Immunofluorescent Verification of Silencing Estrogen Receptor α with siRNA in the Intact Rodent Brain.

The ability to silence the expression of gene products in a chemically, spatially, and temporally specific manner in the brains of animals has enabled key breakthroughs in the field of behavioral neuroscience. Using this technique, estrogen receptor alpha (ERα) has been specifically implicated in a multitude of behaviors in mice, including sexual, aggressive, locomotor, and maternal behaviors, in a variety of brain regions, including the medial preoptic area, ventromedial hypothalamus, and amygdala. In this chapter, we describe the techniques involved in the generation of the small hairpin RNAs (shRNAs) specifically designed to silence ERα, the construction of the adeno-associated viral (AAV) vector for delivery of the shRNA, the procedures to confirm the silencing of ERα (in vitro and in vivo) and in vivo delivery of the shRNAs to the brains of animals.

Estrogen receptors α and ß in the central amygdala and the ventromedial nucleus of the hypothalamus: Sociosexual behaviors, fear and arousal in female rats during emotionally challenging events.

Estrogens receptors (ER) are involved in several sociosexual behaviors and fear responses. In particular, the ERα is important for sexual behaviors, whereas ERß modulates anxiolytic responses. Using shRNA directed either against the ERα or the ERß RNAs (or containing luciferase control) encoded within an adeno-associated viral vector, we silenced these receptors in the ventromedial nucleus of the hypothalamus (VMN) and the central amygdala (CeA). We exposed ovariectomized female rats, sequentially treated with estradiol benzoate and progesterone, to five stimuli, previously reported to elicit positive and negative affect. The subjects were housed in groups of 4 females and 3 males in a seminatural environment for several days before hormone treatment. We analyzed the frequency of a large number of behavior patterns. In addition, we performed analyses of co-occurrence in order to detect changes in the structure of behavior after infusion of the vectors. Silencing the ERα in the VMN disrupted lordosis and showed some anxiolytic properties in aversive situations, whereas silencing of the ERß in this structure had no effect. This was also the case after silencing the ERα in the CeA. Silencing of the ERß in this structure increased risk assessment, an expression of anxiety, and increased olfactory exploration of the environment. We hypothesize that the ERß in the CeA has an important role in the well-established anxiolytic effects of estrogens, and that it may modulate arousal level. Furthermore, it seems that the ERα in the VMN is anxiogenic in aversive or threatening situations, in agreement with other studies.

VGF in the Medial Preoptic Nucleus Increases Sexual Activity Following Sexual Arousal Induction in Male Rats.

The central part of the medial preoptic nucleus (MPNc) is associated with sexual arousal induction in male rats. However, it is largely unclear how males are sexually aroused and achieve their first copulation. We previously reported that more MPNc neurons activate during the first copulation than the second copulation. In this study, to explore the molecules responsible for sexual arousal induction, we performed DNA microarray of the MPNc in sexually naive males and males after they copulated for their first and second times. We then performed quantitative PCR analyses to validate the results of the DNA microarray. Six genes were identified. Their expression increased following copulation and was higher in males after they copulated for the first time than after the second time. The genes encode transcription factors (Fos, Nfil3, and Nr4a3), a serine/threonine kinase (Sik1), an antioxidant protein (Srxn1), and a neuropeptide precursor VGF (Vgf), which may be the candidate genes responsible for sexual arousal induction. We examined the effects of Vgf knockdown in the MPNc on sexual partner preference and sexual behavior in sexually inexperienced and experienced males to determine the role of VGF in sexual arousal induction. A preference for estrous female rats was reinforced, and the latency of mount and intromission became short after sexually inexperienced males copulated for the first time. However, Vgf knockdown disrupted these phenomena. Vgf knockdown did not have any significant effect in sexually experienced males. VGF-derived neuropeptides presumably serve as an effector molecule to increase sexual activity following sexual arousal induction.

The Role of Estrogen Receptor ß in the Dorsal Raphe Nucleus on the Expression of Female Sexual Behavior in C57BL/6J Mice.

