This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Puberty has been described as a critical developmental period susceptible to changes at the hormonal level, which can modulate cellular and molecular substrates leading to particular behaviors. Secretion of male hormones is important to promote growth of the accessory sex organs and to establish adequate reproductive function. Beside actions of sex steroids in the brain regulating reproductive behavior, androgens modulate non-reproductive behaviors such as anxiety, and emotional learning. Interestingly, neuropeptides are cellular substrates that have been shown to be under hormonal control, have been associated with different domains of behaviors and are highly expressed in brain regions mediating these behaviors. Knowledge that androgens can modulate specific cellular substrates such as neuropeptides has raised relevant questions regarding the cellular mechanisms responsible for the observed behavioral changes under supraphysiological concentrations. We have previously found that anabolic androgenic steroids (AAS) decrease neuropeptide Y (NPY) levels in the amygdala (AMY) of female pubertal rats and an increase in NPY in the ventromedial nuclei (VMN) of pubertal males. This proposal aims to determine the role of NPY in androgen modulation of reproductive behaviors and emotional learning during early exposure to AAS. Pubertal rats (PN32) will be chronically exposed to high doses of AAS followed by assessment of anxiety-like behaviors using the elevated plus maze (EPM), and emotional learning, using the passive avoidance task (PAT) in females and sexual behavior in males. Anxiety and emotional learning will be study in females since these behaviors are controlled by the AMY, and sexual motivation will be studied in males given that the VMN plays an important role in this component of sexual behavior. To further investigate if AAS affect emotional learning and reproductive behaviors through NPY mechanisms, peripubertal animals (PN28) will be systemically exposed for one week to androgens and will be implanted with bilateral injection cannulae in the AMY (females) and the VMN (males). After two weeks of AAS exposure, and still in puberty (PN35), pharmacological drugs (NPY ago/antagonists) will be intracranially infused. Five minutes after the infusion EPM, PAT and sexual motivation will be performed. This study will contribute with new cellular insights of androgen effects in puberty, which is the most sensitive age to engage in androgen misuse and most deleterious neuroendocrine and psychiatric symptoms are observed.
SPECIFIC AIMS Data have demonstrated an important role for androgens in sexual behaviors, anxiety and learning &memory. Interestingly, all of these behaviors are modulated throughout biochemical molecules such as neurotransmitters and neuropeptides. The long-term goal of this research program will be focused on providing a better understanding of the role of neuropeptides in reproductive behaviors and emotional learning after in vivo exposure to androgens. Given that anabolic androgenic steroid (AAS) misuse is associated with multiple reproductive-related behaviors and learning and memory, this study will provide critical data of the biochemical aspects of behavioral changes associated with androgen exposure, especially in adolescents, where an increased misuse has been reported. This study propose:
Aim 1. Determine the role of Neuropeptide Y (NPY) in pubertal brain regions involved in anxiety and emotional learning after androgen exposure. The amygdala (AMY) is a complex brain region associated with emotional processing and anxiety disorders (Ledoux, 1998) that has been demonstrated to be under hormonal influence (Cooke et al., 2003;2006). Androgen regulation of peptidergic cellular substrates controlling behavior has been well documented (Clark and Henderson, 2003). In the context of this hormonal control, we have found from results of our first funding cycle that AAS decrease NPY levels in the AMY of female pubertal rats (Barreto-Estrada et al., unpublished results). Since NPY has been related to anxiety and emotional events (Carvajal, et al., 2006; Primeaux et al., 2005), our hypothesis is that in vivo exposure to AAS will elicit changes in anxiety and/or emotional learning through NPY mechanisms in pubertal females. To test this hypothesis, pubertal female rats (PN32) will be chronically exposed to high doses of AAS. Thereafter, assessment of anxiety-like behaviors using the elevated plus maze (EPM), and emotional learning, using the passive avoidance task (PAT) will be performed. To find out if AAS affect anxiety and/or emotional learning through NPY mechanisms, peripubertal females (PN28) will be systemically exposed to androgens and also will be implanted with bilateral injection cannulae in the AMY. After two weeks of AAS exposure, and still in puberty (PN35), NPY agonist will be intracranially infused in the AMY. Five minutes after the infusion EPM and PAT will be performed. AAS-affected behaviors will be reversed with the antagonist.
Aim 2. Determine the role of NPY in pubertal brain regions involved in reproductive-related behaviors after androgen exposure. The VMN is a hypothalamic nuclei principally involved in reproductive-related behaviors. In particular, it is associated with the female sexual response, while in males it is associated with sexual motivation. Similar to the AMY, the VMN has been shown to be under hormonal influence (Harding and McGinnis, 2004). We have found from previous results that AAS increases NPY levels in the VMN of male pubertal rats (Barreto-Estrada et al., unpublished results). Since the VMN has been related to the motivational component of sexual behavior in males, and given that the VMN express NPY (Beck, 2006), our hypothesis is that in vivo exposure to AAS will elicit changes in sexual motivation through NPY mechanisms in pubertal males. To test this hypothesis, pubertal male rats (PN32) will be chronically exposed to high doses of AAS. Sexual motivation parameters such as the latency to the first mount, latency to the first ejaculation and the partner preference will be assessed. To find out if AAS affect sexual motivation through NPY mechanisms, peripubertal males (PN28) will be systemically exposed to androgens and will also be implanted with bilateral injection cannulae in the VMN. After two weeks of AAS exposure, and still in puberty (PN35), NPY antagonist will be intracranially infused in the VMN. Sexual motivation will be assessed five minutes after the antagonist infusion. AASaffected behaviors will be reversed with the antagonist.
Aim 3. Establish the effect of AAS in synaptic plasticity Synaptic connectivity and neuronal branching have been shown to be affected by androgens (Cherry et al., 1992;McEwen, 1992).
This aim will study the effect of AAS in synaptic function, in particular dendritic spines that could be regulated by hormones (Smart and Halpain, 2000), and that are known to be affected in cognitive processes (Julien et al., 2008). Double labeling immunohistochemistry for drebrin, an actin-binding dendritic spine protein (Sekino et al., 2007) and NPY will be performed in the AMY and VMN. Modulation of the synaptic microenvironment by AAS might be responsible for changes in behavior. The present aim investigates the potential synaptic dysfunction after AAS exposure in emotional memory, anxiety and sexual behaviors.
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