The hippocampus is a key brain region regulating complex cognitive and emotional responses, and is implicated in the etiology of depressive and anxiety disorders, many of which exhibit some degree of sex difference. The male rat hippocampus is consistently reported to be slightly but significantly larger than the female. The majority of studies on the development of volumetric sex differences have focused on the effects of estradiol (E 2 ), with relatively few focusing on androgens. We examined the impact of both E 2 and androgens on newly born cells in the developing rat hippocampus, and report that neonatal males have significantly more 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdU)+ cells than females. Both testosterone (T) and dihydrotestosterone treatment of females significantly increased the number of BrdU+ cells, an effect blocked by the androgen receptor antagonist, flutamide. However, only T significantly increased the number of neuronal nuclear antigen+ neurons in the female rat hippocampus. Interestingly, E 2 treatment also increased BrdU+ cells in females, but had no effect on neuron number. Instead, E 2 and T significantly increased the number of newly born glial fibrillary acidic protein or glutamine synthetase+ glial cells in females, indicating that androgens and E 2 may act independently to achieve distinct endpoints. Quantification of pyknotic cells at two different developmental time points indicates no sex difference in the number of cells dying, suggesting, but not proving, that gonadal steroids are promoting cell genesis.

Ovarian hormones can protect against brain injury, neurodegeneration, and cognitive decline. Most attention has focused on estrogens and accumulating data demonstrate that estrogen seems to specifically protect cortical and hippocampal neurons from ischemic injury and from damage due to severe seizures. Although multiple studies demonstrate protection by estrogen, in only a few instances is the issue of how the steroid confers protection known. Here, we first review data evaluating the neuroprotective effects of estrogens, a selective estrogen receptor modulator (SERM), and estrogen receptor alpha- and beta-selective ligands in animal models of focal and global ischemia. Using focal ischemia in ovariectomized ERalphaKO, ERbetaKO, and wild-type mice, we clearly established that the ERalpha subtype is the critical ER mediating neuroprotection in mouse focal ischemia. In rats and mice, the middle cerebral artery occlusion (MCAO) model was used to represent cerebrovascular stroke, while in gerbils the two-vessel occlusion model, representing global ischemia, was used. The gerbil global ischemia model was used to evaluate the neuroprotective effects of estrogen, SERMs, and ERalpha- and ERbeta-selective compounds in the hippocampus. Analysis of neurogranin mRNA, a marker of viability of hippocampal neurons, with in situ hybridization, revealed that estrogen treatment protected the dorsal CA1 regions not only when administered before, but also when given 1 h after occlusion. Estrogen rarely is secreted alone and studies of neuroprotection have been less extensive for a second key ovarian hormone progesterone. In the second half of this review, we present data on neuroprotection by estrogen and progesterone in animal model of epilepsy followed by exploration into ovarian steroid effects on neuronal damage in models of multiple sclerosis and traumatic brain injury.

SummaryMethamphetamine (MA) abuse has reached epidemic proportions in the United States. Users of MA report dramatic increases in sexual drive that have been associated with increased engagement in risky sexual behavior leading to higher rates of sexually transmitted diseases and unplanned pregnancies. The ability of MA to enhance sexual drive in females is enigmatic since related psychostimulants like amphetamine and cocaine appear not to affect sexual drive in women, and in rodents models, amphetamine has been reported to be inhibitory to female sexual behavior. Examination of MA's effects on female sexual behavior in an animal model is lacking. Here, using a rodent model, we have demonstrated that MA enhanced female sexual behavior. MA (5mg/kg) or saline vehicle was administered once daily for three days to adult ovariectomized rats primed with ovarian steroids. MA treatment significantly increased the number of proceptive events and the lordosis response compared to hormonally-primed, saline controls. The effect of MA on the neural circuitry underlying the motivation for sexual behavior was examined using Fos immunoreactivity. In the medial amygdala and the ventromedial nucleus of the hypothalamus, nuclei implicated in Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflicts of InterestThe authors declare that, except for income received from the primary employer, no financial support or compensation has been received from any individual or corporate entity over the past three years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest. NIH Public Access Author ManuscriptPsychoneuroendocrinology. Author manuscript; available in PMC 2011 February 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript motivated behaviors, ovarian hormones and MA independently enhance the neuronal activation, but more striking was the significantly greater activation induced by their combined administration. Increases in dopamine neurotransmission may underlie the MA/hormone mediated increase in neuronal activation. In support of this possibility, ovarian hormones significantly increased tyrosine hyroxylase (the rate limiting enzyme in dopamine synthesis) immunoreactivity in the medial amygdala. Thus our present data suggest that the interactions of MA and ovarian hormones leads to changes in the neural substrate of key nuclei involved in mediating female sexual behaviors, and these changes may underlie MA's ability to enhance these behaviors.

