Elsevier

Neuroscience

Volume 88, Issue 3, February 1999, Pages 899-915
Neuroscience

Cellular and subcellular localization of 5-hydroxytryptamine1B receptors in the rat central nervous system: immunocytochemical, autoradiographic and lesion studies

https://doi.org/10.1016/S0306-4522(98)00256-5Get rights and content

Abstract

The localization of 5-hydroxytryptamine1B receptors in the rat central nervous system was investigated using anti-peptide antibodies that recognize a selective portion of the third intracytoplasmic loop of the receptor protein. At the light microscope level the densest 5-hydroxytryptamine1B receptor-like immunoreactivity was observed in ventral pallidum, globus pallidus, substantia nigra and dorsal subiculum. In addition, moderate immunoreactivity was found in the entopeduncular nucleus, the superficial gray layer of the superior colliculus, the caudate–putamen and the deep nuclei of the cerebellum. This distribution matched perfectly that previously described from radioligand binding studies. At the ultrastructural level, 5-hydroxytryptamine1B receptor-like immunoreactivity was associated with axons and axon terminals in the three areas examined: substantia nigra, globus pallidus and superficial gray layer of the superior colliculus. In all cases, immunostaining was located on the plasma membrane of unmyelinated axon terminals and in the cytoplasm close to the plasmalemma. Synaptic differentiations were never labelled, but, in some cases, 5-hydroxytryptamine1B receptor-like immunoreactivity was found in their close vicinity. Injection of kainic acid into the neostriatum resulted in a marked decrease in receptor-like immunoreactivity in the globus pallidus and the substantia nigra, consistent with the location of 5-hydroxytryptamine1B receptors on terminals of striatopallidal and striatonigral fibres, respectively. A reduction in 5-hydroxytryptamine1B receptor-like immunoreactivity was also noted in the superficial gray layer of the superior colliculus after contralateral enucleation, as expected of the location of 5-hydroxytryptamine1B receptors on the terminals of retinocollicular fibres. In both lesion experiments, immunolabelled degenerating terminals were observed in the projection areas. Anterograde labelling experiments coupled with immunocytochemical detection further showed that 5-hydroxytryptamine1B receptors in the substantia nigra are located on axons of striatal neurons.

These data provide anatomical supports to the idea that 5-hydroxytryptamine1B receptors act as terminal receptors involved in presynaptic regulation of the release of various neurotransmitters, including 5-hydroxytryptamine itself.

Section snippets

Animals

Male Wistar rats weighing 300–350 g were maintained under controlled environmental conditions (22–24°C: 12/12 h light/dark cycle, food and water ad libitum). All efforts were made to minimize animal suffering and to reduce the number of animals used.

Surgical procedures

For lesion of the striatopallidal and striatonigral pathways, rats were anaesthetized with pentobarbital (50 mg/kg, i.p.) and placed in a stereotaxic frame (David Kopf Inc.). Two injections of kainic acid (1 μg dissolved in 1 μl of 0.9% NaCl at each

Results

Extensive characterization of the antibodies raised against the selective Val263–Lys287 portion of the rat 5-HT1B receptor protein, which were used in the present study, has clearly demonstrated their specificity for this receptor.34, 52

Distribution of 5-hydroxytryptamine1B receptor and binding sites in the rat brain

Comparison of the distribution of 5-HT1B receptor-like immunoreactivity in the rat brain with maps derived from the autoradiographic detection of 5-HT1B binding sites using [125I]CYP or serotonin-O-carboxymethylglycyl-[125I]iodotyrosinamide ([125I]GTI)8, 12, 13, 47reveals a remarkable degree of similarity. High densities of both receptor-like immunoreactivity and specific binding sites are present in globus pallidus, ventral pallidum, substantia nigra and dorsal subiculum.8, 12, 34, 52Other

Conclusion

A remarkable similarity exists between the distribution of 5-HT1B receptors established from immunohistochemical and radioligand binding studies.8, 12, 13, 34, 47, 52The high resolution power of immunocytochemical techniques allowed the subcellular visualization of 5-HT1B receptors. At the ultrastructural level, we found that the protein is essentially located on axons and axon terminals in the globus pallidus, the substantia nigra and the superficial gray layer of the superior colliculus. In

Acknowledgements

This work was supported by grants from CNRS, INSERM, DRET (Contract n° 95-142), the MERT (Ministère de l'Education et de la Recherche et de la Technologie), the EEC (Biotech, contract BI04-CT96-0752), and the ECOS Committee (Action n° C94-B03). We are grateful to Mr P. Nguyen for his excellent photographic work.

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