Neonatal Rat Hypothalamus Research Articles

Specific effects of prenatal DEHP exposure on neuroendocrine gene expression in the developing hypothalamus of male rats.

Endocrine disrupting chemicals may disrupt developing neuroendocrine systems, especially when the exposure occurs during a critical period. This study aimed to investigate whether prenatal exposure to di-(2-ethylhexyl) phthalate (DEHP), a major component of plasticizers used worldwide, disrupted the development of a network of genes important for neuroendocrine function in male rats. Pregnant rats were treated with corn oil (vehicle control), 2, 10 or 50mg/kg DEHP by gavage from gestational day 14 to 19. The neuroendocrine gene expressions were quantified using a 48-gene Taqman qPCR array in the whole hypothalamus of neonatal rats (postnatal day 1) and in the anteroventral periventricular nucleus (AVPV), medial preoptic nucleus (MPN) and arcuate nucleus (ARC) of adult rats (postnatal day 70). Immunofluorescent signals of ERα and CYP19 were detected using the confocal microscopy in adult AVPV, MPN and ARC. The results showed that prenatal DEHP exposure perturbed somatic and reproductive development of offspring. Eleven genes were down-regulated in neonatal hypothalamus and showed non-monotonic dose-response relationships, that the 10mg/kg DEHP dosage was associated with the greatest number of gene expression changes. Different from this, 14 genes were altered in adult AVPV, MPN and ARC and most of alterations were found in the 50mg/kg DEHP group. Also, 50mg/kg DEHP reduced ERα expression in the ARC, but no alterations were observed in CYP19 expression. These results indicated that prenatal DEHP exposure may perturb hypothalamic gene programming and the influences are permanent. The effects showed dependence on developmental stages and nuclei region.

Neonatal overexpression of estrogen receptor-α alters midbrain dopamine neuron development and reverses the effects of low maternal care in female offspring.

Maternal behavior is dependent on estrogen receptor-alpha (ERα; Esr1) and oxytocin receptor (OTR) signaling in the medial preoptic area (MPOA) of the hypothalamus, as well as dopamine signaling from the ventral tegmental area (VTA) to forebrain regions. Previous studies in rats indicate that low levels of maternal care, particularly licking/grooming (LG), lead to reduced levels of MPOA ERα and VTA dopamine neurons in female offspring and predict lower levels of postpartum maternal behavior by these offspring. The aim of this study was to determine the functional impact on maternal behavior of neonatal manipulation of ERα in females that had experienced low versus high levels of postnatal maternal LG. Adenovirus expressing ESR1 was targeted to the MPOA in female pups from low and high LG litters on postnatal day 2-3. Overexpression of ESR1 in low LG offspring elevated the level of ERα-immunoreactive cells in the MPOA and of tyrosine hydroxylase cells in the VTA to that observed in high LG females. Amongst juvenile female low LG offspring, ESR1 overexpression also decreased the latency to engage in maternal behavior toward donor pups. These results show that virally mediated expression of ESR1 in the neonatal rat hypothalamus results in lasting changes in ESR1 expression through the juvenile period, and can "rescue" hormone receptor levels and behavior of offspring reared by low LG dams, potentially mediated by downstream alterations within reward circuitry. Thus, the transmission of maternal behavior from one generation to the next can be augmented by neonatal ERα in the MPOA.

Effects of progesterone and RU486 on the development and expression of adult male sexual behaviour and gene expression in the amygdala and preoptic area of the hypothalamus

The number of progesterone receptors is greater in the male than female neonatal rat hypothalamus. The aims of the present study were to determine developmental effects of progesterone on the expression of adult male sexual behaviour and whether changes in behaviour were reflected by altered gene expression within the hypothalamic preoptic area (POA) or medial amygdala. Male rats were treated with progesterone (40 µg kg(-1), i.p.), the progesterone receptor antagonist RU486 (40 µg kg(-1), i.p.) or an equal volume of vehicle (10% ethanol, 90% corn oil) on postnatal Days 1-5. Treatment with either progesterone or RU486 inhibited (P ≤ 0.07) the initial expression of consummatory sexual behaviour at 10.5 weeks of age without influencing growth or serum concentrations of testosterone. Sexual interest, as measured by latency to exhibiting mounting behaviour or the number of mounts achieved, was not influenced by treatment with either progesterone or RU486. The effects of treatment with progesterone or RU486 on sexual behaviour were diminished by experience. Microarray analysis of the POA indicated 61 genes that were upregulated and 49 that were downregulated (P ≤ 0.01) following RU486 treatment of male rats. However, the altered expression of selected genes was not confirmed by real-time reverse transcription-polymerase chain reaction. The expression of targeted genes within the amygdala was not influenced by treatment with either progesterone or RU486. Neonatal treatment with RU486, but not progesterone, decreased testes weight (P=0.02) without affecting testes morphology. The results indicate that altering the progesterone environment during a critical developmental period affects the expression of behaviour, but that changes in behaviour are not mirrored by the altered expression of selected genes.

