Elsevier

Neuroscience

Volume 100, Issue 1, 7 September 2000, Pages 141-153
Neuroscience

Comparative immunohistochemical distributions of carboxy terminus epitopes from the mu-opioid receptor splice variants MOR-1D, MOR-1 and MOR-1C in the mouse and rat CNS

https://doi.org/10.1016/S0306-4522(00)00248-7Get rights and content

Abstract

The present study examined immunohistochemically the CNS distributions of a splice variant of the mu-opioid receptor, MOR-1D, in both rats and mice. In MOR-1D, exon 4 of MOR-1 is replaced by two additional exons that code for seven amino acids. Using rabbit antisera, we compared immunohistochemically the regional distribution of a C-terminal epitope of MOR-1D to that of a C-terminal epitope from MOR-1 and a C-terminal epitope from another splice variant, MOR-1C. The general distribution of MOR-1D-like immunoreactivity was similar in both mouse and rat. MOR-1D-like immunoreactivity was seen in the dentate gyrus and in the mossy fibers of the hippocampal formation, the nucleus of the solitary tract and the area postrema, the inferior olivary nucleus, the nucleus ambiguous, the spinal trigeminal nucleus and the spinal cord. MOR-1D-like immunoreactivity was not observed in some regions containing dense MOR-1-like immunoreactivity, such as the striatum or the locus coeruleus. In regions containing MOR-1, MOR-1C and MOR-1D, the pattern of each variant was unique.

MOR-1D and MOR-1C are splice variants of the cloned mu-opioid receptor MOR-1. Although they differ only at the tip of the carboxy terminus, they show marked differences in their regional distributions, as determined immunohistochemically by epitopes in their unique carboxy termini. Since the splice variants are derived from the same gene, these differences in regional distribution imply region-specific messenger RNA processing.

Section snippets

Experimental procedures

All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the MSKCC and conform to NIH guidelines on the ethical use of animals. Care was taken to minimize the number of animals used and to minimize any suffering.

Transfected cells

The MOR-1D antiserum intensely labeled HEK-293 cells transiently transfected with a cDNA encoding MOR-1D (Fig. 2A), but not cells transfected with MOR-1 (Fig. 2H). No MOR-1D-LI was detected in non-transfected cells (Fig. 2F). Immunolabeling was abolished when the incubation was performed with either preimmune serum (Fig. 2E) or with immune serum preabsorbed with the peptide antigen (10 nM; Fig. 2C) or when the primary antibody was omitted (Fig. 2D). As a control, we also transfected HEK-293

Discussion

Splice variants of MOR-1 have unique distributions in the CNS. Previous studies looking at MOR-1B-LI observed a dense, but limited, presence in the olfactory bulb.52 MOR-1C-LI is particularly abundant in the lateral septum, the hypothalamic nuclei and the spinal cord. In the present study, the distribution of MOR-1D-LI was mainly restricted to the hippocampal formation, the nucleus of the solitary tract and the spinal cord. Within these regions, each splice variant has a unique pattern of

Conclusion

These immunohistochemical results demonstrate a differential localization of the splice variants of the MOR-1 gene in the CNS, consistent with the various physiological and pharmacological functions mediated by the opioid receptors. The recent identification of five novel exons within the MOR-1 gene and four new splice variants40., 41. illustrates the complexity of the MOR family. With the two variants reported several years ago, there are now seven different MOR-1 variants differing only in

Acknowledgements

This work was supported, in part, by a Mentored Scientist Award (DA00296) to Y.-X.P., research grants (DA02615 and DA06241) and a Senior Scientist Award (DA00220) to G.W.P. from the National Institute on Drug Abuse, as well as a core grant from the National Cancer Institute to the MSKCC (CA08748) and a research grant (HL18974) to the Laboratory of Neurobiology at CUMC.

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