Characterization of Opioid Receptors in Cultured Neurons

The appearance of μ‐, δ‐, and k‐opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high‐affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (‐)‐[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax= 210 ± 40 fmol/mg protein); this density is approximately two‐thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the μ‐specific ligand [3H]D‐Ala2,N‐Me‐Phe4,Gly‐ol5‐enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 ± 0.1 nM and a density (Bmax) of 160 ± 20 fmol/mg of protein. Specific binding of (‐)‐[3H]bremazocine under conditions in which μ‐ and δ‐receptor binding was suppressed (k‐receptor labeling conditions) occurred to an apparent single class of sites (KD= 2 ± 1 nM;Bmax= 40 ± 15 fmol/mg of protein). There was no detectable binding of the selective δ‐ligand [3H]D‐Pen2,d‐Pen5‐enkephalin. Thus, cultured striatal neurons expressed μ‐ and k‐receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not δ‐receptors.