Genistein Induces Phosphorylation of cAMP Response Element‐binding Protein in Neonatal Hypothalamus In Vivo

We investigated the effect of the phytoestrogen, genistein and 17beta-oestradiol on cAMP response element-binding protein (CREB) phosphorylation in the neonatal female rat hypothalamus in vivo using western blot analysis and immunohistochemistry. Although CREB expression was insensitive to the compounds we tested, administration of genistein and 17beta-oestradiol induced rapid CREB phosphorylation (< 15 min) in the hypothalamus and its level remained elevated at 4 h. Quantitative immunohistochemical analysis showed that genistein and 17beta-oestradiol had no effect on CREB phosphorylation in the magnocellular subdivision of paraventricular nucleus. By contrast, genistein induced a dose-dependent increase in CREB phosphorylation in the medial preoptic area (mPOA) and anteroventral periventricular nucleus (AVPV). Administration of 17beta-oestradiol also caused a rapid, dose-dependent increase in CREB phosphorylation in the hypothalamus, mPOA and AVPV. These results demonstrate that genistein induces oestrogen-like rapid action on CREB phosphorylation in the neonatal central nervous system in vivo.

Disappearance of Sex Difference in the Anxiety-Like Behavior in Progranulin-Deficient Mice.

It is well established that the mammalian brain possesses sexually dimorphic structure, which is reflected by functional sexual differences in the control of specific types of behavior. Sexual differentiation of the brain in rodents is achieved by estrogens, which are converted from androgens in the brain, during the perinatal period. We have identified the progranulin (PGRN) gene as one of the sex steroid-inducible genes in the hypothalamus of neonatal rats, and suggested that PGRN and/or PGRN-derived peptides (granulins) are involved in the organization of neuronal circuits responsible for the expression of male sexual behavior. We have further generated a line of mice with targeted disruption of the PGRN gene, and observed a decrease in ejaculation incidence and an increase in aggression. Recently, we also suggested the involvement of PGRN in estrogen-induced neurogenesis in adult rat hippocampus. To further clarify the role of PGRN in the sexual differentiation of the brain, we analyzed the anxiety-like behavior and anxiety-related brain nuclei using PGRN-deficient mice. We used 7-week old male and female wild type (Grn+/+) and PGRN-deficient (Grn-/-) mice of C57BL/6J background. To evaluate the anxiety-like behavior, open field test was performed. The open field was composed of 50 x 50 cm square surrounded by 40 cm-height walls. Each mouse was introduced in the center of the square and its behavior was recorded on videotape for 10 min. The duration when the mouse stayed in the peripheral zone (7.5 cm from the wall) or the center, and the distance traveled were analyzed using a computerized technique with Ethovision 3.0 software. Then, 50-µm thick sections were made from 4% paraformaldehyde-fixed brain and stained with 0.1% cresyl violet. The volume and number of cells in the locus ceruleus (LC) and paraventricular nucleus (PVN), both of which are known to be involved in exhibiting anxiety-like behavior, were determined from the digital images of sections using Image J software. Male Grn+/+ mice spent significantly longer time in the center of open filed than female Grn+/+ mice, suggesting that the anxiety is lower in males than in females. Male Grn-/- mice spent significantly shorter time in the center of open field than male Grn+/+ mice, and the duration staying in the center was not different between male and female Grn-/- mice. Distances traveled in both areas were not significantly different between the sexes nor genotypes. Histological analysis revealed that the volume and the number of cells in the LC of Grn-/- mice were significantly larger than those of Grn+/+ mice. On the other hand, there were no significant differences in the volume and cell numbers in the PVN between the sexes nor genotypes. These results suggest that there are sex differences in anxiety to the novel environment in wild type mice of the strain used in the present study, i.e., females exhibit enhanced anxiety than males, and that PGRN plays an important role in the formation of this sex difference. It is probable that PGRN produces the sex difference in anxiety by affecting the organization of neuronal structure in the LC. Given that PGRN is involved in estrogen-induced neurogenesis, PGRN may suppress proliferation and/or the survival of neural cells in the LC during the perinatal period, thereby affects the behaviors such as aggression and anxiety in adulthood.

