Differentiation of Fetal Mouse Hypothalamic Cells in Serum-Free Medium

Abstract

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.

A serum-free culture of the neurons in the septal, preoptic, and hypothalamic region. Effects of triiodothyronine and estradiol.

“…The preincubation requirement of our cells in a serum-containing medium (plating medium) agrees well with those of Bottenstein et al (1980) and FaivreBauman et al (1981), yet we tried to shorten its incubation time (less than 3 hours), for serum has an ability to induce a cholinergic di Terentiation in neurons, even though they might be exposed to it only briefly (Higgins et al, 1984). The possibility of such induction seems unlikely in our cultures, because our incubation time is too short to allow induction to occur (Higgins et al, 1984) and the ChAT activity in our cultures is significantly low in comparison with that of Faivre-Bauman et al (1981). In comparison with enzymatic harvesting by trypsin, mechanical harvesting by scraper (Faivre-Bauman et al, 1981) may be disruptive of cells to release their cytosolic enzymes (Sorimach and Yasumura, 1983).…”

“…The possibility of such induction seems unlikely in our cultures, because our incubation time is too short to allow induction to occur (Higgins et al, 1984) and the ChAT activity in our cultures is significantly low in comparison with that of Faivre-Bauman et al (1981). In comparison with enzymatic harvesting by trypsin, mechanical harvesting by scraper (Faivre-Bauman et al, 1981) may be disruptive of cells to release their cytosolic enzymes (Sorimach and Yasumura, 1983). The direct cell-to-substratum contact of our cells thus requires enzymatic harvesting for a higher recovery of our cultured cells.…”

17α-Estradiol Exerts Neuroprotective Effects on SK-N-SH Cells

“…These data indicate that E2 concentrations observed in rodents and in women can serve a neuroprotective effect. In rats, neurotrophic (Toran-Allerand, 1976, 1980Faivre-Bauman et al, 1981;Nishizuka and Arai, 1981;Toran-Allerand et al, 1983;Morse et al, 1986;Wooley et al, 1990;Wooley and McEwen, 1992) and neuroprotective Singh et al, 1994;Behl et al, 1995;Goodman et al, 1996) effects of 17␤-E2 have been described. However, to our knowledge, 17␣-E2 has not been demonstrated previously to exert either neurotrophic or neuroprotective effects.…”

Estrogen Actions in the Central Nervous System1

“…However, it has been difficult to obtain satisfactory binding data for putative steroid receptors on rat brain membranes. Approaches using progesterone linked to 125 I-labeled BSA have met with some success in labeling putative progestin sites on brain membranes (90). However, the majority of the evidence to date is based on functional studies using electrophysiology or other endpoints, and some of this will be summarized below as it applies to the coupling to second messenger systems.…”

“…The first neuroprotective actions of estradiol were described in relation to the effects of serum deprivation on neuronal survival in cell culture (125)(126)(127)(128)(129). In one of these studies (127), picomolar levels of 17␤-estradiol enhanced fetal rat hypothalamic neuronal survival in a serum-free medium in the presence or absence of glial cells by a mechanism that was blocked by tamoxifen and that, presumably, involves intracellular ERs.…”