Ruth I. Wood - US grants

The objectives of this research are to understand the actions of gonadal steroids on androgen and estrogen receptors in the brain that control male sexual behavior. The Syrian hamster is an appropriate animal model for these studies beacuse the male is dependent upon both chemosensory stimuli and gonadal hormones to elicit mating, and the basic pathways through the brain that mediate this behavior are known. Olfactory and vomeronasal chemosensory stimuli are processed through a chain of neurons that selectively take up androgens and estrogens from the bloodstream, and both of these signals (odors and hormones) are essential for normal mating to occur. Our preliminary studies have described the locations of steroid receptor- containing neurons and the repartitioning of the receptor from nucleus to cytoplasm after hormone withdrawal using immunocytochemistry. In the first Specific Aim, we will determine essential neurobiological mechanisms by which gonadal hormones stimulate copulation through autoregulation of their receptor. First, we will determine how androgens influence the levels of their receptor in the brain using in situ hybridization and receptor binding to quantify androgen receptor mRNA and protein in brains of gonad-intact and castrate males. Secondly, the time-course and dose- response to testosterone activation of neural andogen receptors will be determined using immunocytochemistry. Finally, we will localize unbound cytoplasmic androgen receptor at the ultrastructural level within neurons of castrated males. In Specific Aim 2, we will focus on functional populations of androgen and estrogen receptor-containing neurons controlling sexual behavior. In particular, our studies will focus on the medial amygdaloid nucleus (Me), bed nucleus of the stria terminalis (BNST), and the medial preoptic area (MPOA) as potential sites for the integration of chemosensory and hormonal signals for copulation. We will determine the role of neonatal steroid exposure in the sexually dimorphic pattern of androgen receptors in MPOA. In addition, colocalization of androgen and estrogen receptor immunoreactivity will determine if individual neurons can respond to both gonadal steroids. Finally, we will use intracerebral implants of estrogen and DHT in Me and MPOA to determine the identity (androgen or estrogen) of steroid signals that induce copulation in these brain areas. The proposed studies have significance to understanding neural function under the influence of anabolic steroids. Abuse of these substances is becoming a serious health problem in our society. These observations on the location and hormonal regulation of androgen receptors in the brain will provide an essential basis for studying the behavioral effects of exogenous androgens.

DESCRIPTION (Adapted from applicant's abstract): Mating is a complex motivated behavior whose expression requires the interaction of odors and gonadal steroids. These studies have relevance to understanding the regulation of complex behaviors in humans and animals because sensory and hormonal stimuli are seldom present in isolation. Instead, an organism in a natural setting is confronted with multiple signals from its internal and external environment, and the product of multimodal integration transforms the individual characteristics of these stimuli to produce a rich behavioral response. The Syrian hamster is used as a model for these studies because the male is dependent upon both odors and hormones to mate, and the basic neural circuits that mediate this behavior are known. These studies will contribute to a growing literature that hormones interact with chemosensory, visual, nutritional, and somatosensory cues to regulate reproduction. One hallmark of hormone-sensitive brain regions is the dense interconnections between steroid receptor-containing nuclei to form a hormone-responsive neural network. This network structure has profound implications for steroidal regulation of neural function. In 1986, Cottingham and Pfaff proposed that properties of the hormone-responsive neural network could include redundancy, amplification, stability and selective filtering of steroid cues to regulate reproduction, concepts which can be applied to the steroidal control of male hamster sexual behavior. The PI's laboratory recently determined that communication between odors and hormones is required for mating, and Specific Aim 1 will investigate where in the brain this is accomplished. In particular Aim 1A will use intracerebral implants of testosterone to test the hypothesis that there exist separate parallel subcircuits for transmission of odors and hormones, and that the hormonal subcircuit is functionally redundant. Aim 1B will combine intracerebral implants with unilateral olfactory bulbectomy to determine sites for integration of hormonal cues with odors. The implication is that hormones act as a selective filter for odor cues to permit mating. Specific Aim 2 will test the hypothesis that multifocal steroid action amplifies male sexual behavior using multiple implants of testosterone in the mating behavior pathway. Finally, Specific Aim 3 will investigate stability of the mating behavior circuitry through reciprocal connections of steroid-responsive subnuclei.