17ß-Estradiol (E2) regulates the expression of female sexual behavior by acting through estrogen receptor (ER) α and ß. Previously, we have shown that ERß knockout female mice maintain high level of lordosis expression on the day after behavioral estrus when wild-type mice show a clear decline of the behavior, suggesting ERß may be involved in inhibitory regulation of lordosis. However, it is not identified yet in which brain region(s) ERß may mediate an inhibitory action of E2. In this study, we have focused on the dorsal raphe nucleus (DRN) that expresses ERß in higher density than ERα. We site specifically knocked down ERß in the DRN in ovariectomized mice with virally mediated RNA interference method. All mice were tested weekly for a total of 3 weeks for their lordosis expression against a stud male in two consecutive days: day 1 with the hormonal condition mimicking the day of behavioral estrus, and day 2 under the hormonal condition mimicking the day after behavioral estrus. We found that the level of lordosis expression in ERß knockdown (ßERKD) mice was not different from that of control mice on day 1. However, ßERKD mice continuously showed elevated levels of lordosis behavior on day 2 tests, whereas control mice showed a clear decline of the behavior on day 2. These results suggest that the expression of ERß in the DRN may be involved in the inhibitory regulation of sexual behavior on the day after behavioral estrus in cycling female mice.

Inhibiting ERα expression in the medial amygdala increases prosocial behavior in male meadow voles (Microtus pennsylvanicus).

This study tested the hypothesis that site-specific estrogen receptor alpha (ERα) expression is a critical factor in the expression of male prosocial behavior and aggression. Previous studies have shown that in the socially monogamous prairie vole (Microtus ochrogaster) low levels of ERα expression, in the medial amygdala (MeA), play an essential role in the expression of high levels of male prosocial behavior and that increasing ERα expression reduced male prosocial behavior. We used an shRNA adeno-associated viral vector to knock down/inhibit ERα in the MeA of the polygynous male meadow vole (M. pennsylvanicus), which displays significantly higher levels of ERα in the MeA than its monogamous relative. Control males were transfected with a luciferase expressing AAV vector. After treatment males participated in three social behavior tests, a same-sex dyadic encounter, an opposite-sex social preference test and an alloparental test. We predicted that decreasing MeA ERα would increase male meadow vole's prosocial behavior and reduce aggression. The results generally supported the hypothesis. Specifically, MeA knockdown males displayed lower levels of defensive aggression during dyadic encounters and increased levels of overall side-x-side physical contact with females during the social preference test, eliminating the partner preference observed in controls. There was no effect on pup interactions, with both treatments expressing low levels of alloparental behavior. Behaviors affected were similar to those in male prairie voles with increased ERα in the BST rather than the MeA, suggesting that relative changes of expression within these nuclei may play a critical role in regulating prosocial behavior.

Pubertal activation of estrogen receptor α in the medial amygdala is essential for the full expression of male social behavior in mice.

Testosterone plays a central role in the facilitation of male-type social behaviors, such as sexual and aggressive behaviors, and the development of their neural bases in male mice. The action of testosterone via estrogen receptor (ER) α, after being aromatized to estradiol, has been suggested to be crucial for the full expression of these behaviors. We previously reported that silencing of ERα in adult male mice with the use of a virally mediated RNAi method in the medial preoptic area (MPOA) greatly reduced sexual behaviors without affecting aggressive behaviors whereas that in the medial amygdala (MeA) had no effect on either behavior. It is well accepted that testosterone stimulation during the pubertal period is necessary for the full expression of male-type social behaviors. However, it is still not known whether, and in which brain region, ERα is involved in this developmental effect of testosterone. In this study, we knocked down ERα in the MeA or MPOA in gonadally intact male mice at the age of 21 d and examined its effects on the sexual and aggressive behaviors later in adulthood. We found that the prepubertal knockdown of ERα in the MeA reduced both sexual and aggressive behaviors whereas that in the MPOA reduced only sexual, but not aggressive, behavior. Furthermore, the number of MeA neurons was reduced by prepubertal knockdown of ERα. These results indicate that ERα activation in the MeA during the pubertal period is crucial for male mice to fully express their male-type social behaviors in adulthood.

Effects of Prepubertal or Adult Site-Specific Knockdown of Estrogen Receptor ß in the Medial Preoptic Area and Medial Amygdala on Social Behaviors in Male Mice.

