A role for hypocretin (orexin) in male sexual behavior.

The role of hypocretin (orexin; hcrt/orx) neurons in regulation of arousal is well established. Recently, hcrt/orx has been implicated in food reward and drug-seeking behavior. We report here that in male rats, Fos immunoreactivity (ir) in hcrt/orx neurons increases markedly during copulation, whereas castration produces decreases in hcrt/orx neuron cell counts and protein levels in a time course consistent with postcastration impairments in copulatory behavior. This effect was reversed by estradiol replacement. Immunolabeling for androgen (AR) and estrogen (ER alpha) receptors revealed no colocalization of hcrt/orx with AR and few hcrt/orx neurons expressing ER alpha, suggesting that hormonal regulation of hcrt/orx expression is via afferents from neurons containing those receptors. We also demonstrate that systemic administration of the orexin-1 receptor antagonist SB 334867 [N-(2-methyl-6-benzoxazolyl)-N''-1,5-naphthyridin-4-yl urea] impairs copulatory behavior. One locus for the prosexual effects of hcrt/orx may be the ventral tegmental area (VTA). We show here that hcrt-1/orx-A produces dose-dependent increases in firing rate and population activity of VTA dopamine (DA) neurons in vivo. Activation of hcrt/orx during copulation, and in turn, excitation of VTA DA neurons by hcrt/orx, may contribute to the robust increases in nucleus accumbens DA previously observed during male sexual behavior. Subsequent triple immunolabeling in anterior VTA showed that Fos-ir in tyrosine hydroxylase-positive neurons apposed to hcrt/orx fibers increases during copulation. Together, these data support the view that hcrt/orx peptides may act in a steroid-sensitive manner to facilitate the energized pursuit of natural rewards like sex via activation of the mesolimbic DA system.

Testosterone and nucleus accumbens dopamine in the male Syrian hamster.

Most drugs of abuse increase dopamine (DA) in nucleus accumbens (Acb). However, the effects of anabolic androgenic steroids (AAS) on Acb DA have not been examined. We determined the effects of subcutaneous (sc) testosterone (T) on Acb DA in male hamsters. The effects of sc amphetamine were also examined for comparison. In addition, Acb DA was evaluated during intracerebroventricular (ICV) T infusion, designed to mimic T intake during ICV T self-administration in drug-naïve and drug-preexposed animals. Acb DA was measured using in vivo microdialysis and HPLC-EC. T (7.5 or 37.5 mg/kg), amphetamine (1 or 5 mg/kg), or vehicle was injected sc and Acb DA monitored for 4h. In the ICV experiment, T (1 or 2 microg/infusion) or vehicle was infused ICV every 6 min for 4h and Acb DA monitored. ICV T preexposure was accomplished by repeating the same ICV T infusion (1 microg/infusion) daily for 14 days, and T infusion was accompanied by microdialysis on 15th day. Neither sc nor ICV T administration increased Acb DA. At high dose (2 microg/infusion), ICV T decreased Acb DA. Likewise, daily ICV infusion of T for 15 days did not alter Acb DA. In contrast, sc amphetamine significantly increased Acb DA at both doses. Therefore, unlike many drugs of abuse, AAS does not increase Acb DA levels. The reduction in DA at high T doses is likely due to autonomic depressant effects of AAS. We suggest that AAS act via mechanism distinct from those of stimulants, but may share neural substrates with other drugs of abuse.

Acute inhibition of neurosteroid estrogen synthesis suppresses status epilepticus in an animal model.

Status epilepticus (SE) is a common neurological emergency for which new treatments are needed. In vitro studies suggest a novel approach to controlling seizures in SE: acute inhibition of estrogen synthesis in the brain. Here, we show in rats that systemic administration of an aromatase (estrogen synthase) inhibitor after seizure onset strongly suppresses both electrographic and behavioral seizures induced by kainic acid (KA). We found that KA-induced SE stimulates synthesis of estradiol (E2) in the hippocampus, a brain region commonly involved in seizures and where E2 is known to acutely promote neural activity. Hippocampal E2 levels were higher in rats experiencing more severe seizures. Consistent with a seizure-promoting effect of hippocampal estrogen synthesis, intra-hippocampal aromatase inhibition also suppressed seizures. These results reveal neurosteroid estrogen synthesis as a previously unknown factor in the escalation of seizures and suggest that acute administration of aromatase inhibitors may be an effective treatment for SE.

