Androgens increase survival of adult-born neurons in the dentate gyrus by an androgen receptor-dependent mechanism in male rats.

Gonadal steroids are potent regulators of adult neurogenesis. We previously reported that androgens, such as testosterone (T) and dihydrotestosterone (DHT), but not estradiol, increased the survival of new neurons in the dentate gyrus of the male rat. These results suggest androgens regulate hippocampal neurogenesis via the androgen receptor (AR). To test this supposition, we examined the role of ARs in hippocampal neurogenesis using 2 different approaches. In experiment 1, we examined neurogenesis in male rats insensitive to androgens due to a naturally occurring mutation in the gene encoding the AR (termed testicular feminization mutation) compared with wild-type males. In experiment 2, we injected the AR antagonist, flutamide, into castrated male rats and compared neurogenesis levels in the dentate gyrus of DHT and oil-treated controls. In experiment 1, chronic T increased hippocampal neurogenesis in wild-type males but not in androgen-insensitive testicular feminization mutation males. In experiment 2, DHT increased hippocampal neurogenesis via cell survival, an effect that was blocked by concurrent treatment with flutamide. DHT, however, did not affect cell proliferation. Interestingly, cells expressing doublecortin, a marker of immature neurons, did not colabel with ARs in the dentate gyrus, but ARs were robustly expressed in other regions of the hippocampus. Together these studies provide complementary evidence that androgens regulate adult neurogenesis in the hippocampus via the AR but at a site other than the dentate gyrus. Understanding where in the brain androgens act to increase the survival of new neurons in the adult brain may have implications for neurodegenerative disorders.

Distribution of androgen receptor immunoreactivity in the brainstem of male rats.

Gonadal steroids such as testosterone and estrogen are necessary for the normal activation of male rat sexual behavior. The medial preoptic area (MPOA), an important neural substrate regulating mating, accumulates steroids and also expresses functional androgen receptors (AR). The MPOA is intimately connected with other regions implicated in copulation, such as the bed nucleus of the stria terminalis and medial amygdala. Inputs to the MPOA arise from several areas within the brainstem, synapsing preferentially onto steroid sensitive MPOA cells which are activated during sexual activity. Given that little is known about the distribution of AR protein in the brainstem of male rats, we mapped the distribution of AR expressing cells in the pons and medulla using immunocytochemistry. In agreement with previous reports, AR immunoreactivity (AR-ir) was detected in ventral spinal motoneurons and interneurons. In addition, AR-ir was detected in areas corresponding to the solitary tract, lateral paragigantocellular and alpha and ventral divisions of the gigantocellular reticular nuclei, area postrema, raphe pallidus, ambiguus nucleus, and intermediate reticular nucleus. Several regions within the pons contained AR-ir, such as the tegmental and central gray, parabrachial nucleus, locus coeruleus, Barrington's nucleus, periaqueductal gray, and dorsal raphe. In contrast with in situ hybridization studies, auditory and somatosensory areas were AR-ir negative, and, except for very light staining in the prepositus nucleus, areas carrying vestibular information did not display AR-ir. Additionally, cranial nerve motoneurons of the hypoglossal, facial, dorsal vagus, and spinal trigeminal did not display AR-ir in contrast to previous reports. The data presented here indicate that androgens may influence numerous cell groups within the brainstem. Some of these probably constitute a steroid sensitive circuit linking the MPOA to motoneurons in the spinal cord via androgen responsive cells in the caudal ventral medulla.

Estrogen-inducible progesterone receptors in the rat lumbar spinal cord: regulation by ovarian steroids and fluctuation across the estrous cycle.

Ovarian hormones influence the physiology of the spinal cord through incompletely understood cellular mechanisms. To date, there has been little compelling evidence for progesterone receptors in spinal cord neurons. Using two antibodies specific for progesterone receptors in an immunohistochemical investigation, we now report the presence of estrogen-inducible progesterone receptors in the spinal cord. Estrogen-inducible progesterone receptors were observed in the neurons of lamina X and the interomedialateral cell column, which are also known to express estrogen receptors. Estrogen-inducible progesterone receptors similar to those observed in females were also apparent in lamina X and interomediolateral cell column neurons in the spinal cords of males treated with estradiol. Furthermore, the density of progesterone receptors in lamina X was observed to fluctuate across the estrous cycle in female rats, with the highest progesterone receptor expression levels occurring late in proestrus, following the estradiol surge and coincident with high circulating progesterone levels. The lowest progesterone receptor expression levels were observed late in estrus following the progesterone surge. Together, these results demonstrate that estrogen-sensitive progestin targets exist in the spinal cord, and their possible role in the nervous control of reproduction and ovarian steroid modulation of nociception is discussed.

