Investigation of the ontogenetic patterns of rat hypothalamic dopaminergic neurone morphology and function in vitro

Using fetal rat hypothalamic cells in primary culture maintained in a serum-free defined medium we have investigated the morphological and functional development of the dopamine (DA)-containing neurones intrinsic to the hypothalamus. Immunocytochemical studies demonstrated the presence of three morphologically distinct subtypes of tyrosine hydroxylase-immunopositive neurones. On day 3 in vitro unipolar, bipolar and multipolar cell types were apparent. The latter two subtypes persisted to later days in culture and increased both in perikarya size and neurite length. All subtypes have been shown to have correlates in vivo. Biochemical studies employing [3H]DA demonstrated a time- and temperature-dependent uptake mechanism within the cultures which was significantly attenuated by the uptake inhibitors benztropine and nomifensine in a dose-dependent manner. [3H]DA was also released under both basal and 56 mmol K+/l-stimulated conditions and the magnitude of the response was reduced by exclusion of calcium from the release medium. The amount of [3H]DA accumulated and released by the cultural cells increased with the age of the culture, suggesting functional maturation of the DA-containing neurones within this preparation. The role of oestradiol-17 beta in regulating hypothalamic dopaminergic function was also investigated both indirectly with the use of [3H]DA and by direct measurement of endogenously synthesized DA using high-performance liquid chromatography coupled with electrochemical detection. Both uptake and release of [3H] and release of endogenous DA were significantly modulated by the concentration of steroid in the defined medium. These results demonstrate that hypothalamic dopaminergic neurones, when maintained in primary culture, undergo morphological and functional maturation which have several correlates in vivo. In addition, we have demonstrated that at least one sub-population of dopaminergic neurones within this preparation is responsive to oestradiol-17 beta. As DA is considered to be a vital component in the regulation of neuroendocrine activity we suggest that this model is valuable for the investigation of the functional development of the DA systems of the hypothalamus and the relationship existing between neurotransmitters, neuropeptides and neuroactive steroids.