Developmental Sex Differences in Calbindin-D 28K and Calretinin Immunoreactivity in the Neonatal Rat Hypothalamus Darrin H. Brager, Micah J. Sickel, Margaret M. McCarthy Department of Physiology and Center for Studies in Reproduction, University of Maryland, School of Medicine, Baltimore, Maryland, 21201 Received 8 March 1999; accepted 1 July 1999 ABSTRACT: The proteins calbindin-D 28K and cal- retinin buffer intracellular calcium and are speculated to be involved in the integration of neuronal signaling. Using Western blot analysis, we compared the levels of calbindin-D 28K and calretinin in the developing male and female rat hypothalamus on postnatal days (PN) 0, PN2, PN4, PN6, PN8, and PN10. Analysis of variance (ANOVA) of mean calbindin levels indicated a signifi- cant effect of sex (p < .001) and age (p < .0001) and a significant interaction (p < .02). Post-hoc Neuman-Keuls analysis revealed that PN0 and PN2 males had signifi- cantly elevated calbindin levels over PN0 and PN2 fe- males (p < .05). ANOVA of mean calretinin levels from the same animals also indicated a significant effect of sex (p < .002) and a significant interaction between sex and age (p < .001). Post-hoc analysis indicated males had significantly elevated calretinin levels over PN0, PN4 (p < .05) and PN6 (p < .01) females. Immunocytochemical analyses indicated calbindin-immunopositive staining for cell bodies in the central subdivision of the medial preoptic nucleus, paraventricular nucleus, arcuate nu- cleus, and dorsomedial nucleus, and an area immedi- ately surrounding the ventromedial nucleus (VMN). Calbindin immunoreactivity was absent from the ven- trolateral VMN, but lightly stained cell bodies were observed in the dorsomedial VMN. The sex differences observed in calcium binding proteins parallel our pre- viously observed sex differences in excitatory -ami- nobutyric acid and glutamate early in development and may be related to mechanisms of sexual differentiation of the brain. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 315–322, 2000 Keywords: development; GABA; ventromedial nucleus; Western blot; calcium Sexual differentiation of the brain is a steroid-mediated phenomenon that occurs during a restricted developmen- tal window referred to as the sensitive or critical period. In rats, the onset of this critical period is gestational day 18 (GD18) as a result of activation of the embryonic testis and production of copious quantities of testoster- one. The sensitive period continues through postnatal day 7–10 (PN7–10), after which time either the admin- istration or removal of testosterone is without substantial effect on the developing brain (Gerall et al., 1992; Ja- cobson and Gorski, 1981). At completion of the sensi- tive period, the brain is either masculinized or remains feminized, resulting in adult sexual dimorphisms in re- productive behavior, brain morphology, and control of gonadotropin release. An important question has been the nature of the neuroanatomical underpinnings that regulate sex dif- ferences in adult brain functioning. Early work fo- cused on the sexually dimorphic nucleus of the pre- optic area (SDN-POA), which is significantly larger in males than females as a result of differential steroid exposure (Gorski et al., 1978). Females treated peri- natally with testosterone propionate throughout the critical period develop an SDN-POA equivalent in size to that of normal males (Do ¨ hler et al., 1984a) and removal of the testis in males (Davis et al., 1995) or treatment with an estrogen receptor antagonist Correspondence to: D. H. Brager © 2000 John Wiley & Sons, Inc. CCC 0022-3034/00/030315-08 315