N-cadherin is regulated by gonadal steroids in adult sexually dimorphic spinal motoneurons.

Gonadal steroids influence the morphology and function of neurons in the adult spinal cord through cellular and molecular mechanisms that are largely unknown. The cadherins are cell adhesion molecules that participate in the formation and organization of the CNS during embryonic development, and recent evidence suggests that the cadherins continue to regulate neural structure and function in adulthood. Using degenerate oligonucleotides coding conserved regions of the catenin-binding domain of classical cadherins in a RT-PCR cloning strategy, we identified several cadherin subtypes, the most frequently cloned being N-, E-, and R-cadherin, suggesting that these are the major classical cadherin subtypes present in the adult male rat lumbosacral spinal cord. We then examined cadherin expression levels of these cadherin subtypes under steroid conditions known to induce plastic changes in spinal motoneurons. Semiquantitative PCR revealed that mRNA levels of N-cadherin, but not E-cadherin or R-cadherin, are elevated in castrated rats treated with testosterone, 17 beta-estradiol, or dihydrotestosterone relative to castrate rats not treated with steroids. Immunolocalization of N-cadherin revealed that steroid treatment increased N-cadherin expression levels in functionally related neural populations whose morphology and function are regulated by steroids. These results suggest a role for N-cadherin in steroid-induced neuroplastic change in the adult lumbar spinal cord.

N-cadherin expression in motoneurons is directly regulated by androgens: a genetic mosaic analysis in rats.

We have recently reported that systemic androgens regulate adult N-cadherin (N-cad) expression in spinal motoneurons. However, the mechanism through which androgen mediates this effect remains undetermined. Androgen may act directly on motoneurons to regulate N-cad expression, or indirectly, via effects on androgen-sensitive afferent or efferent structures. Here, we describe a genetic mosaic investigation of this site-of-action indeterminacy. Following developmental random X chromosome inactivation, androgenized female rats heterozygous for the tfm androgen receptor mutation (X(WT)X(tfm)) are phenotypic mosaics of androgen-sensitive wild-type (WT) and androgen-insensitive (tfm) motoneurons. We compared steroid effects on WT and tfm cells in two sexually-dimorphic motoneuron pools, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN), as well as a less steroid responsive motoneuron pool, the sexually monomorphic retrodorsolateral nucleus (RDLN). Independent of steroid treatment, a greater proportion of wild-type cells were N-cad immunoreactive (IR) in the DLN and RDLN. Following testosterone treatment, increased N-cad expression was observed in both cell types in the DLN, but in the SNB only the androgen-competent WT cells increased N-cad expression. Testosterone treatment did not significantly alter N-cad expression in the mosaic RDLN. The results indicate both cell autonomous and cell non-autonomous androgenic regulation of N-cad expression in spinal motoneurons.

Sex differences in throwing: monkeys having a fling.

Fast and accurate throwing was undoubtedly important to ancestral hominids, and was subject to sexual-selection pressures that generated a male advantage in throwing accuracy that persists in modern humans. The balance of evidence, including that from a recent comparative study of throwing in humans and capuchin monkeys, suggests that high-performance throwing involves unique adaptations in the domains of spatial targeting, precision timing, and multi-joint motor control.

Neuronal size in the spinal nucleus of the bulbocavernosus: direct modulation by androgen in rats with mosaic androgen insensitivity.

