Morphological and Functional Evidences for In Vitro Embryonic Hypothalamic Neuron Development.

MORENO G; CARRI NG; REYNALDO M; SPINEDI E

Toronto, Canada

Congreso; 89th Endocrine Society Meeting; 2007

The Endocrine Society

There are only few evidences on the ontogeny and the differentiation process of the hypothalamic supraoptic-paraventriculo-neurohypophysial (HSPN) neurosecretory system. In vitro neuron survival improves if cells come from embryonic origin. However, it was found that surviving hypothalamic neurons in culture express small and minimal amounts of arginine vasopressin (AVP) and oxytocin (OT), respectively. Thus the in vitro use of embryonic hypothalamic cells could result in a valuable tool for evaluation of HSPN system development. The aim of the present study was to develop a primary neuronal culture design able to be used for the study of the magnocellular hypothalamic system functionality and interaction with glial cells. For this aim, a primary neuronal culture was set up after mechanical dissociation of sterile hypothalamic blocks from, Sprague-Dawley, rat embryos (E18) of both sexes. Isolated hypothalamic cells were cultured with supplemented (B27)-NeuroBasal medium, containing an agent inhibiting non-neuron cell proliferation (AraC) and antibiotics. The characterization of the neurosecretory process was achieved by detecting AVP and OT (by specific radioimmunoassays) secreted into the medium on different days (d8, d10, d13, d15 and d17) of culture. Morphological identification of cells was assessed by inmunocytochemical analysis using anti-glial fibrillary acid protein (GFAP), for staining of glial cells, and specific anti-AVP and anti-OT sera for labeling peptidergic neurons. Functional data indicate that spontaneous AVP and OT release occurred in a culture day-dependent fashion. Moreover, the in vitro neurosecretory activity was maximal (p<0.05 vs. respective d8-values) on d13 for AVP (25,41±2.93 vs. 15.56±1.02 pg/ml) and on d10 for OT (94.02±14.73 vs. 45.73±3.82 pg/ml). Additionally, on d17, unspecific (56 mM KCl) and specific (10 nM Angiotensin II, AII) stimuli (for 8 hours) were able to significantly (p<0.05) enhance neuropetide output (32.33±4.58 and 28.92 ± 5.99 pg AVP/ml, and 60.52 ± 13.62 and 41.97 ± 4.34 pg OT/ml, after KCl and AII respectively) over the respective baseline (AVP: 20.53 ± 1.99, and OT: 29.13 ± 3.54 pg/ml). On d17, AVP mRNA expression was greater (approximately 3 fold) than that of OT. Finally, only a 6.67 ± 2.08 % of glial cells was detected. This study supports that our experimental design is useful for the study of both HSPN system function, and interaction of AVP- and OT-ergic neurons with glial cells.Supported by PIP6176; CICPBA; and FNSR 3200BO-105657/1.Date: Saturday, June 2, 2007Session Info: POSTER SESSION: BASIC/TRANSLATIONAL - Neuroendocrinology I: Models of Hormone Therapy, Brain Sex Differences & Development (11:00 AM-12:00 PM and 2:30 PM-3:30 PM)Presentation Time: 11:00 AMRoom: Hall D/E