Quantitative analysis of long-form aromatase mRNA in the male and female rat brain.

In vitro studies show that estrogens acutely modulate synaptic function in both sexes. These acute effects may be mediated in vivo by estrogens synthesized within the brain, which could fluctuate more rapidly than circulating estrogens. For this to be the case, brain regions that respond acutely to estrogens should be capable of synthesizing them. To investigate this question, we used quantitative real-time PCR to measure expression of mRNA for the estrogen-synthesizing enzyme, aromatase, in different brain regions of male and female rats. Importantly, because brain aromatase exists in two forms, a long form with aromatase activity and a short form with unknown function, we targeted a sequence found exclusively in long-form aromatase. With this approach, we found highest expression of aromatase mRNA in the amygdala followed closely by the bed nucleus of the stria terminalis (BNST) and preoptic area (POA); we found moderate levels of aromatase mRNA in the dorsal hippocampus and cingulate cortex; and aromatase mRNA was detectable in brainstem and cerebellum, but levels were very low. In the amygdala, gonadal/hormonal status regulated aromatase expression in both sexes; in the BNST and POA, castration of males down-regulated aromatase, whereas there was no effect of estradiol in ovariectomized females. In the dorsal hippocampus and cingulate cortex, there were no differences in aromatase levels between males and females or effects of gonadal/hormonal status. These findings demonstrate that long-form aromatase is expressed in brain regions that respond acutely to estrogens, such as the dorsal hippocampus, and that gonadal/hormonal regulation of aromatase differs among different brain regions.

Quantitative mapping of cocaine-induced ΔFosB expression in the striatum of male and female rats.

ΔFosB plays a critical role in drug-induced long-term changes in the brain. In the current study, we evaluated locomotor activity in male and female rats treated with saline or cocaine for 2 weeks and quantitatively mapped ΔFosB expression in the dorsal striatum and nucleus accumbens of each animal by using an anti-FosB antibody that recognizes ΔFosB isoforms preferentially. Behavioral analysis showed that while there was little difference between males and females that sensitized to cocaine, nonsensitizing rats showed a large sex difference. Nonsensitizing males showed low behavioral activation in response to cocaine on the first day of treatment, and their activity remained low. In contrast, nonsensitizing females showed high activation on the first day of treatment and their activity remained high. Western blot and immunohistochemical analyses indicated that basal levels of ΔFosB were higher in the nucleus accumbens than the dorsal striatum, but that the effect of cocaine on ΔFosB was greater in the dorsal striatum. Immunostaining showed that the effect of cocaine in both the dorsal striatum and nucleus accumbens was primarily to increase the intensity of ΔFosB immunoreactivity in individual neurons, rather than to increase the number of cells that express ΔFosB. Detailed mapping of ΔFosB-labeled nuclei showed that basal ΔFosB levels were highest in the medial portion of the dorsal striatum and dorsomedial accumbens, particularly adjacent to the lateral ventricle, whereas the cocaine-induced increase in ΔFosB was most pronounced in the lateral dorsal striatum, where basal ΔFosB expression was lowest. Sex differences in ΔFosB expression were small and independent of cocaine treatment. We discuss implications of the sex difference in locomotor activation and regionally-specific ΔFosB induction by cocaine.

Membrane androgen receptors may mediate androgen reinforcement.