The motoneurons of the spinal nucleus of the bulbocavernosus (SNB) and its target muscles, the bulbocavernosus and levator ani, form a sexually dimorphic circuit that is developmentally dependent on androgen exposure and exhibits numerous structural and functional changes in response to androgen exposure in adulthood. Castration of male adult rats causes shrinkage of SNB somata, and testosterone replacement reverses this effect, but the site at which androgen is acting to cause this change is undetermined. We exploited the X-chromosome residency of the androgen receptor (AR) gene to generate androgenized female rats that were heterozygous for the testicular feminization mutant (tfm) AR mutation and that, as a consequence of ontogenetic random X-inactivation, expressed a blend of androgen-sensitive wild-type cells and tfm-affected androgen-insensitive cells in the SNB. Chronic testosterone treatment of adult mosaics increased soma sizes only in androgen-competent wild-type SNB cells. The size of tfm-affected SNB somata in the same animals did not differ from the size of either the wild-type or tfm-affected SNB neurons in control mosaics that did not receive androgen treatment in adulthood. Because the muscle targets of the SNB are known to be uniformly androgen-sensitive in tfm mosaics, this mosaic analysis provides unambiguous evidence that androgenic effects on motoneuron soma size are mediated locally in the SNB. It is possible that the neuronal AR plays a permissive role in coordinating the actions of androgen.

N-cadherin is regulated by gonadal steroids in the adult hippocampus.

In the adult hippocampus, gonadal steroids induce neural remodeling through cellular and molecular mechanisms that are largely unknown. The calcium-dependent cell adhesion molecule N-cadherin, which participates in the developmental organization of the nervous system, has recently been localized to hippocampal synapses and is suspected to participate in adult synaptic physiology. Little is currently known about the regulation of cadherins in the adult central nervous system, although posttranslational modifications are thought to account for variability in N-cadherin expression levels. To evaluate the possibility that gonadal steroids regulate N-cadherin in the adult hippocampus, we examined hippocampal N-cadherin mRNA levels and protein expression in castrated adult male rats treated with testosterone, or its metabolites 17beta-estradiol or dihydrotestosterone. Northern blot analysis indicated increased hippocampal N-cadherin mRNA levels in the adult rat hippocampus after treatment with 17beta-estradiol but not testosterone or dihydrotestosterone. Increased N-cadherin immunoreactivity was observed in CA1 and CA3 pyramidal cells after 17beta-estradiol treatment. Additionally, both 17beta-estradiol and testosterone treatment increased N-cadherin immunoreactivity in the neuropil of the stratum lacunosum-moleculare, which includes apical dendrites from pyramidal cells. In contrast, dihydrotestosterone treatment had no effect on levels of N-cadherin protein expression in CA1 or CA3 pyramidal cells or in the stratum lacunosum-moleculare. These results demonstrate that, in the hippocampus, expression levels of N-cadherin are dynamic in adulthood. To our knowledge, there have been no other demonstrations of steroid regulation of cadherin expression in neural populations. These results suggest a possible adhesive mechanism for steroid-induced plasticity of the adult nervous system.

Direct androgenic regulation of calcitonin gene-related peptide expression in motoneurons of rats with mosaic androgen insensitivity.

The spinal nucleus of the bulbocavernosus (SNB) and its target muscles, bulbocavernosus and levator ani (BC/LA), form a sexually dimorphic neuromuscular circuit whose development and maintenance are androgen-dependent. The mechanisms whereby androgen regulates gene expression in the SNB of adult rats are largely unknown, although a retrograde influence from the BC/LA muscles has been suggested to underlie the suppression of calcitonin gene-related peptide (CGRP) expression observed in SNB motoneurons after systemic androgen treatment. A mosaic paradigm was used to determine the site of action of androgen in the regulation of CGRP expression in SNB motoneurons. As a consequence of random X chromosome inactivation, androgenized female rats heterozygous for the tfm androgen receptor (AR) mutation (XwtXtfm-mosaics) express a mosaic of androgen-sensitive and androgen-insensitive motoneurons in the SNB, whereas the BC/LA target musculature appears to be uniformly sensitive to androgens. In adult mosaics, testosterone administration resulted in a reduction in the proportion of androgen-sensitive cells expressing CGRP, whereas no such reduction was observed in the androgen-insensitive population, indicating that neuronal AR plays an essential role in the neuromuscular regulation of CGRP expression in these motoneurons. This provides the first in vivo demonstration of AR regulation of gene expression unambiguously localized to a neuronal population.