Ethanol Induces Apoptotic Death of β‐Endorphin Neurons in the Rat Hypothalamus by a TGF‐β1‐Dependent Mechanism

We have previously shown that developing beta-endorphin neurons, in the arcuate nucleus of the hypothalamus become increasingly apoptotic when exposed to ethanol. As in the previous study we have observed an involvement in transforming growth factor beta 1 (TGF-beta1) in mediation of the apoptotic process, the present study was conducted to determine the ethanol-induced changes in this apoptotic regulatory peptide signaling in the arcuate nucleus of the hypothalamus of neonatal rats. Pups were exposed to 11.34% ethanol in a milk-based diet or control diet on postnatal day (PND) 3 to PND7. Two hours after the last daily feeding, brains were collected and frozen in liquid nitrogen for analysis of various apoptosis regulatory proteins in the arcuate tissue by Western blots. Some animals were fixed in 4% paraformaldehyde and analyzed immunohistochemically. Ethanol exposure increased apoptotic death of beta-endorphin neurons in the arcuate nucleus of the hypothalamus. The cell death was associated with an increase in the tissue levels of TGF-beta1 in the mediobasal hypothalamus. This was correlated with a reduction in the arcuate level of retinoblastoma protein (Rb) phosphorylation. The reduced level of Rb phosphorylation was associated with an increased protein level of the cyclin dependent kinase inhibitor p27/kip but with a decreased protein level of cyclin dependent kinase 4 and cyclin D3. In addition, the apoptotic cell death was positively correlated with the level of Bclxs but negatively correlated with the level of the Bcl2. These results suggest that ethanol exposure increases TGF-beta1 signaling involving Bcl2 and Rb repression that may lead to apoptotic death of cells including beta-endorphin neurons in the arcuate nucleus of the hypothalamus.

Expression of functional Angiotensin II (Ang II) receptors types, AT1R and AT2R, in RVLM neuronal cultures from adult rat brain

Our previous studies have demonstrated that AT1R signaling pathway regulates RVLM neuronal activity. Their increased expression and coupling to PI3K has been linked to increased Ang II‐mediated neuronal activity in the SHR‐RVLM. These findings, obtained from mixed cultures of neonatal rat hypothalamus, may not reflect those of the RVLM in adults. Thus, our objectives in this study were to establish neuronal cultures from the RVLM of adult rats, and use them to study the effects of Ang II. GFP‐AAV viral vector was injected into the RVLM of SD rats two weeks prior to isolation of neurons for anatomical confirmation. Cells were dissociated, plated onto culture dishes and allowed to establish for 7–14 days. Presence of neuron specific markers, neuN and synaptophysin in GFP expressing cells confirmed the presence of RVLM neurons. Certain fluorescent neurons expressed AT1R. Ang II effect was tested on Ca2+ currents (IBa) recorded from GFP expressing neurons. Ang II caused a 30% increase in IBa that was attenuated by 2 μM losartan. However, pretreatment with AT2R antagonist, PD123319, resulted in 2.5 fold augmentation of Ang II‐mediated increase in IBa. Together, these observations demonstrate that both AT1R and AT2R are present on the same neuron and that inhibition of the AT2R accentuates the AT1R‐mediated stimulation of IBa. They suggest a functional coupling of AT1R and AT2R in the RVLM. (Supported by NIH HL 33610 and HLT6312)

Effects of Perinatal Exposure to Phthalate/Adipate Esters on Hypothalamic Gene Expression and Sexual Behavior in Rats