[unreadable] DESCRIPTION (provided by applicant): The purpose of this proposal is to investigate the effects of neurotoxic injury to the nigrostriatal dopaminergic system on the serotonergic system to understand how these alterations influence affective and motor dysfunction in Parkinson's Disease (PD). It is becoming evident that PD is not simply a motor disorder. Patients with PD are significantly more depressed than other similarly-disabled patients. At the molecular level, interactions between the serotonergic and dopaminergic systems contribute to affective disorders including anxiety and depression, and may underlie the close relationship between affective and motor dysfunction in PD. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse will be used here as a model since there is return of striatal DA and intrinsic plasticity of the dopaminergic system over time. In the proposed studies, we will test the hypothesis that there are dynamic behavioral, molecular, and morphological alterations in the serotonergic system in the MPTP-lesioned mouse. Mice will receive MPTP or saline and analyzed at post-lesioned days 5 (early, acute depletion when MPTP-induced cell death is complete), and 35 (partial DA recovery). Analyses will include (i) depression and anxiety; (ii) striatal dopamine and serotonin by HPLC, (iii) cell counts of dopaminergic neurons in substantia nigra pars compacta and serotonergic neurons in the dorsal raphe, and (iv) mRNA expression of 5-HT receptors 1a, 1b, 2a, and 2c in midbrain, striatum, limbic system, and frontal cortex. We hypothesize that there is an acute increase in the serotonergic system after MPTP-lesioning that is then down-regulated in an attempt to facilitate dopaminergic neurotransmission. As DA returns 5-HT neurotransmission normalizes. Furthermore, increases or decreases in the 5-HT system drive changes in anxiety and depression. A secondary goal of our studies is to determine the role of steroid hormones in susceptibility to affective disorders in PD. We hypothesize that castration will increase anxiety and depression in the MPTP-lesioned mouse. Working with Dr. Michael Jakowec of the Department of Neurology at the Keck School of Medicine of USC, Dr. Wood will receive training in molecular techniques for measurement of serotonin and its receptors. In addition, she will spend time in the laboratory of Dr. George F. Koob at The Scripps Research Institute to learn additional approaches to study anxiety and depression in mice. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Although anabolic-androgenic steroids (AAS) have legitimate medical uses, they are also drugs of abuse. AAS are taken in large quantities by athletes and others to increase performance, with negative long-term health consequences. In 1991, testosterone was declared a controlled substance. Nonetheless, illicit use of AAS continues to increase, particularly among adolescents. Indeed, the incidence of steroid use among high school seniors is comparable to that for cocaine or heroin. Although users defend performance enhancing substances as a healthy lifestyle choice, clinical studies and anecdotal reports present a different picture. Inappropriate and excessive agonistic behavior ('roid rage) is the most widely-reported psychiatric side effect of AAS in humans. Heightened sexuality and sexual violence have also been noted. Furthermore, AAS are linked with polydrug abuse, most notably opioids. We hope to understand why AAS abuse predisposes individuals to engage in inappropriate and excessive aggression, sexual behavior and drug use, and to unravel the underlying neural mechanisms. In particular, we aim to address a major public misconception that 'roid rage represents a loss of control, a sudden and exaggerated response to a minimal provocation. Instead, research suggests that AAS-treated rats remain sensitive to the context (individual and environment) of social interactions. This suggests a new explanation for behavioral effects of AAS abuse. The current proposal focuses on our hypothesis that chronic exposure to AAS during adolescence inappropriately increases responsiveness to rewarding stimuli, both natural rewards (sex, aggression) and drugs of abuse. The proposed studies will investigate how high-dose androgens in adolescent rats alter reward processes. Understanding behavioral effects of AAS use in humans is complicated by the user's motivation for increased strength and muscle mass. Animal studies can evaluate responses to AAS in an experimental context where appearance and athletic performance are irrelevant. In this regard, animals also demonstrate AAS-induced aggression and sexual behavior. Yet, these studies have emphasized consummatory aspects of behavior, not the appetitive motivation for mating or fighting. Importantly, sex, fighting and drug use are each reinforcing, and each is sensitive to androgens. Accordingly, we hypothesize that AAS increase expression of social behaviors and drug self-administration by increasing the reward value of these steroid-sensitive behaviors. Aim 1 will determine if AAS increase motivation for aggression (Aim 1A), mating (Aim 1B) and morphine self- administration (Aim 1C). Aim 2 will explore neural mechanisms for these effects. We will determine if testosterone is a permissive signal to enhance dopamine activity in the nucleus accumbens in response to sexual stimuli (Aim 2A). Aim 2B will determine fundamental mechanisms underlying these responses by measuring levels of tyrosine hydroxylase, dopamine D1 and D2 receptors, and the dopamine transporter. Together, these studies will provide insight into the potential for AAS to enhance motivation and reward, and the mechanisms through which this occurs.