Testosterone, after being converted to estradiol in the brain, acts on estrogen receptors (ERα and ERß) and controls the expression of male-type social behavior. Previous studies in male mice have revealed that ERα expressed in the medial preoptic area (MPOA) and medial amygdala (MeA) are differently involved in the regulation of sexual and aggressive behaviors by testosterone action at the time of testing in adult and/or on brain masculinization process during pubertal period. However, a role played by ERß in these brain regions still remains unclear. Here we examined the effects of site-specific knockdown of ERß (ßERKD) in the MPOA and MeA on male social behaviors with the use of adeno-associated viral mediated RNA interference methods in ICR/Jcl mice. Prepubertal ßERKD in the MPOA revealed that continuous suppression of ERß gene expression throughout the pubertal period and adulthood decreased aggressive but not sexual behavior tested as adults. Because ßERKD in the MPOA only in adulthood did not affect either sexual or aggressive behaviors, it was concluded that pubertal ERß in the MPOA might have an essential role for the full expression of aggressive behavior in adulthood. On the other hand, although neither prepubertal nor adult ßERKD in the MeA had any effects on sexual and aggressive behavior, ßERKD in adulthood disrupted sexual preference of receptive females over nonreceptive females. Collectively, these results suggest that ERß in the MPOA and MeA are involved in the regulation of male sexual and aggressive behavior in a manner substantially different from that of ERα.

Silencing Estrogen Receptor-α with siRNA in the Intact Rodent Brain.

The ability to silence the expression of gene products in a chemically, spatially, and temporally specific manner in the brains of animals has enabled key breakthroughs in the field of behavioral neuroscience. Using this technique, estrogen receptor alpha (ERα) has been specifically implicated in a multitude of behaviors in mice, including sexual, aggressive, locomotor, and maternal behaviors. ERα has been identified in a variety of brain regions, including the medial preoptic area, ventromedial hypothalamus, and amygdala. In this chapter we describe the techniques involved in the generation of the small hairpin RNAs (shRNAs) specifically designed to silence ERα, the construction of the adeno-associated viral (AAV) vector for delivery of the shRNA, the procedures to confirm the silencing of ERα (in vitro and in vivo) and in vivo delivery of the shRNAs to the brains of animals.

The tumor suppressor PTEN regulates motor responses to striatal dopamine in normal and Parkinsonian animals.

Phosphatase and Tensin homolog deleted on chromosome 10 (PTEN) is a dual lipid-protein phosphatase known primarily as a growth preventing tumor suppressor. PTEN is also expressed in neurons, and pathways modulated by PTEN can influence neuronal function. Here we report a novel function of PTEN as a regulator of striatal dopamine signaling in a model of Parkinson's disease (PD). Blocking PTEN expression with an adeno-associated virus (AAV) vector expressing a small hairpin RNA (shRNA) resulted in reduced responses of cultured striatal neurons to dopamine, which appeared to be largely due to reduction in D2 receptor activation. Co-expression of shRNA-resistant wild-type and mutant forms of PTEN indicated that the lipid-phosphatase activity was essential for this effect. In both normal and Parkinsonian rats, inhibition of striatal PTEN in vivo resulted in motor dysfunction and impaired responses to dopamine, particularly D2 receptor agonists. Expression of PTEN mutants confirmed the lipid-phosphatase activity as critical, while co-expression of a dominant-negative form of Akt overcame the PTEN shRNA effect. These results identify PTEN as a key mediator of striatal responses to dopamine, and suggest that drugs designed to potentiate PTEN expression or activity, such as cancer chemotherapeutics, may also be useful for improving striatal responses to dopamine in conditions of dopamine depletion such as PD. This also suggests that strategies which increase Akt or decrease PTEN expression or function, such as growth factors to prevent neuronal death, may have a paradoxical effect on neurological functioning by inhibiting striatal responses to dopamine.

Nitric oxide regulates synaptic transmission between spiny projection neurons.

Recurrent axon collaterals are a major means of communication between spiny projection neurons (SPNs) in the striatum and profoundly affect the function of the basal ganglia. However, little is known about the molecular and cellular mechanisms that underlie this communication. We show that intrastriatal nitric oxide (NO) signaling elevates the expression of the vesicular GABA transporter (VGAT) within recurrent collaterals of SPNs. Down-regulation of striatal NO signaling resulted in an attenuation of GABAergic signaling in SPN local collaterals, down-regulation of VGAT expression in local processes of SPNs, and impaired motor behavior. PKG1 and cAMP response element-binding protein are involved in the signal transduction that transcriptionally regulates VGAT by NO. These data suggest that transcriptional control of the vesicular GABA transporter by NO regulates GABA transmission and action selection.

Visualisation and characterisation of oestrogen receptor α-positive neurons expressing green fluorescent protein under the control of the oestrogen receptor α promoter.

