Regional, developmental, and cell cycle-dependent differences in mu, delta, and kappa-opioid receptor expression among cultured mouse astrocytes.

The diversity of opioid receptor expression was examined in astrocytes in low-density and non-dividing (confluent) cultures from the cerebral cortex, hippocampus, cerebellum, and striatum of 1-day-old mice. Mu, delta, and kappa opioid receptor expression was assessed in individual cells immunocytochemically, by using flow cytometry, and functionally by examining agonist-induced changes in intracellular calcium ([Ca2+]i). Significant spatial and temporal differences were evident in the pattern of expression of mu, delta, and kappa receptors among astrocytes. In low-density cultures, greater proportions of astrocytes expressed mu-opioid receptor immunoreactivity in the cerebral cortex and hippocampus (26-34%) than in the cerebellum or striatum (7-12%). At confluence, a greater percentage of astrocytes in cerebellar (26%) and striatal (30%) cultures expressed mu-immunoreactivity. Fewer astrocytes possessed delta-immunoreactivity in low-density striatal cultures (8%) compared to other regions (16-22%). The proportion of delta receptor-expressing astrocytes declined in the cerebellum but increased in the hippocampus. Kappa-opioid receptors were uniformly expressed by 27-34% of astrocytes from all regions, except in cortical cultures, where the proportion of kappa expressing cells was 38% at low-density and decreased to 22% at confluence. Selective mu (PLO 17; H-Tyr-Pro-Phe (N-Me) -D-Pro-NH2, delta ([D-Pen2, D-Pen5] enkephalin), or kappa (U50,488H; trans-(+/-)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeneacetamide methanesulfonate) opioid receptor agonists increased [Ca2+]i in subpopulations of astrocytes indicating the presence of functional receptors. Lastly, opioid receptor immunofluorescence varied during the cell division cycle. A greater proportion of astrocytes in the G2/M phase of the cell cycle were mu or delta receptor immunofluorescent than at G0/G1. When astrocytes were reversibly arrested in G1, significantly fewer cells expressed delta receptor immunofluorescence; however, upon reentry into the cell cycle immunofluorescent cells reappeared. In conclusion, opioid phenotype varies considerably among individual cultured astrocytes, and this diversity was determined by regional and developmental (age and cell cycle dependent) differences in the brain. These in vitro findings suggest astroglia contribute to regional and developmental idiosyncrasies in opioid function within the brain.