Cortical localization of dopamine D4 receptors in the rat brain--immunocytochemical study.

Using polyclonal antibody against dopamine D4 receptor we investigated cortical distribution of D4 receptors, with the special emphasis on regions of the prefrontal cortex. Prefrontal cortex is regarded as a target for neuroleptic drugs, and engaged in the regulation of the psychotic effects of various substances used in the experimental modeling of schizophrenia. Western blot analysis performed on samples from the rat cingulate, parietal, piriform cortices and also striatum revealed that antibody recognized one main band of approximately 40 kD, which corresponds to the predicted molecular weight of D4 receptor protein. In immunocytochemical studies we found D4 receptor-positive neurons in all regions of prefrontal cortex (cingulate, agranular/insular and orbital cortices) and all cortical regions adjacent to prefrontal cortex, such as frontal, parietal and piriform cortex. Substantial number of D4 receptor-positive neurons has also been observed within the striatum and nucleus accumbens. In general, a clear stratification of the D4 receptor-positive neurons was observed in the cortex with the highest density seen in layers II/III and V/VI. D4 immunopositive material was also found in the dendritic processes, particularly clearly visible in the layer II/III. At the cellular level D4 receptor immunoreactivity was seen predominantly on the periphery of the cell body, but a certain population of neurons with clear cytoplasmatic localization was also identified. In addition to cortical distribution of D4 receptor-positive neurons we tried also to define types of neurons expressing D4 receptor protein. In double-labeling experiments, D4 receptor protein was found in nonphosphorylated neurofilament H-positive, calbindin-D28k-positive, as well as parvalbumin-positive cells. Since, used proteins are markers of certain populations of pyramidal neurons and GABA-ergic interneurons, respectively, our data indicate that D4 receptors are located on cortical pyramidal output neurons and their dendritic processes as well as on interneurons. Above localization indicates that D4 receptors are not only directly influencing excitability of cortical inter- and output neurons but also might be engaged in dendritic spatial and temporal integration, required for the generation of axonal messages. Additionally, our data show that D4 receptors are widely distributed throughout the cortex of rat brain, and that their cortical localization exceeds the localization of dopaminergic terminals.