Both neuropeptide Y and serotonin are necessary for entrainment of circadian rhythms in mice by daily treadmill running schedules.

This study investigated the role of the suprachiasmatic nucleus (SCN) circadian pacemaker and its neuropeptide Y (NPY) and serotonin (5-HT) afferents in entrainment (synchronization) of mouse circadian rhythms by treadmill running. Blind C57BL/6j mice were run in treadmills for 3 hr/d for 3-10 weeks after receiving radio-frequency lesions of the SCN or the intergeniculate leaflet (IGL, the source of SCN NPY) or infusions of the 5-HT neurotoxin 5,7-DHT into the SCN area. Of 25 intact mice, 22 entrained and three showed period (tau, the mean duration of the circadian cycle) modulations to scheduled running. Arrhythmic SCN-ablated mice did not synchronize to scheduled running in a way suggestive of circadian pacemaker mediation. Of 15 mice with IGL lesions, only two with partial lesions entrained. Mice with complete IGL lesions (five), confirmed by immunocytochemistry, showed no entrainment or tau changes. Of 19 mice with 5-HT lesions, only two with partial lesions entrained. All but two mice with complete (10) or nearly complete (4) 5-HT denervation, confirmed by immunocytochemistry, showed tau modulations during the treadmill schedule. Failure to entrain was not explained by group differences in tau before the treadmill schedules. The results indicate that the SCN and both NPY and 5-HT are necessary for entrainment to 24 hr schedules of forced running but that complete loss of 5-HT does not prevent modulations of pacemaker motion by behavioral stimuli. Treadmill entrainment in mice may involve synergistic interactions between 5-HT and NPY afferents at some site within the circadian system.

Androgen spares androgen-insensitive motoneurons from apoptosis in the spinal nucleus of the bulbocavernosus in rats.

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic motor nucleus in the rat lumbar spinal cord. The sex difference arises through the androgenic sparing of the motoneurons and their target muscles from ontogenetic cell death. Indirect evidence suggests that androgen acts on the target muscles rather than directly on SNB motoneurons to spare them from death. The testicular feminization mutation (Tfm), a defect in the androgen receptor (AR), blocks androgenic sparing of SNB motoneurons and their targets. The pattern of AR immunocytochemistry was previously found to be different in adult Tfm and wild-type rats: immunostaining was nuclear in most SNB cells of wild-type rats, but very few SNB cells display nuclear AR immunostaining in affected Tfm rats. Because the Tfm mutation is carried on the X chromosome, random X inactivation during development makes female carriers of Tfm (+/Tfm) genetic mosaics for androgen sensitivity. Tfm carriers, their wild-type sisters, and affected Tfm males were treated with perinatal testosterone and immunocytochemistry was used to detect androgen receptor in the SNB when the rats reached adulthood. Mosaic females could be distinguished from their wild-type sisters by external morphology. In such perinatally androgenized mosaics, adult SNB cells were equally divided between wild-type and Tfm genotype, as indicated by AR immunocytochemistry. In contrast, the pattern of AR immunocytochemistry in target muscles of mosaics appeared similar to that of wild-type females. These results indicate that early androgen spared both androgen-sensitive and -insensitive motoneurons from cell death, confirming a site of androgen action other than the motoneurons themselves.

Effects of 5-HT3 agonists on reproductive behaviors in rats.

Activity at 5-HT1 and 5-HT2 receptor sites influences sexual behavior in male and female rats. 5-HT3 antagonists reportedly have no effect on copulatory activity in rats of either sex although they influence a variety of other behaviors. The effects of 5-HT3 agonists on sexual behavior are unknown. The following experiments were undertaken to assess the influence of the 5-HT3 agonists 1-phenylbiguanide (PBG) and 2-methyl-serotonin (2-Me-5-HT) on sexual behavior, when administered intracerebroventricularly. Consistent with earlier reports indicating that 5-HT1 and 5-HT2 receptor activity influences reproductive activity in a sex-dependent manner, PBG was found to facilitate male, but not female, rat sexual behavior. 2-Me-5-HT, however, failed to modify either female or male rat sexual activity. Evidence that PBG, but not 2-Me-5-HT, induces carrier-mediated dopamine release suggests that the effect of PBG in male rats is due to dopaminergic mediation. Overall, the present data indicate that 5-HT3 receptor activation has only slight effects on rat sexual behavior.

