Elsevier

Neuroscience

Volume 113, Issue 1, 2 August 2002, Pages 23-35
Neuroscience

Similar ultrastructural distribution of the 5-HT2A serotonin receptor and microtubule-associated protein MAP1A in cortical dendrites of adult rat

https://doi.org/10.1016/S0306-4522(02)00146-XGet rights and content

Abstract

As visualized by light and electron microscopic immunocytochemistry, the distribution of the neuronal serotonin-2A (5-HT2A) receptor is mainly intracellular throughout adult rat brain. This localization is particularly striking in the pyramidal cells of cerebral cortex, the dendrites of which are intensely immunoreactive, but without any labeling of their spines. In view of recent yeast two-hybrid and biochemical results suggesting an association of 5-HT2A receptors with the cytoskeletal microtubule-associated protein MAP1A, the respective subcellular distributions of the receptors and of MAP1A were compared by quantitative electron microscopic immunocytochemistry in dendrites of adult rat frontoparietal cortex. Counts of silver-intensified immunogold particles revealed a higher density of 5-HT2A receptors in smaller rather than larger dendrites, and an apportionment between pre-defined compartments representing the plasma membrane and the cytoplasm that was proportional to the relative surface area of these compartments. MAP1A immunoreactivity also predominated in smaller versus larger dendrites, but with a slightly lower proportion of labeling in the plasma membrane versus cytoplasmic compartment. The co-localization of 5-HT2A receptors and MAP1A protein in the same dendrites could be demonstrated in double immunolabeling experiments. These results confirmed the predominantly somato-dendritic, intracellular localization of 5-HT2A receptors in cerebral cortex, showed their higher concentration in distal as opposed to proximal dendrites, and suggested their potential association to the cytoskeleton in cortical neurons in vivo.

Such a distribution of 5-HT2A receptors reinforces our earlier hypothesis that 5-HT2A receptors participate in intraneuronal signaling processes involving the cytoskeleton, and raises the possibility that their activation could be dependent upon that of another co-localized, plasma membrane-bound, 5-HT receptor.

Section snippets

5-HT2A and MAP1A antibodies

Three 5-HT2A receptor antibodies were used. (1) A monoclonal antibody commercially available at Pharmingen (San Diego, CA, USA). This antibody was produced and characterized by Wu et al. (1998), using a GST fusion protein antigen containing the entire N-terminal domain (1–76) of the cloned human 5-HT2A receptor sequence. The targeted epitope is the sequence comprised between residues 64 and 76 (TCLSILHLQEKNW; C. Wu, personal communication). (2) A polyclonal antibody directed against a peptide

Subcellular distribution of 5-HT2A receptors in cortical dendrites

At the light microscopic level, the regional distribution of the immunoreactivity after labeling with every one of the three 5-HT2A receptor antibodies was the same as previously described in detail following immunostaining with the monoclonal N-terminal antibody (Cornea-Hébert et al., 1999a). In the frontoparietal cortex, a laminar pattern was observed. In layer V strong labeling of pyramidal neuronal somata and of their apical dendrites, spanning across the upper layers and branching

Discussion

The present immunoelectron microscopic study confirmed the prominent intracellular localization of 5-HT2A receptors in dendrites of the cerebral cortex. After immunogold labeling with two antibodies against their N-terminal portion, and a third against their C-terminal portion, the apportionment of 5-HT2A receptors in a plasma membrane and an intracellular compartment was equivalent to the relative surface area of these compartments, excluding any significant enrichment of the plasma membrane.

Acknowledgements

This work was supported by a studentship from the Groupe de recherche sur le système nerveux central (FCAR) to V.C.-H., Grant MT-3544 from the Medical Research Council of Canada (now NRF 3544 from CIHR) to L.D., and Grants RO1MH 61887 and KO2MH 01366 from NIH to B.L.R. The authors are indebted to Chun Wu and Sujay K. Singh for their generous gift of the Pharmingen 5-HT2A antibody. They also thank Gaston Lambert and Jean Léveillé for expert photographic work.

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