No abstract

We have previously demonstrated that differentiation of hypothalamic dopaminergic (DA) neurons can be induced in culture by their pituitary intermediate lobe target cells, through both membrane and diffusible factors. We also showed that subpopulations of DA neurons from the arcuate nucleus only, not the periventricular area, can respond to the target. Here we investigated the possibility that both neuronal subsets could also respond differentially to brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT3). Addition of NT3, but not BDNF, enhanced growth and branching of neurites, tyrosine hydroxylase (TH) as well as increasing levels of cultured arcuate DA neurons. Conversely, BDNF, but not NT3, affected the same parameters in cultured periventricular DA neurons. The neurotrophins thus affect DA neurons in a structure and neuronal type-selective manner, since general neuronal markers were not affected by either neurotrophin. Neurotrophin effects were reversed by addition of specific antibodies directed against them or their respective receptors, TrkB or TrkC. By themselves, the antibodies inhibited development of DA neurons below that of control cultures, suggesting involvement of endogenous neurotrophins. BDNF and NT3 were indeed found in both arcuate and periventricular neurons and in the intermediate lobe. BDNF was always present as the mature peptide. The mature form of NT3 was only detected in the periventricular area; a precursor-like heavier form was present in all tissues studied. The present data suggest that NT3, but not BDNF, could participate in the differentiating action of intermediate lobe cells on arcuate DA neurons.

We show here that cells dissociated from fetal mouse hypothalamus and cerebral hemispheres can be grown in primary culture in a serum-free medium (SFM). We describe several properties of these cultures and compare them to those in serum-supplemented medium (SSM). The SFM used is a modification of that described for neuroblastoma cells: neuronal survival is improved when 17 β-estradiol is added. Initial events in culture development were similar to those observed in SSM. However, after 1 week, several differences could be noted: in SFM, the proportion of neuron-like cells was increased while the basal glial layer was noticeably reduced, and the neurite network remained less developed than in SSM. These findings demonstrate that the use of SFM permits manipulation of the types and proportions of cells in these primary cultures. This point has been already made. Several neuronal activities were studied. In cultures from both hypothalamus and cerebral hemispheres, thyroliberin (TRH)-immunoreactive cells were visualized by immunohistochemistry, and TRH was radioimmunoassayed in cell extracts and in the medium. In hypothalamic cultures, tyrosine hydroxylase was shown to remain stable for 1 week, and then declined. Glutamic acid decarboxylase disappeared very quickly in vitro, whereas choline acetyltransferase activity increased more rapidly in SFM than in SSM. It is concluded that the use of an SFM for growing normal fetal hypothalamic cells offers a promising model for studying neuroendocrine regulatory mechanisms in culture.