Space and cognition: the measurement of behavioral functions during a 6-day space mission.

We measured nonspecific (attention, mental flexibility, psychomotor speed) and visuospatial cognitive processing in a single case study during a 6-d visit on the Russian orbital complex MIR, using computer-based psychometric tasks. Reaction times and accuracy scores showed only minor, nonsignificant changes between preflight, flight, and postflight assessments. These results suggest that several behavioral functions, among them complex visuospatial processing skills, remain essentially intact on short space visits, provided that the performing subject experiences no symptoms of space motion sickness or other physical impairments. Computerized psychometric tasks are a sensitive and flexible tool to measure behavioral functions in space life sciences.

Lack of effects of 5-HT3 antagonists on normal and morphine-attenuated sexual behaviours in female and male rats.

Although 5-HT1 and 5-HT2 receptor activity is known to influence copulation, the effects of 5-HT3 receptor-selective drugs on sexual activity have yet to be systematically studied. The following experiments investigated the effects of the 5-HT3-selective antagonists MDL 72222, ondansetron and ICS 205-930 on female sexual behaviour; male rats were studied using ondansetron and granisetron. These compounds influenced neither male nor female copulatory behaviours, suggesting that 5-HT3 receptors contribute little to the modulation of sexual activity. 5-HT3 receptor antagonists block certain opioid-induced behaviours and opioids selectively inhibit sexual behaviours; therefore, the ability of ondansetron and ICS 205-930 to modify morphine-attenuated copulatory activity was also tested. While morphine inhibited copulation, 5-HT3 antagonists failed to reverse the effects.

Serotonin-dependent cerebral activation: effects of methiothepin and other serotonergic antagonists.

In scopolamine-treated (5.0 mg/kg, s.c.) rats hippocampal rhythmical slow activity (RSA) and neocortical low voltage fast activity (LVFA) occur only in close correlation with head movements, spontaneous changes in posture, or locomotion (Type I behavior). Previous work indicates that such scopolamine-resistant RSA and LVFA are dependent on ascending serotonergic projections. A test of 9 serotonergic antagonists (methiothepin; ritanserin; ketanserin; pizotifen; mianserin; pirenperone; ICS-205-930; metoclopramide; methysergide) showed that methiothepin produces a partial reduction in RSA and LVFA in scopolamine-treated rats, while the other antagonists are completely inactive over a wide range of doses. It may be that serotonergic cerebral activation depends on both 5-HT1 and 5-HT2 receptors.

Concurrent wet dog shaking and inhibition of male rat copulation after ventromedial brainstem injection of the 5-HT2 agonist DOI.

Systemic administration of drugs that augment 5-HT2 activity generally induces 'wet dog' shaking (WDS) in rats. This suggests that the naturally occurring form of WDS seen in untreated rats may also serve as a behavioral index of 5-HT2 receptor activation, during the performance of other behaviors. Indeed, spontaneously occurring WDS has previously been reported to be inversely related to male rat copulatory proficiency. In order to examine a potential central nervous system mechanism subsuming these behaviors, male rats were tested for WDS and sexual behavior after brainstem administration of the 5-HT2 agonist DOI. Male Long-Evans rats were implanted with cannulae terminating in the region of the nucleus raphe obscurus/inferior olive, through which they received injections of DOI (0.1-10 micrograms). DOI produced a dose-dependent decrease in sexual behavior and concurrent increase in WDS. Pretreatment with the 5-HT2 antagonist ritanserin effectively blocked the effects of DOI. The results suggest that WDS and copulatory behaviors are modulated by a shared brainstem substrate. It is possible that the results may be the behavioral concomitant of recently described brainstem cells, with bifurcating axons, that project to both the medial preoptic area and the cervical spinal cord.

Aggression by a female rat cohabitating with a sterile male: termination of pseudopregnancy does not abolish aggression.

