Regional, developmental, and cell cycle-dependent differences in mu, delta, and kappa-opioid receptor expression among cultured mouse astrocytes - PubMed (original) (raw)
Regional, developmental, and cell cycle-dependent differences in mu, delta, and kappa-opioid receptor expression among cultured mouse astrocytes
A Stiene-Martin et al. Glia. 1998 Mar.
Abstract
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.
Figures
Figure 1
(A-F) Opioid receptor immunofluorescence (green FITC product) in cerebral cortical astrocytes counterstained with propidium iodide (red nuclear product) at 6 days _in vitro_—similar to those used for flow cytometry. Astrocytes can express μ, δ, and/or κ opioid receptor immunoreactivity (A-F). μ-Opioid receptor immunoreactivity is reticular and highly polarized within individual astroglia (A), δ receptor immunoproduct is more diffuse and uniformly distributed throughout the cytoplasm (B), while κ-Opioid receptor immunoreactivity has both punctate and diffuse attributes (C); scale bar in A = 25 μm (A-C). Figure 1A-C is intended to illustrate the cellular pattern of opioid receptor immunoreactivity, rather than the actual proportion of receptor positive cells in these cultures. Astrocytes possessing opioid receptor immunoreactivity were not distributed uniformly in cell clusters. Specific immunoreactivity was not seen in preabsorbed controls (D-F). Scale bar in C = 20 μm (D-F). (G-H) μ, δ, and/or κ opioid receptor activation increased [Ca2+]i in some astrocytes. Functional changes in intracellular Ca2+ ([Ca2+]i, in response to opioid agonist treatment, varied greatly among individual astrocytes (repeated measures in cerebral forebrain astrocytes at 6 days in culture). (G) Exposure to the δ opioid receptor agonist, DPDPE, caused transient increases in [Ca2+]i within a subset of astrocytes (arrows). Alternatively, treatment with a κ agonist (U50,488H) did not increase [Ca2+]i; while PL017, a μ receptor agonist, elevated [Ca2+]i in cells (arrow) that had previously responded to DPDPE. (H) DPDPE exposure elevated [Ca2+]i in a subpopulation of astrocytes (hatched arrow), while U50,488H treatment recruited sustained [Ca2+]i increases in several additional cells (arrows) that also responded to PL017 treatment. Drug concentrations were 100 nM (G-H).
Figure 2
Effect of opioid receptor agonists on free intracellular Ca2+ ([Ca2+]i) in individual flat, polyhedral astrocytes (A-F). Astrocytes do not respond uniformly to μ, δ, or κ opioid agonists—individual cells display unique responses to μ, δ, and/or κ receptor activation. The effects of opioid receptor stimulation on [Ca2+]i are selective, often only a single astrocyte within a field was affected by a particular agonist type (B,C, D), and there was little cross reactivity among agonist types (A-D). The topography of the response was generally similar irrespective of whether μ, δ, or κ opioid receptors were activated. Agonist concentrations were 100 nM and agonist effects could be prevented by opioid antagonists (E-F). For example, naloxone pretreatment attenuated the response to U50,488 (F). Responsive cells (—) or (– - – - –); Non-responsive cells (..........).
Figure 3
Effect of regional or age-related (low-density vs. confluent) differences on the percentage of astrocytes expressing μ, δ or κ opioid receptor immunoreactivity. The mean ± SEM was determined from at least 3 cultures. About 500 astrocytes were assessed per culture; each culture consisted of an independent sample of cells from separate mice. The proportion of μ or δ receptor-expressing astrocytes differed significantly across regions (P < 0.05), and differed among low density and confluent cultures (*P < 0.05 vs. confluent astrocytes).
Figure 4
Effect of regional or age-related (low-density vs. confluent) differences on the percentage of astrocytes exhibiting changes in [Ca2+]i in response to μ, δ, or κ opioid agonists. The mean ± SEM was determined from at least five cultures. Forty-sixty astrocytes were assessed in each culture; each culture consisted of an independent sample of cells from separate mice. The proportion of astrocytes displaying increases in [Ca2+]i in response to μ receptor activation differed significantly across regions (P < 0.05), and low density vs. confluent cultures of the hippocampus (*P < 0.05 vs. confluent astrocytes). Because, opioid receptors may or may not couple to increases in [Ca2+]I, a strict one-to-one correspondence was not expected when comparing the proportion of immunoreactive cells (Figure 3) to the proportion of cells displaying functional changes in [Ca2+]i.
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