Our previous research has identified the granulin (grn) and p130 genes as sex steroid-regulated genes in the neonatal rat hypothalamus that might be involved in sexual differentiation of the brain. Since phthalate/adipate esters such as di-n-butyl phthalate (DBP), diisononyl phthalate (DINP), and di-2-ethylhexyl adipate (DEHA) are suspected to interfere with the endocrine system as environmental endocrine disruptors having estrogenic or antiandrogenic properties, these chemicals may affect sexual differentiation of the brain. The present study assessed the effects of perinatal exposure to DBP, DINP, and DEHA on grn and p130 mRNA expressions in the hypothalamus on postnatal day (PND) 7 and sexual behaviors after maturation in rats. Maternal rats were given a phytoestrogen-free diet containing different doses of DBP (20, 200, 2,000, and 10,000 ppm), DINP (40, 400, 4,000, and 20,000 ppm) and DEHA (480, 2,400, and 12,000 ppm) from gestational day 15 to the day of weaning (PND 21). DBP and DINP exposure during the perinatal period resulted in an increase in hypothalamic grn and p130 mRNA levels in females and males, respectively, but DEHA exposure decreased expression levels of grn in males and p130 in females, although the effects were not dose-dependent. After maturation, male rats that were exposed to several doses of DBP, DINP, and DEHA displayed decreased copulatory behavior. The lordosis quotient was decreased in females perinatally exposed to DBP, DINP, and DEHA at all the doses used. On the other hand, serum levels of LH and FSH in both sexes and the estrous cycles in females were not affected by the treatments. These results suggest that inappropriate expression of grn and/or p130 genes in the brains of male and female neonatal rats by perinatal exposure to these chemicals may exert permanent effects on the hypothalamus, thereby decreasing sexual behavior after maturation.

Enhanced nicotinic acetylcholine receptor-mediated [ 3H]norepinephrine release from neonatal rat hypothalamus

Nicotinic acetylcholine receptor (nAChR)-evoked release of norepinephrine (NE) has been demonstrated in a number of brain regions that receive sole noradrenergic innervation from the locus coeruleus (LC). Many of these structures display enhanced nicotine-stimulated NE release in the neonate. We have examined the hypothalamus in order to determine if this region, which receives NE projections from both the LC and medullary catecholaminergic nuclei, also demonstrates maturational changes in nAChR-mediated NE release. Quantification of radiolabeled-NE release from rat hypothalamus slices by a maximally effective dose of nicotine revealed a peak response during the first postnatal week. This was followed by a decrease at postnatal day (P) 14, and a second peak at P21. Thereafter, release was equivalent to that observed at P14. Comparison of the pharmacological properties of nAChRs mediating NE release in neonatal (P7) and mature hypothalamus suggested involvement of different nAChR subtypes at the two ages. Using the selective toxin, DSP-4, nAChR-mediated NE release in the neonatal hypothalamus was shown to be from LC terminals. Our findings demonstrate an early sensitivity of hypothalamic LC terminals to nAChR regulation that may be associated with development of systems controlling critical homeostatic functions such as stress, feeding and cardiovascular regulation.

Neuronal expression of catechol O-methyltransferase mRNA in neonatal rat suprachiasmatic nucleus.

We examined the expression profile of catechol O-methyltransferase (COMT) mRNA and its protein in the neonatal rat hypothalamus by in situ hybridization and immunohistochemistry to clarify the sites of dopamine degradation. Strong COMT mRNA expression was observed in the suprachiasmatic nucleus (SCN) throughout its rostrocaudal extent at postnatal day 1 (P1) and P2, and the mRNA levels decreased gradually until P16. COMT mRNA was predominantly localized to the ventral and medial parts of the SCN. Intense COMT immunoreactivity was demonstrated in the ventral SCN and was detected in neuronal perikarya and processes at P1. Ependymal and microglial cells also exhibited strong COMT immunoreactivity. These results indicate that COMT may directly be involved in dopaminergic signaling in the neonatal SCN.

Angiotensin II induction of AP-1 in neurons requires stimulation of PI3-K and JNK

Angiotensin II (Ang II) acts via its type 1 (AT 1) receptor in neurons to regulate the activity of multiple intracellular signaling molecules, including intracellular Ca 2+, protein kinase C, phosphatidylinositol 3-kinase (PI3-K), and c-Jun NH 2-terminal kinase (JNK). The present studies investigated the upstream signaling molecules involved in the Ang II stimulation of activator protein-1 (AP-1) DNA binding in neurons. Treatment of neurons cultured from neonatal rat hypothalamus and brainstem with Ang II (100 nM) showed a time-dependent increase in AP-1 DNA binding and this effect was inhibited by the AT 1 receptor antagonist, losartan (1 μM), the PI3-K inhibitor, LY294002 (10 μM), and the JNK inhibitor, JNK inhibitor II (100 nM). Furthermore, Ang II (100 nM) causes a time-dependent increase in JNK activity which was attenuated by PI3-K inhibition. These data establish, for the first time, a signaling cascade involved in the Ang II activation of AP-1 DNA binding in neurons.