At the end of that time, each female was assessed for aggressiveness toward an unfamiliar female intruder once each week for 3 weeks. Those females displaying a high level of aggression had their male cagemate changed. For half of the females, the new male cagemate was a castrated male with a testosterone implant. For the other half, the new cagemate was a castrated male without a testosterone implant. Replacement males had been subjected to surgery 9 weeks previously. There were no differences in the aggressiveness of females of the two groups on any of 3 subsequent weekly tests of aggression. In a 3-h evaluation of male sexual behavior, none of the 9 castrated males without testosterone replacement displayed sexual activity with an estrogen/progesterone primed ovariectomized female, but 6 of 9 males with testosterone replacement did. Reanalysis of the aggression data comparing the females whose males had no testosterone replacement and females housed with the 6 males that were sexually active also revealed no differences in aggression over the 21-day test period. Since pseudopregnancy is known to last 13 days, these results indicate that the continuous presence of pseudopregnancy is not required for maintenance of aggression by a female cohabiting with a sterile male.

DOI-induced inhibition of copulatory behavior in male rats: reversal by 5-HT2 antagonists.

Relatively little is known regarding the role of 5-HT2 receptor activity in male rat sexual behavior. Previous work has yielded equivocal results, and both facilitation and inhibition of copulation have been reported to follow administration of selective 5-HT2 antagonists. In the present series of experiments, the ability of a variety of 5-HT2 antagonists to block inhibition induced by the 5-HT2/5-HT1C agonist DOI was examined. Systemic ritanserin, pirenperone and ketanserin all potently blocked DOI-induced (1.0 mg/kg SC) inhibition of mounts, intromissions and ejaculations. None of these drugs influenced the sexual behavior of the male rats when given alone in doses that effectively blocked DOI-induced inhibition. In addition, unlike ritanserin and ketanserin, pirenperone produced a biphasic effect on DOI-induced inhibition, exhibiting a diminished blockade at higher doses. This may be due to activity at receptors other than 5-HT2. Overall, the present data suggest that activity at 5-HT2 receptors mediates an inhibition of male rat sexual behavior.

Relation of spontaneous wet dog shakes and copulatory behavior in male rats.

Pharmacological manipulation of 5-HT2 activity has yielded equivocal effects on male rat sexual behavior. Both facilitation and inhibition of copulation have been reported following treatment with 5-HT2 antagonists. "Wet dog shake" (WDS), a component of the Serotonin Behavioral Syndrome, is largely mediated by 5-HT2 receptors. The present series of experiments were aimed at determining whether WDS might yield a spontaneous behavioral measure of 5-HT2 activity that converges on the pharmacological data. In Experiment 1, spontaneous WDS was recorded in 58 male rats paired with receptive females. Normal copulators (Studs) exhibited significantly less WDS than did noncopulators (Duds). In Experiment 2, the males were additionally paired with males and nonreceptive females. In these situations, WDS did not discriminate Duds from Studs, but Studs scores differed from each other across the three different partner conditions. Lastly, in Experiment 3, treatment with the selective 5-HT2 agonist DOI potently inhibited copulatory responses and increased WDS. Overall, the data from the three experiments suggest that 5-HT2 activity mediates an inhibition of male rat sexual behavior.

Hormone-dependent aggression in male rats is proportional to serum testosterone concentration but sexual behavior is not.

Male hooded rats were castrated and implanted with Silastic capsules (1.57 mm i.d.; 3.18 mm o.d.) having a testosterone-filled space 0, 7, 22, 60, or 90 mm long. All animals were returned to their original group cages for a three-week period to allow hormone concentrations and behavioral tendencies to stabilize. Each male was then housed with an intact female in a large cage. Aggression by the male toward an unfamiliar male was tested at weekly intervals for three weeks. Sexual behavior with an estrogen/progesterone-primed ovariectomized female was tested on each of the subsequent two weeks. Serum testosterone was measured during the following week. The frequency of aggression was correlated with serum testosterone concentration up to the normal level and did not increase with higher serum testosterone concentrations. In contrast, sexual behavior was virtually absent in animals with no testosterone replacement and normal in all other groups. These results demonstrate a clear dissociation in the dependence of hormone-dependent aggression and sexual behavior on serum testosterone concentration. In a male cohabiting with a female, sexual experience activates hormone-dependent aggression toward an unfamiliar male but the level of aggression that develops depends on the serum testosterone concentration in the resident male.