Characterization of opioid-dependent glial development in dissociated and organotypic cultures of mouse central nervous system: critical periods and target specificity

Abstract

Opioid-dependent changes in glial growth were characterized in primary dissociated and organotypic explant cultures of the developing mouse central nervous system (CNS) continuously grown in the presence of an endogenous opioid, [Met 5]enkephalin, or an opiate drug, morphine. The glia in dissociated, astrocyte-enriched cultures derived from the cerebra of postnatal day 1, 3, or 5 mice, respectively, displayed age-dependent reductions in glial numbers that occurred at 3, 7, or 9 days in vitro (DIV) in response to continuous [Met 5]enkephalin (10 −6 M) exposure. In contrast, in cultures derived from gestational day 19 mice, glial numbers were not reduced following continuous exposure to 10 −6 M [Met 5]enkephalin during the first 7 days in vitro. An examination of [ 3H]thymidine incorporation by glial fibrillary acidic protein-(GFAP) immunoreactive astrocytes with flat (type 1) morphology in dissociated cultures derived from postnatal day 1 mice revealed that the reduction in glial numbers at 3 DIV was not immediately preceded by a reduction in the rate of [ 3H]thymidine incorporation at 2 DIV, although previous studies have shown that opioids inhibit the rate of [ 3H]thymidine incorporation by more mature astrocytes at 4 or 6 DIV. Early (i.e., at 2 to 3 DIV) changes in glial numbers may result from an inhibition of the proliferative rate of non-GFAP-containing glia or astrocyte precursors, or an enhanced rate of glial death. The rate of [ 3H]thymidine incorporation by GFAP-immunoreactive astrocytes with process-bearing (type 2) morphology was unchanged by opioid treatment. In separate experiments, a comparison of the area of growth of GFAP-immunoreactive astrocytes in paired symmetrical (right vs left) organotypic explant cultures demonstrated that opiates (i.e., 10 −5 M morphine) can inhibit astrocyte growth when the normal histiotypic organization of neurons and glia are maintained, and that there are regional differences in astrocyte responsiveness. Opioid-dependent alterations in astrocyte growth were mediated through specific opioid receptors since they were prevented by simultaneous treatment with (−)naloxone. The results suggest that the ability of opioids to modify glial growth is highly selective and varies depending on astrocyte type, as well as temporal and regional factors. Spatial and temporal differences in the response of developing glia to opioids may determine critical periods of CNS vulnerability to opioids in the maturing brain.