Oestrogen receptor (ER)α plays important roles in the development and function of various neuronal systems through activation by its ligands, oestrogens. To visualise ERα-positive neurons, we generated transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the ERα promoter. In three independent tg lines, GFP-positive neurons were observed in areas previously reported to express ERα mRNA, including the lateral septum, bed nucleus of the stria terminalis, medial preoptic nucleus (MPO), hypothalamus, and amygdala. In these areas, GFP signals mostly overlapped with ERα immunoreactivity. GFP fluorescence was seen in neurites and cell bodies of neurons. In addition, the network and detailed structure of neurites were visible in dissociated and slice cultures of hypothalamic neurons. We examined the effect of oestrogen deprivation by ovariectomy on the structure of the GFP-positive neurons. The area of ERα-positive cell bodies in the bed nucleus of the stria terminalis and MPO was measured by capturing the GFP signal and was found to be significantly smaller in ovariectomy mice than in control mice. When neurons in the MPO were infected with an adeno-associated virus that expressed small hairpin RNA targeting the ERα gene, an apparent induction of GFP was observed in this area, suggesting a negative feedback mechanism in which ERα controls expression of the ERα gene itself. Thus, the ERα promoter-GFP tg mice will be useful to analyse the development and plastic changes of the structure of ERα-expressing neurons and oestrogen and its receptor-mediated neuronal responses.

Differential effects of site-specific knockdown of estrogen receptor α in the medial amygdala, medial pre-optic area, and ventromedial nucleus of the hypothalamus on sexual and aggressive behavior of male mice.

Testosterone is known to play an important role in the regulation of male-type sexual and aggressive behavior. As an aromatised metabolite of testosterone, estradiol-induced activation of estrogen receptor α (ERα) may be crucial for the induction of these behaviors in male mice. However, the importance of ERα expressed in different nuclei for this facilitatory action of testosterone has not been determined. To investigate this issue, we generated an adeno-associated virus vector expressing a small hairpin RNA targeting ERα to site-specifically knockdown ERα expression. We stereotaxically injected either a control or ERα targeting vector into the medial amygdala, medial pre-optic area (MPOA), or ventromedial nucleus of the hypothalamus (VMN) in gonadally intact male mice. Two weeks after injection, all mice were tested biweekly for sexual and aggressive behavior, alternating between behavior tests each week. We found that suppressing ERα in the MPOA reduced sexual but not aggressive behavior, whereas in the VMN it reduced both behaviors. Knockdown of ERα in the medial amygdala did not alter either behavior. Additionally, it was found that ERα knockdown in the MPOA caused a parallel reduction in the number of neuronal nitric oxide synthase-expressing cells. Taken together, these results indicate that the testosterone facilitatory action on male sexual behavior requires the expression of ERα in both the MPOA and VMN, whereas the testosterone facilitatory action on aggression requires the expression of ERα in only the VMN.

siRNA silencing of estrogen receptor-α expression specifically in medial preoptic area neurons abolishes maternal care in female mice.

The medial preoptic area has been shown to be intricately involved in many behaviors, including locomotion, sexual behavior, maternal care, and aggression. The gene encoding estrogen receptor-α (ERα) protein is expressed in preoptic area neurons, and a very dense immunoreactive field of ERα is found in the preoptic region. ERα knockout animals show deficits in maternal care and sexual behavior and fail to exhibit increases in these behaviors in response to systemic estradiol treatment. In the present study, we used viral-vector mediated RNA interference to silence ERα expression specifically in the preoptic area of female mice and measured a variety of behaviors, including social and sexual aggression, maternal care, and arousal activity. Suppression of ERα in the preoptic area almost completely abolished maternal care, significantly increasing the latency to pup retrieval and significantly reducing the time the moms spent nursing and licking the pups. Strikingly, maternal aggression toward a male intruder was not different between control and preoptic ERα-silenced mice, demonstrating the remarkably specific role of ERα in these neurons. Reduction of ERα expression in preoptic neurons significantly decreased sexual behavior in female mice and increased aggression toward both sexual partners and male intruders in a seminatural environment. Estrogen-dependent increases in arousal, measured by home cage activity, were not mediated by ERα expression in the preoptic neurons we targeted, as ERα-suppressed mice had increases similar to control mice. Thus, we have established that a specific gene in a specific group of neurons is required for a crucially important natural behavior.

Hypothalamic Fkbp51 is induced by fasting, and elevated hypothalamic expression promotes obese phenotypes.