Expression analyses of sex steroid-regulated genes in neonatal rat hypothalamus.

Estrogen plays an important role in sexual differentiation of the brain in rats during the perinatal period. To elucidate molecular mechanisms underlying sexual differentiation of the brain, in this study we investigated genes differentially expressed between sexes or induced to express by estrogen in neonatal rat hypothalamus using DNA microarray analysis in combination with real-time RT-PCR. It was found that the levels of expression of the genes encoding glutamic acid decarboxylase 65 and coronin 1b were higher in male than female hypothalamus on postnatal day (PN) 5 and those of collagen type 3 alpha1 and thioredoxin reductase 2 genes in female hypothalamus on PN5 were decreased and increased, respectively, by treatment with estradiol on PN2. Then the developmental changes in the expression of these 4 genes were examined from 1 day before the parturition to PN9, and they all showed sexual dimorphic patterns. In addition, dependence of the expression of these genes on either estradiol, testosterone or dihydrotestosterone during the neonatal period was confirmed. These results suggest that these four genes are involved in sexual differentiation of the rat brain, and that androgen per se as well as estrogen may take part in the processes.

Androgen induces p130 mRNA expression in the neonatal rat hypothalamus

Our previous research using cDNA microarray analysis demonstrated that female rats displayed a higher p130 mRNA level than males in the hypothalamus at postnatal day (PN) 5. In the present study, it was shown that at PN3 males had a significantly elevated mRNA level over females, whereas at PN7 females displayed a higher expression level using a real-time reverse transcription-polymerase chain reaction. In situ hybridization analysis indicated relatively strong p130 mRNA signals in the ventromedial nucleus and the arcuate nucleus in the neonatal hypothalamus. Subcutaneous injection of 5α-dihydrotestosterone as well as testosterone propionate to PN2 neonatal rats significantly increased p130 gene expression at PN3, whereas estradiol benzoate did not have a significant effect. These results suggest that expression of the p130 gene in the neonatal rat hypothalamus is responsive to androgens and may be involved in sexual differentiation of the brain.

Effects of early undernutrition on the brain insulin-like growth factor-I system.

Undernutrition reduces circulating concentrations of insulin-like growth factor (IGF)-I, but how it affects the brain IGF system, especially during development, is largely unknown. We have studied IGF-I, IGF-II, IGF receptor and IGF binding protein (BP)-2 mRNA expression in the hypothalamus, cerebellum and cerebral cortex of neonatal rats that were food restricted beginning on gestational day 16. One group was refed starting on postnatal day 14. Rats were killed on postnatal day 8 or 22. Undernutrition did not produce an overall reduction in brain weight at either age but, at 22 days, both the cerebellum and hypothalamus weighed significantly less. At 8 days, no change was detected in the central IGF axis in response to undernutrition. However, in 22-day-old undernourished rats, IGF-I and IGF receptor mRNA expression were increased in both the hypothalamus and cerebellum, while IGFBP-2 was decreased, but only in the hypothalamus. Refeeding had no effect on any of these parameters. These results suggest that the hypothalamus and cerebellum respond to malnutrition and the decrease in circulating IGF-I, a peptide fundamental for growth and development, by increasing the local production of both the growth factor and its receptor in attempt to maintain normal development.

GABA mediates steroid-induced astrocyte differentiation in the neonatal rat hypothalamus.

