Morphlogical and functional evidences for in vitro embryonic hypothalamic neuron development.

GRISELDA MORENO; NESTOR GABRIEL CARRI; MIRTA REYNALDO; EDUARDO SPINEDI

Toronto, Ontario Canada

Congreso; 89th The Endocrine Society´s Annual Meeting; 2007

Endocrine Society

&lt;!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:595.3pt 841.9pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --&gt; There are only few evidences on the ontogeny and differentiation process of the hypothalamic supraoptic-paraventriculo-neurohypophysial (HSPN)neurosecretory system. In vitro neuron survival improves if cells come from embryionic 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 wih glial cells. For this aim, a primary neuronal culture was setup after mechanical dissociation of sterile hypothalamic blocks from Sprague Dawley rat, rat embryos (E18) of both sexes. Isolated hypothalamic cell were cultured with supplement (B27)-NeuroBasal medium was assessed on different days (d8, d10, d13, d15 and d17) of culture. Morphological identification of cells was assessed by immunocytochemical analysis using anti glial fibrillary acid protei (GFAP), for staining of glial cell, 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 dependant fashion. Moreover the in vitro neurosecretory activity was maximal (p<0.05 vs 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 (56mM KCl) and specific (10 nM Angiotensin II, AII) stimuli (for 8 hs) were able to significantly (p<0.05)enhance neuropeptide output (32.33 +/-4.58 and 28.93+/-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 OT. Finally only 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 iteraction of AVP and OT ergic neurons with glial cells.