Neonatal maternal separation up-regulates protein signalling for cell survival in rat hypothalamus

Abstract

We have previously reported that in response to early life stress, such as maternal hyperthyroidism and maternal separation (MS), the rat hypothalamic vasopressinergic system becomes up-regulated, showing enlarged nuclear volume and cell number, with stress hyperresponsivity and high anxiety during adulthood. The detailed signaling pathways involving cell death/survival, modified by adverse experiences in this developmental window remains unknown. Here, we report the effects of MS on cellular density and time-dependent fluctuations of the expression of pro- and anti-apoptotic factors during the development of the hypothalamus. Neonatal male rats were exposed to 3 h-daily MS from postnatal days 2 to 15 (PND 2–15). Cellular density was assessed in the hypothalamus at PND 21 using methylene blue staining, and neuronal nuclear specific protein and glial fibrillary acidic protein immunostaining at PND 36. Expression of factors related to apoptosis and cell survival in the hypothalamus was examined at PND 1, 3, 6, 9, 12, 15, 20 and 43 by Western blot. Rats subjected to MS exhibited greater cell-density and increased neuronal density in all hypothalamic regions assessed. The time course of protein expression in the postnatal brain showed: (1) decreased expression of active caspase 3; (2) increased Bcl-2/Bax ratio; (3) increased activation of ERK1/2, Akt and inactivation of Bad; PND 15 and PND 20 were the most prominent time-points. These data indicate that MS can induce hypothalamic structural reorganization by promoting survival, suppressing cell death pathways, increasing cellular density which may alter the contribution of these modified regions to homeostasis.

• Apoptosis
• Akt/protein kinase B
• Bad/Bcl-2-associated death promoter protein
• caspase 3
• early life stress
• extracellular regulated kinase

Acknowledgements

We thank Surendra P. Verma for help in statistical analysis and for providing UDASYS program, to Nanjing Gao and Hernán Barrio for help in Western blots, Luz Navarro and Laura Escobar for equipment, Guadalupe Domínguez and Enrique Pinzón for technical assistance and Nicolás Villegas-Sepúlveda and Carmen Sánchez-Torres for generous antibody donation.