Immunocytochemical visualization of kappa-opioid receptors on chick embryonic neurons differentiating in vitro

The present paper is the first immunocytochemical demonstration of kappa-opioid receptors in neurons isolated from seven-day-old chick embryonic forebrains and cultivated for one to seven days. The monoclonal antibody KA8 (IgG1-k) [Maderspach et al. (1991) J. Neurochem. 56, 1897-1904] was raised against the frog brain kappa-opioid receptor as an antigen and recognizes an epitope in or near the ligand binding site. The KA8 immunostaining of the neurons displayed individual variations and changed with the in vitro differentiation. Receptors often appeared at the pole of the primary outgrowing process, later on in the whole soma and finally on the branched processes. Specific radioligand binding and KA8 immunocytochemistry both presented an increase in the receptor concentration with development. The equilibrium binding values that were measured at 1 nM [3H]naloxone concentration were 2.9 and 6.1 fmol/10(6) cells on the first and sixth cultivation days, respectively. Neurons were treated with 10(-7) M bremazocine or dynorphine (agonists with relative specificity to kappa-opioid receptors) on the second and third cultivation days. The agonist promoted the morphological differentiation which was already visible within 24 h. It also promoted the expression of the 200,000 mol. wt neurofilament protein, this became pronounced after two to three days. The changes provoked by the agonist were reduced by the opioid antagonist norbinaltorphimine (10(-7) M) or naloxone (10(-5) M) indicating that the effect was receptor-mediated. The hypothesis that kappa-opioid agonists through their receptors may function as regulatory signals in the early neuronal differentiation is discussed.