Our previous work has demonstrated that astrocytes in the developing arcuate nucleus of the rat hypothalamus are sexually dimorphic as a result of differential exposure to oestradiol. Moreover, our experiments in neonatal rats suggest an absence of oestrogen receptors (ER) in arcuate nucleus astrocytes in vivo. This, along with the conspicuous lack of evidence in the literature confirming the presence of ER in arcuate nucleus astrocytes of the neonatal rat brain, led us to question the mechanism by which oestrogen induces changes in arcuate nucleus astrocyte morphology. Based on our previous findings that oestradiol increases gamma-aminobutyric acid (GABA) levels in the neonatal rat arcuate, we hypothesize that GABA is released from neighbouring oestrogen-sensitive neurones and alters arcuate nucleus astrocyte morphology. Here, we report that in vivo reduction of GABA synthesis in the neonatal rat brain by antisense oligodeoxynucleotides to glutamic acid decarboxylase prevented gonadal steroid-induced astrocyte differentiation in males and testosterone-treated females. Conversely, activation of GABAA receptors with the agonist muscimol increased astrocyte differentiation in females in the absence of gonadal steroids. Given that GABA is made only in neurones and that its synthesis is increased by oestradiol, we conclude that it acts as a diffusible factor inducing the differentiation of neighbouring astrocytes.

Estrogen Affects Gene Expression of Estrogen Receptors, Androgen Receptor, and Aromatase in the Neonatal Rat Hypothalamus.

Androgen, largely through its aromatized estrogen, plays an important role in sexual differentiation of the rat brain during the perinatal period. To demonstrate the regulation of estrogen receptors α (ERα), and β (ER β), androgen receptor (AR), and aromatase (AROM) gene expression, the mRNA levels of these mRNAs were determined in the hypothalamus of male and female rats at 2, 6, and 10 days of age using the competitive RT-PCR method. ERα mRNA level did not differ between males and females, except that gene expression on day 6 was lower in males than in females. There were no significant differences in gene expressions of ERβ and AR in the hypothalamus between males and females throughout the neonatal period. AROM mRNA levels were higher on males for all 3 days of determination. Subcutaneous estrogen treatment at 2 days of age decreased ERα mRNA levels, while it increased ERβ, AR and AROM gene expressions. Our results suggest that sexual differences and steroid-dependent regulation of these genes might be important in the sexual differentiation of the rat brain.

Sex-Related Differences in Gene Expression in Neonatal Rat Hypothalamus Assessed by cDNA Microarray Analysis.

Sexual differentiation of the rodent brain is recognized to involve transcriptional activation of multiple genes induced by gonadal steroids at developmental stages. To identify the genes differing in expression level between sexes, we analyzed gene expression in male and female rat hypothalami at postnatal day 5 by means of a cDNA microarray consisting of 2352 genes. By comparing the expression pattern between sexes, we identified 12 male-enriched genes and 20 female-enriched genes. Among them, the expression pattern of 1 male-enriched gene, jagged homolog 1, and those of 2 female-enriched genes, p27Kip1 and p130, were confirmed to be consistent with microarray data by RT-PCR. Investigation of these genes should help to elucidate the molecular and cellular mechanisms underlying sexual differentiation of the rodent central nervous system.

Induction of granulin precursor gene expression by estrogen treatment in neonatal rat hypothalamus

Our previous research has demonstrated that androgen treatment during the perinatal period increases granulin (grn) precursor mRNA levels in the neonatal rat hypothalamus. To elucidate whether exogenous estrogen increases grn mRNA in the neonatal hypothalami, expression of grn gene in the neonatal hypothalamus was studied by the competitive reverse transcription-polymerase chain reaction method. At 6 and 10 days of age, grn gene expression was significantly increased in the hypothalamus of pups whose dam has been dietarily administrated ethinyl estradiol from day 15 of gestation to the day of sampling. The subcutaneous injection of estradiol benzoate to neonatal rats at 2 days of age significantly increased grn gene expression on day 10. It was shown that estrogen, as well as androgen, was able to induce grn gene expression in the neonatal hypothalamus.

Developmental sex differences in calbindin-D28K and calretinin immunoreactivity in the neonatal rat hypothalamus

Developmental sex differences in calbindin-D28K and calretinin immunoreactivity in the neonatal rat hypothalamus

Developmental sex differences in calbindin-D28K and calretinin immunoreactivity in the neonatal rat hypothalamus

The proteins calbindin-D(28K) and calretinin 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 significant 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 significantly elevated calbindin levels over PN0 and PN2 females (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 nucleus, and dorsomedial nucleus, and an area immediately surrounding the ventromedial nucleus (VMN). Calbindin immunoreactivity was absent from the ventrolateral VMN, but lightly stained cell bodies were observed in the dorsomedial VMN. The sex differences observed in calcium binding proteins parallel our previously observed sex differences in excitatory gamma-aminobutyric acid and glutamate early in development and may be related to mechanisms of sexual differentiation of the brain.