Certain anesthetics exhibit neurotoxicity in the brains of immature but not mature animals. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, is excitatory on immature neurons via its action at the GABAA receptor, due to a reversed transmembrane chloride gradient. GABAA receptor activation in immature neurons is sufficient to open L-type voltage gated calcium channels. As propofol is a GABAA agonist, we hypothesized that it and more specific GABAA modulators would increase intracellular free calcium ([Ca2+]i), resulting in the death of neonatal rat hippocampal neurons. Neuronal [Ca2+]i was monitored using Fura2-AM fluorescence imaging. Cell death was assessed by double-staining with propidium iodide and Hoechst 33258 at 1 h (acute) and 48 h (delayed) after 5 h exposure of neurons to propofol or the GABAA receptor agonist, muscimol, in the presence and absence of the GABA receptor antagonist, bicuculline, or the L-type Ca2+ channel blocker, nifedipine. Fluorescent measurements of caspase-3,-7 activities were performed at 1 h after exposure. Both muscimol and propofol induced a rapid increase in [Ca2+]i in day in vitro (DIV) 4, but not in DIV 8 neurons, that was inhibited by nifedipine and bicuculline. Caspase-3,-7 activities and cell death increased significantly in DIV 4 but not DIV 8 hippocampal neuronal cultures 1 h after a 5 h exposure to propofol, but not muscimol, and were inhibited by the presence of bicuculline or nifedipine. We conclude that an increase in [Ca2+]i, due to activation of GABAA receptors and opening of L-type calcium channels, is necessary for propofol-induced death of immature rat hippocampal neurons but that additional mechanisms not elicited by GABAA activation alone also contribute to cell death.

Several sex differences in the nervous system depend on differential cell death during development in males and females. The anti-apoptotic protein, Bcl-2, promotes the survival of many types of neurons during development and in response to injury. To determine whether Bcl-2 might similarly control cell death in sexually dimorphic regions, we compared neuron number in wild-type mice and transgenic mice overexpressing Bcl-2 under the control of a neuron-specific promoter. Three neural areas were examined: the spinal nucleus of the bulbocavernosus (SNB), in which neuron number is greater in males; the retrodorsolateral nucleus (RDLN) of the spinal cord, which exhibits no sex difference in neuron number; and the anteroventral periventricular nucleus (AVPV) of the hypothalamus, in which both overall cell density and the number of tyrosine hydroxylase immunoreactive (TH-ir) neurons are greater in females. Bcl-2 overexpression significantly increased SNB cell number in females, overall cell density of AVPV in males, and RDLN cell number in both sexes. Bcl-2 overexpression did not alter the number of TH-ir neurons in AVPV of males or females. These findings indicate that Bcl-2 can regulate sexually dimorphic cell number in the brain and spinal cord and suggest that Bcl-2 may mediate effects of testosterone on cell survival during neural development. In contrast to the regulation of overall cell density in AVPV, the sex difference in TH cell number apparently is not caused by a Bcl-2-dependent mechanism.

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Steroid-mediated sexual differentiation of the brain is a developmental process that permanently organizes the brain into a male or female phenotype. Previous studies in the rodent have examined the steroid-mediated mechanisms of male brain development. In an effort to identify molecules involved in female brain development, a high-throughput proteomics approach called PowerBlot was used to identify signaling proteins differentially regulated in the neonatal male and female rat hypothalamus during the critical period for brain sexual differentiation. Focal adhesion kinase (FAK) and paxillin, both members of the focal adhesion complex family of proteins, were significantly elevated in the newborn female compared with the male hypothalamus. Sex differences in these proteins were not detected in brain regions that are not subject to substantial organizational effects of steroids. Estrogens, the aromatized products of testosterone in the male, can both masculinize and defeminize the male brain. Daily estradiol administration to neonatal females significantly reduced FAK and paxillin in the hypothalamus, and aromatase inhibition increased paxillin in males to levels comparable with females. Androgens also appear to modulate paxillin levels in combination with estrogen action. Across development, hypothalamic levels of FAK were significantly elevated in females compared with males on postnatal d 6. Synaptic circuits in the hypothalamus develop sex differences perinatally. Estradiol treatment of cultured hypothalamic neurons significantly enhanced axon branching (P<0.01), consistent with the phenotype of FAK-deficient neurons. Together, these data implicate FAK and paxillin as regulators of sex differences in neuronal morphology.

Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) and their target muscles, bulbocavernosus and levator ani (BC/LA), constitute an androgen-sensitive neuromuscular system. Testosterone regulates SNB soma size, SNB dendritic length, and BC/LA muscle mass in adult male rats. Recent evidence indicates that the cell death-regulatory protein, Bcl-2, may also play a role in adult neural plasticity. The present study examined whether gonadal hormones and/or the Bcl-2 protein influence the morphology of the SNB neuromuscular system in adult B6D2F1 mice. In Experiment 1, adult wild-type and Bcl-2 overexpressing males were castrated and implanted with silastic capsules containing testosterone or left blank. Six weeks after castration, cholera toxin-horseradish peroxidase was injected into the BC muscle to label SNB dendrites. Animals were killed 48 h later, and BC/LA muscle mass, SNB soma size, and SNB dendritic arbors were examined. In Experiment 2, wild-type and Bcl-2 overexpressing males were castrated or sham castrated, implanted with testosterone-filled or blank capsules, and examined 12 weeks later. In both experiments, BC/LA muscle mass and SNB soma size were significantly reduced in castrates receiving blank capsules. Surprisingly, however, there was no effect of hormone manipulation on any of several measures of dendritic length. Thus, the dendritic morphology of SNB motoneurons appears to be relatively insensitive to circulating androgen levels in B6D2F1 mice. Bcl-2 overexpression did not influence BC/LA muscle mass, SNB soma size, or SNB dendritic length, indicating that the morphology of this neuromuscular system and the response to castration are not altered by forced expression of the Bcl-2 protein.