An improved method for primary culture of hypothalamic tanycytes

DE FRANCESCO N; CASTROGIOVANNI D; URIARTE M; FRASSA V; AGOSTI F; REYNALDO M; RAINGO J; PERELLO M

Mar del Plata

Congreso; XXX reunion anual de la Sociedad Argentina de investigaciones en neurociencias; 2015

Sociedad Argentina de investigaciones en neurociencias

Hypothalamic tanycytes are specialized bipolar ependymal cells lining the floor of the third ventricle. Given its strategic location, tanycytes play several key functions acting as a selective blood-cerebrospinal fluid barrier and controlling many neuroendocrine axes. The in vitro culture of this cell population has proved to be difficult due to several factors including their limited amount in vivo, and the particular anatomical location and cellular complexity of their normal environment. Here, we report an improved method for generating primary cultures of rat hypothalamic tanycytes. Ependymal cultures were derived from tissue dissected out of the median eminence region of 10-day old rats and cultured in a chemically defined media. After 10 day in vitro, a significant fraction of the cells in culture exhibited morphological features of tanycytes as observed by phase contrast or scanning electron microscopy. All tanycyte-like cells were immuno-reactive for markers of this cell type, including vimentin, GFAP and DARPP32. Whole-cell patch clamp recordings of these cells showed their resting membrane potential became more negative with increased cell length. Current injections failed to produce spike-like responses. Thus, the current method allows obtaining cultures highly enriched in tanycyte-like cells that resemble in vivo tanycytes in terms of morphology, molecular markers and electricalactivity.