found in the caudate-putamen, the lateral and medial septal areas, the nucleus accumbens, the bed nucleus of the stria terminalis, the ventral pallidum, and the hypothalamus. The distribution of neurons containing a i subunit mRNA in the forebrain was very similar to that of neurons expressing t2 subunit mRNA, suggesting that these two subunits frequently coexist in the same neurons in the forebrain.
strated t2"3°'34'35. Cells containing the a~ subunit variant
INTRODUCTION
m R N A have been demonstrated in the cortex, the hippocampus, and the olfactory bulb 12'3°'34'35, but no infor-
9-Aminobutyric acid ( G A B A ) is the major inhibitory neurotransmitter in the central nervous system 5"27. Its inhibitory action is mediated via the activation of its spc ~ cific receptor, G A B A A and G A B A e receptors. U p to now, five subunits of the G A B A A receptor with some varzants have been found and their primary structures have been cloned. These are at_ 5, subunitsS,9.tl,2t,22,25,28,29,31,33,36.37.
ill-3,
91, 92,
6,
mation is yet available about other forebrain areas. In the present study, we used in situ hybridization histochemistry with an oligonudeotide probe unique to the sequence of the a~ subunit to investigate the detailed localization of cells expressing G A B A A receptor a 1 subunit m R N A in the rat forebrain.
and e
To analyze the functions of the variants of these subunits, their cellular localization needs to be explored. Recently, in situ hybridization histochemistry was used to determine the details of the region-specific expression of m R N A s encoding the fl subunit (~1, f12, and f13) and the 92 subunit of the G A B A A receptor in the rat brain~°" 40.4~. In the case of the a subunit variants, several sites have been shown to contain each of these vaI~anLs 6"12"30" 34,35, but the overall distribution of the cells expressing these a subunits is still not established. F o r example, although detailed mapping of a~ subunit m R N A expression in the lower brainstem has been reported 6, its localization in the forebrain has not been fully demon-
MATERIALS AND METHODS Animals and tissue preparation Male Wistar rats weighing approximately 100 g were decapitated under sodium pentoi~arbita! anesthesia (50 mg/kg, i.p.). The fresh brains were quickly remov¢~4 and immediately frozen on powdered dry ice. Serial sections 10-15 pm thick were cut on a cryostat and thaw-mounted onto gelatin-coated sl,,'des, which were stored at -80 °C until use (up to 1 month). Microscopic analysis revealed no appreciable change in the hybridization signals with storage. In situ hybridization histochemistry The procedure for in situ hybridization histochemistry was essentially the same as that described previously 14.41. After being warmed to room temperature, the slide-mounted sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 5 min. (All the following steps were performed at room temperature unless otherwise indicated.) The sections were then rinsed three times
* J.-H.Z. is a visiting researcher from the Department of Anatomy of the Fourth Military Medical College, Shaan Xi, Xi'an, People's Republic of China. Correspondence: M. Tohyama, Department of Anatomy and Neuroscience, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka, 565, Japan.
240
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