DESCRIPTION (provided by applicant): Drinking behavior and social context are intimately intertwined, particularly among young adults. Peer relations can promote drinking. At the same time, alcohol consumption promotes social bonding, as in the popular concept of a drinking buddy. Ultimately, to combat unhealthy patterns of social drinking, it is important to understand how ethanol shapes the neurochemistry of affiliative behavior. We have developed a mouse model of conditioned partner preference, and we have obtained pilot data to demonstrate ethanol (EtOH)-induced social preference in female mice. Conditioned partner preference is similar to conditioned place preference, but it incorporates social aspects of approach, recognition, and affiliation. This has relevance to drinking behavior in humans. In our pilot studies thus far, female mice prefer conspecifics with whom they have previously been intoxicated. There is a further interaction of EtOH and estradiol to promote social preference, since EtOH-induced partner preference is enhanced in estrogen- treated ovariectomized females (OVX+E) vs ovariectomized females without estrogen (OVX). The proposed studies will use C57Bl/6 female mice to extend our initial observations. Aim 1a will determine the range of EtOH doses which facilitate conditioned partner preference in OVX, OVX+E, and OVX+E females with progesterone. Aim 1b will examine sex differences in EtOH- induced conditioned partner preference by testing orchidectomized males with and without testosterone. Aim 2 will expand the conditioned partner preference model to test the effects of other drugs of abuse (amphetamines, morphine) on social bonding. Finally, Aim 3 will begin to explore underlying mechanisms for EtOH-induced conditioned partner preference. In this regard, pair bonding and affiliative behavior are sensitive to vasopressin mediated through the vasopressin V1a receptor. Furthermore, the vasopressin system is sensitive to both EtOH and estradiol. Aim 3 will test the ability of a V1a receptor antagonist to block EtOH-induced conditioned partner preference. Together, these studies represent an essential first-step to understand substance abuse and social bonding in mice.

? DESCRIPTION (provided by applicant): Although anabolic-androgenic steroids (AAS) have legitimate medical uses, they are also drugs of abuse. AAS are taken in large quantities by athletes and others to increase performance, with negative long- term health consequences. In 1991, testosterone was declared a controlled substance. Nonetheless, illicit use of AAS continues to increase, particularly among adolescents. Indeed, the incidence of steroid use among high school seniors is comparable to that for cocaine or heroin. Although users defend performance enhancing substances as a healthy lifestyle choice, clinical studies and anecdotal reports present a different picture. Many AAS users meet DSM criteria for psychoactive substance dependence, including continued use despite negative side effects, and withdrawal symptoms when steroids are discontinued. Ultimately, unlike other illicit drugs, AAS have only a limited capacity for acute intoxication. Instead, a key danger of AAS abuse reflects the likelihood that users will engage in behaviors that pose risks to themselves and those around them. Understanding behavioral effects of AAS use in humans is complicated by the user's motivation for increased strength and muscle mass. Furthermore, we cannot control for preexisting psychopathology or for variability in the type or dose of AAS, and it is unethical to test high doses of AAS in normal volunteers. Animal studies can evaluate responses to AAS in an experimental context where appearance and athletic performance are irrelevant. Therefore, we use rats treated chronically with high-dose testosterone to model human AAS use. In humans, AAS increase risk-taking: fighting, unsafe sex, drinking and driving, carrying a weapon. Our studies in rats demonstrate that AAS separately modify decision making on tests of effort, punishment, delay and probability discounting. The proposed studies will build on these recent findings to test the hypothesis that AAS impair complex decision making and cooperation, and these effects are mediated by dopamine (DA) acting via D1- (D1R) and D2-like receptors (D2R) in subnuclei of the nucleus accumbens (Acb). In operant discounting tasks, rats choose between a small reward (1 sugar pellet) vs a large reward (3-4 pellets) which is discounted (made less desirable) by some cost. Chronic high-dose testosterone makes rats less sensitive to physical effort, punishment or delay, but more sensitive to uncertainty. Studies in Aim 1 go beyond simple discounting tasks to model decision-making in a natural environment, presenting cognitively-demanding choices with conflicting costs, and incorporating social interaction. Aim 2 will explore neurobiologic mechanisms for impaired decision making with AAS. As part of the mesolimbic DA system, Acb is central to motivated behavior and decision-making, and DA dysfunction impairs decision making. We will test intra-Acb implants of testosterone (Aim 2A), and pharmacologic manipulation of DAR in Acb during effort- and probability discounting (Aim 2B) and reciprocal cooperation (Aim 2C). Together, these studies will provide insight into the cognitive changes induced by AAS, and the mechanisms through which these occur.