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Ontogeny of 5HT2 receptor gene expression in developing rat brain
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ACNP Symposium Serotonin Receptor Subtypes as Targets for Psychotherapeutics Tuesday, 20 October 1992
Ontogeny of 5HT 2 receptor gene expression in developing rat brain
C i a r a n e l l o , R . D . , G a r l o w , S.J. a n d M o r i l a k , D.A. Nancy Pritzker Laboratory of Developmental and Molecular Neurobiology, Department of Psychiatry, Stanford University School of Medicine, Stanford, CA 94305, USA
Previous work from our laboratory has shown that whole brain 5HT 2 receptor mRNA appears around embryonic day 17 (El7) in the rat. Messenger RNA levels increase slowly to postnatal day 4 (P4), then undergo a burst in transcription from P4 until P12, when mRNA levels have increased by 13-fold. Message levels then decline by about 25-50% between P12 and P27, when they attain adult levels. Quantitative mdioligand binding exhibits the same profile as receptor mRNA. The changes taking place in 5HT 2 mRNA over this period correspond to a time when the cerebral cortex is undergoing serotonergic hyperinnervation followed by axonal pruning. To explore further the developmental control of 5HT 2 receptor gene expression, we prepared an antibody to the receptor immunizing rabbits with 20-mer oligopeptides from different receptor domains, based on antigenic analysis of the translated cDNA sequence. Best results where obtained with an antibody directed against a region of the N-terminal sequence of the receptor, and this antibody was used to map the developmental profile of receptor protein. Receptor mRNA was visualized during development using a 230 bp AluI fragment from the receptor eDNA in situ hybridization mapping studies. Our data indicate that, in contrast to serotonergic innervation, which makes its appearance early in brain development, 5HT 2 receptors appear considerably later. 5HT 2 receptor development in the brain proceeds in a caudalto-rostral pattern. 5HT~-expressing cells in the pontine dorsal tegmental nucleus appear as early as El8, and attain adult numbers and morphology by P4. In the caudate nucleus, some receptor labelling can be seen at P0, and the adult pattern is seen at P12. In the hippocampus, no receptor labelling is seen until P4, and a functional adult pattern of cell morphology and number is not seen until some time after PI2. Finally, in the cerebral cortex, receptor labelling begins to appear around P7, and reaches a peak of complexity at P12. Cortical 5HT2-bearing neurons exhibit an extensive and complex arbodzation, but only a few cells actually show immunopositivity. Between P12 and P27, the number of 5HT2-expressing neurons remains constant, but there is a decline in the extent of cellular arborization, consistent with the previously observed decrease in receptor binding and mRNA. Among the principal conclusions from this study are that a surprisingly small number of neurons in the brain express 5HT 2 receptors, although in most brain regions these all exhibit considerable dendritic arborization. In most cases, 5HT 2 receptor expression appears to take place on either cholinergic or GABA-ergic intemeurons. Despite the extensive ramification of the 5HT2-bearing neurons, our results suggest that disease processes which effect the absolute number of 5HT2-expressing neurons could have a significant impact on the total brain concentration of these receptors. Supported by NMH grants MH 39437 and MH 00219, by the Spunk Fund, the Edward and Marjorie Gray Fund, the Dana Neurosciences Foundation Fellowship Fund and the endowment to the Nancy Pritzker Laboratory.
ACNP Symposium Serotonin Receptor Subtypes as Targets for Psychotherapeutics Tuesday, 20 October 1992
Ontogeny of 5HT 2 receptor gene expression in developing rat brain
C i a r a n e l l o , R . D . , G a r l o w , S.J. a n d M o r i l a k , D.A. Nancy Pritzker Laboratory of Developmental and Molecular Neurobiology, Department of Psychiatry, Stanford University School of Medicine, Stanford, CA 94305, USA
Previous work from our laboratory has shown that whole brain 5HT 2 receptor mRNA appears around embryonic day 17 (El7) in the rat. Messenger RNA levels increase slowly to postnatal day 4 (P4), then undergo a burst in transcription from P4 until P12, when mRNA levels have increased by 13-fold. Message levels then decline by about 25-50% between P12 and P27, when they attain adult levels. Quantitative mdioligand binding exhibits the same profile as receptor mRNA. The changes taking place in 5HT 2 mRNA over this period correspond to a time when the cerebral cortex is undergoing serotonergic hyperinnervation followed by axonal pruning. To explore further the developmental control of 5HT 2 receptor gene expression, we prepared an antibody to the receptor immunizing rabbits with 20-mer oligopeptides from different receptor domains, based on antigenic analysis of the translated cDNA sequence. Best results where obtained with an antibody directed against a region of the N-terminal sequence of the receptor, and this antibody was used to map the developmental profile of receptor protein. Receptor mRNA was visualized during development using a 230 bp AluI fragment from the receptor eDNA in situ hybridization mapping studies. Our data indicate that, in contrast to serotonergic innervation, which makes its appearance early in brain development, 5HT 2 receptors appear considerably later. 5HT 2 receptor development in the brain proceeds in a caudalto-rostral pattern. 5HT~-expressing cells in the pontine dorsal tegmental nucleus appear as early as El8, and attain adult numbers and morphology by P4. In the caudate nucleus, some receptor labelling can be seen at P0, and the adult pattern is seen at P12. In the hippocampus, no receptor labelling is seen until P4, and a functional adult pattern of cell morphology and number is not seen until some time after PI2. Finally, in the cerebral cortex, receptor labelling begins to appear around P7, and reaches a peak of complexity at P12. Cortical 5HT2-bearing neurons exhibit an extensive and complex arbodzation, but only a few cells actually show immunopositivity. Between P12 and P27, the number of 5HT2-expressing neurons remains constant, but there is a decline in the extent of cellular arborization, consistent with the previously observed decrease in receptor binding and mRNA. Among the principal conclusions from this study are that a surprisingly small number of neurons in the brain express 5HT 2 receptors, although in most brain regions these all exhibit considerable dendritic arborization. In most cases, 5HT 2 receptor expression appears to take place on either cholinergic or GABA-ergic intemeurons. Despite the extensive ramification of the 5HT2-bearing neurons, our results suggest that disease processes which effect the absolute number of 5HT2-expressing neurons could have a significant impact on the total brain concentration of these receptors. Supported by NMH grants MH 39437 and MH 00219, by the Spunk Fund, the Edward and Marjorie Gray Fund, the Dana Neurosciences Foundation Fellowship Fund and the endowment to the Nancy Pritzker Laboratory.