Anabolic-androgenic steroid (AAS) abuse is widespread. Moreover, AAS are reinforcing, as shown by self-administration in rodents. However, the receptors that transduce the reinforcing effects of AAS are unclear. AAS may bind to classical nuclear androgen receptors (ARs) or membrane receptors. We used two approaches to examine the role of nuclear ARs in AAS self-administration. First, we tested androgen self-administration in rats with the testicular feminization mutation (Tfm), which interferes with androgen binding. If nuclear ARs are essential for AAS self-administration, Tfm males should not self-administer androgens. Tfm males and wild-type (WT) littermates self-administered the non-aromatizable androgen dihydrotestosterone (DHT) or vehicle intracerebroventricularly (ICV) at fixed-ratio (FR) schedules up to FR5. Both Tfm and WT rats acquired a preference for the active nose-poke during DHT self-administration (66.4+/-9.6 responses/4 h for Tfm and 79.2+/-11.5 for WT responses/4 h), and nose-pokes increased as the FR requirement increased. Preference scores were significantly lower in rats self-administering vehicle (42.3+/-5.3 responses/4 h for Tfm and 19.1+/-4.0 responses/4 h for WT). We also tested self-administration of DHT conjugated to bovine serum albumin (BSA) at C3 and C17, which is limited to actions at the cell surface. Hamsters were allowed to self-administer DHT, BSA and DHT-BSA conjugates for 15 days at FR1. The hamsters showed a significant preference for DHT (18.0+/-4.1 responses/4 h) or DHT-BSA conjugates (10.0+/-3.7 responses/4 h and 21.0+/-7.2 responses/4 h), but not for BSA (2.5+/-2.4 responses/4 h). Taken together, these data demonstrate that nuclear ARs are not required for androgen self-administration. Furthermore, androgen self-administration may be mediated by plasma membrane receptors.

Adolescents and androgens, receptors and rewards.

Adolescence is associated with increases in pleasure-seeking behaviors, which, in turn, are shaped by the pubertal activation of the hypothalamo-pituitary-gonadal axis. In animal models of naturally rewarding behaviors, such as sex, testicular androgens contribute to the development and expression of the behavior in males. To effect behavioral maturation, the brain undergoes significant remodeling during adolescence, and many of the changes are likewise sensitive to androgens, presumably acting through androgen receptors (AR). Given the delicate interaction of gonadal hormones and brain development, it is no surprise that disruption of hormone levels during this sensitive period significantly alters adolescent and adult behaviors. In male hamsters, exposure to testosterone during adolescence is required for normal expression of adult sexual behavior. Males deprived of androgens during puberty display sustained deficits in mating. Conversely, androgens alone are not sufficient to induce mating in prepubertal males, even though brain AR are present before puberty. In this context, wide-spread use of anabolic-androgenic steroids (AAS) during adolescence is a significant concern. AAS abuse has the potential to alter both the timing and the levels of androgens in adolescent males. In hamsters, adolescent AAS exposure increases aggression, and causes lasting changes in neurotransmitter systems. In addition, AAS are themselves reinforcing, as demonstrated by self-administration of testosterone and other AAS. However, recent evidence suggests that the reinforcing effects of androgens may not require classical AR. Therefore, further examination of interactions between androgens and rewarding behaviors in the adolescent brain is required for a better understanding of AAS abuse.

The effects of nitric oxide-cGMP pathway stimulation on dopamine in the medial preoptic area and copulation in DHT-treated castrated male rats.

Dopamine (DA) in the medial preoptic area (MPOA) provides important facilitative influence on male rat copulation. We have shown that the nitric oxide-cGMP (NO-cGMP) pathway modulates MPOA DA levels and copulation. We have also shown that systemic estradiol (E(2)) maintains neuronal NO synthase (nNOS) immunoreactivity in the MPOA of castrates, as well as relatively normal DA levels. This effect of E(2) on nNOS probably accounts for at least some of the previously demonstrated behavioral facilitation by intra-MPOA E(2) administration in castrates. Therefore, we hypothesized that stimulation of the MPOA NO-cGMP pathway in dihydrotestosterone (DHT)-treated castrates should restore DA levels and copulatory behaviors. Reverse-dialysis of a NO donor, sodium nitroprusside (SNP), increased extracellular DA in the MPOA of DHT-treated castrates and restored the ability to copulate to ejaculation in half of the animals. A cGMP analog, 8-Br-cGMP, also increased extracellular DA, though not as robustly, but did not restore copulatory ability. The effectiveness of the NO donor in restoring copulation and MPOA DA levels is consistent with our hypothesis. However, the lack of behavioral effects of 8-Br-cGMP, despite its increase in MPOA DA, suggests that NO may have additional mediators in the MPOA in the regulation of copulation. Furthermore, the suboptimal copulation seen in the NO donor-treated animals suggests the importance of extra-MPOA systems in the regulation of copulation.