To discover hypothalamic genes that might play a role in regulating energy balance, we carried out a microarray screen for genes induced by a 48-h fast in male C57Bl/6J mouse hypothalamus. One such gene was Fkbp51 (FK506 binding protein 5; Locus NP_034350). The product of this gene is of interest because it blocks glucocorticoid action, suggesting that fasting-induced elevation of this gene in the hypothalamus may reduce glucocorticoid negative feedback, leading to elevated glucocorticoid levels, thus promoting obese phenotypes. Subsequent analysis demonstrated that a 48-h fast induces Fkbp51 in ventromedial, paraventricular, and arcuate hypothalamic nuclei of mice and rats. To assess if hypothalamic Fkbp51 promotes obesity, the gene was transferred to the hypothalamus via an adeno-associated virus vector. Within 2 wk following Fkbp51 overexpression, mice on a high-fat diet exhibited elevated body weight, without hyperphagia, relative to mice receiving the control mCherry vector. Body weight remained elevated for more than 8 wk and was associated with elevated corticosterone and impaired glucose tolerance. These studies suggest that elevated hypothalamic Fkbp51 promotes obese phenotypes.

The role of the estrogen receptor α in the medial preoptic area in sexual incentive motivation, proceptivity and receptivity, anxiety, and wheel running in female rats.

Ovariectomized females were given an infusion in the medial preoptic area (MPOA) of a viral vector carrying either a shRNA directed against the estrogen receptor α (ERα) or luciferase. The females were subjected to a test for sexual incentive motivation immediately followed by a test for receptivity and proceptive behaviors. Two weeks later they were tested in the light/dark choice procedure, and after another 2 weeks they were subjected to a test in a brightly lit open field. Finally, the females were given free access to a running wheel for 88h. The females were treated with estradiol benzoate (EB), 18 or 1.5µg/kg, in randomized order 52h before each test except the running wheel. In that experiment, they were given EB 48h after introduction into the wheel cage. They were given progesterone, 1mg/rat, about 4h before all tests, except the running wheel. The shRNA reduced the number of ERα with 83%. Females with few ERα in the MPOA showed increased lordosis quotient after the 1.5µg/kg dose of EB. There was no effect on proceptive behaviors or on rejections. When given the 18µg/kg EB dose, there was no difference between females with few preoptic ERα and controls. In the test for sexual incentive motivation, females with few preoptic ERα approached the castrated male incentive more than controls, regardless of EB dose. They also moved a shorter distance. In the light/dark choice test as well as in the open field, females with few ERα in the MPOA showed signs of reduced fear/anxiety, since they spent more time in the light part of the dark/light box and in the center of the open field. Finally, the data from the running wheel showed that females with few preoptic ERα failed to show enhanced activity after treatment with EB. These data show that the preoptic ERα inhibits lordosis in females with an intermediate level of receptivity while it fails to do so in fully receptive females. The ERα in the MPOA seems to be necessary for selective approach to a sexual incentive. Finally, activation of this receptor appears to have anxiogenic effects in the procedures employed here. A hypothesis for how all these actions of the preoptic ERα contributes to efficient reproductive behavior is outlined.

Adeno-associated viral gene delivery in neurodegenerative disease.

The advent of viral gene therapy technology has contributed greatly to the study of a variety of medical conditions, and there is increasing promise for clinical translation of gene therapy into human treatments. Adeno-associated viral (AAV) vectors provide one of the more promising approaches to gene delivery, and have been used extensively over the last 20 years. Derived from nonpathogenic parvoviruses, these vectors allow for stable and robust expression of desired transgenes in vitro and in vivo. AAV vectors efficiently and stably transduce neurons, with some strains targeting neurons exclusively in the brain. Thus, AAV vectors are particularly useful for neurodegenerative diseases, which have led to numerous preclinical studies and several human trials of gene therapy in patients with Parkinson's disease, Alzheimer's disease, and pediatric neurogenetic disorders. Here, we describe an efficient and reliable method for the production and purification of AAV serotype 2 vectors for both in vitro and in vivo applications.

Reversal of depressed behaviors in mice by p11 gene therapy in the nucleus accumbens.

The etiology of major depression remains unknown, but dysfunction of serotonergic signaling has long been implicated in the pathophysiology of this disorder. p11 is an S100 family member recently identified as a serotonin 1B [5-hydroxytryptamine 1B (5-HT(1B))] and serotonin 4 (5-HT(4)) receptor-binding protein. Mutant mice in which p11 is deleted show depression-like behaviors, suggesting that p11 may be a mediator of affective disorder pathophysiology. Using somatic gene transfer, we have now identified the nucleus accumbens as a key site of p11 action. Reduction of p11 with adeno-associated virus (AAV)-mediated RNA interference in the nucleus accumbens, but not in the anterior cingulate, of normal adult mice resulted in depression-like behaviors nearly identical to those seen in p11 knockout mice. Restoration of p11 expression specifically in the nucleus accumbens of p11 knockout mice normalized depression-like behaviors. Human nucleus accumbens tissue shows a significant reduction of p11 protein in depressed patients when compared to matched healthy controls. These results suggest that p11 loss in rodent and human nucleus accumbens may contribute to the pathophysiology of depression. Normalization of p11 expression within this brain region with AAV-mediated gene therapy may be of therapeutic value.

The role of the estrogen receptor alpha in the medial amygdala and ventromedial nucleus of the hypothalamus in social recognition, anxiety and aggression.

Social recognition manifests itself in decreased investigation of a previously encountered individual. Estrogen receptor alpha (ERalpha) knock out mice show deficient social recognition and anxiety. These data show that the ERalpha is involved in these effects, but they do not say anything about the brain sites important for these effects. In this study, an shRNA encoded within an AAV viral vector directed against the ERalpha receptor gene (or containing luciferase control), was injected bilaterally into the posterodorsal amygdala (MePDA) or the ventromedial nucleus of the hypothalamus (VMN) of female rats. An 81% reduction of ERalpha expression in the MePDA eliminated social recognition. Moreover, this diminution of ERalpha in the MePDA reduced anxiety in the light/dark choice test. In contrast, social recognition was unaffected after ERalpha knockdown in the VMN while aggressiveness against the juvenile was enhanced. In conclusion, social recognition and anxiety in female rats are modulated by the ERalpha in the amygdala. Moreover, aggression against juveniles but not against adults could, at least partly, depend on the ERalpha in the VMN.

Estrogen receptor-alpha in the bed nucleus of the stria terminalis regulates social affiliation in male prairie voles (Microtus ochrogaster).

Estrogen receptor alpha (ERalpha) typically masculinizes male behavior, while low levels of ERalpha in the medial amygdala (MeA) and the bed nucleus of the stria terminalis (BST) are associated with high levels of male prosocial behavior. In the males of the highly social prairie vole (Microtus ochrogaster), increasing ERalpha in the MeA inhibited the expression of spontaneous alloparental behavior and produced a preference for novel females. To test for the effects of increased ERalpha in the BST, a viral vector was used to enhance ERalpha expression in the BST of adult male prairie voles. Following treatment, adult males were tested for alloparental behavior with 1-3-day-old pups, and for heterosexual social preference and affiliation. Treatment did not affect alloparental behavior as 73% of ERalpha-BST males and 62.5% of control males were alloparental. Increasing ERalpha in the BST affected heterosexual affiliation, with ERalpha-BST males spending significantly less total time in side-by-side contact with females relative to time spent with control males. ERalpha-BST males did not show a preference for either the familiar or novel female. These findings differed significantly from those reported in ERalpha-MeA enhanced males, where ERalpha inhibited alloparental behavior and produced a preference for a novel female. The findings from this study suggest two things: first, that increased ERalpha in the BST decreases social affiliation and second, that altering ERalpha in different regions of the social neural circuit differentially impacts the expression of social behavior.

Estrogen-induced sexual incentive motivation, proceptivity and receptivity depend on a functional estrogen receptor alpha in the ventromedial nucleus of the hypothalamus but not in the amygdala.

The display of copulatory behaviors usually requires the presence of a mate and is, therefore, preceded by a search for and approach to a potential partner. The intensity of approach behaviors is determined by a process labeled sexual incentive motivation. Although it is known that female sexual motivation depends on estrogens, their site of action within the brain is unknown. In the present experiment, we obtained data relevant to this issue. An shRNA encoded within an adeno-associated viral (AAV) vector directed against the estrogen receptor alpha (ERalpha) gene (or containing a nonsense base sequence as a control treatment) was injected bilaterally into the ventromedial nucleus of the hypothalamus (VMN) or the posterodorsal amygdala (MePDA) of female rats. After an 80% reduction of the number of ERalpha in the VMN, sexual incentive motivation was absent after treatment with estradiol and progesterone. Proceptivity and receptivity were also much reduced, while the number of rejections was enhanced. Suppression of the ERalpha in the MePDA lacked these effects. Likewise, the inactive control AAV vector failed to modify any behavior. Thus, the ERalpha in the VMN, but not in the MePDA, is important for proceptivity and receptivity as well as for sexual incentive motivation. These results show that ERalpha in the VMN is crucial for the entire sequence of behavioral events from the processes leading to the establishment of sexual contact until the accomplishment of copulatory behaviors.