Estrogen-Induced Sexual Incentive Motivation, Proceptivity and Receptivity Depend on a Functional Estrogen Receptor 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  (ER ) 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 ER 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 ER  in the Received: October 21, 2008 Accepted after revision: May 23, 2009 Published online: November 4, 2009 Anders Ågmo Department of Psychology, University of Tromsø NO–9037 Tromsø (Norway) Tel. +47 77 64 63 65, Fax +47 77 64 52 91 E-Mail andersa @ psyk.uit.no © 2009 S. Karger AG, Basel 0028–3835/10/0912–0142$26.00/0 Accessible online at: www.karger.com/nen Sexual Behavior Effects of ER  in the VMN Neuroendocrinology 2010;91:142–154 143 pothalamus (VMN) [for reviews, see 6, 7 ]. Additional evidence for this was provided in a recent study where it was shown that infusion of a shRNA encoded within an adeno-associated viral (AAV) vector directed against the ER gene into the VMN strongly reduced sexual receptivity and proceptive behaviors in female mice [8] . Displays of receptivity and proceptivity occur when the female is in close physical proximity to the male. In fact, lordosis is a tactile reflex [9] and proceptive behaviors are strongly stimulated by tactile stimuli provided by the male [reviewed in 10, 11 ]. Consequently, few or none of the copulatory behaviors are displayed at a distance from the male. Approach to a male is, then, an event normally preceding displays of copulatory behaviors [12] . There are many observations suggesting that sexual approach behaviors are controlled differently from receptivity and proceptivity. Lesion studies have revealed that receptivity can be abolished without affecting approach to a sexually active male [13] while approach can be affected without reducing receptivity [14] . Some lesions may even enhance receptivity while reducing sexual approach [15] . Thus, the fact that the brain structures and the ER subtype involved in receptivity and proceptivity are quite well known does not mean that this applies to sexual approach behaviors. Furthermore, much of the recent data stem from studies in mice, and it is not evident that the hormone dependence of sexual approach behaviors is identical in rats and mice. In rats, approach to sexually relevant stimuli emitted by an intact male varies during the estrous cycle, with a peak around proestrus [16, 17] . No such variation occurs in mice [18] . There are even data suggesting that the approach of female mice to stimuli emitted by males is entirely independent of ovarian hormones [19] . These observations indicate that it may be risky to extrapolate the results from mice to rats. Although the brain areas involved in the control of receptivity and proceptivity as well as the crucial ER are reasonably well known, knowledge concerning sexual approach behaviors is scant. However, data from lesion studies [20, 21] suggest that the VMN may be important for these behaviors, in addition to receptivity/proceptivity as mentioned above. However, lesion data do not allow for any conclusion as to the role of ERs, and even less as to the specific ER involved. Another potentially important structure for sexual approach is the amygdala. This structure receives projections both from the main and accessory olfactory systems, and olfactory stimuli are crucial for the activation of approach behaviors [reviewed in 22 ]. Lesion of the medial posterodorsal amygdala (MePDA) reduces these behaviors in female rats [23, 24] . Furthermore, there is a substantial projection from the amygdala to the VMN [25] , suggesting that olfactory stimulation reaches the VMN via the amygdala. Thus, among all brain areas potentially involved in sexual approach behaviors, the case for a role of the VMN and the MePDA seems particularly strong. In the present experiments we suppressed the ER in the VMN and in the MePDA in ovariectomized female rats with the help of a shRNA directed against the ER receptor gene [8] . Receptivity, proceptivity and sexual incentive motivation after treatment with estradiol and progesterone were then evaluated. These experiments provide data essential for our understanding of the hormonal control of sexual approach behaviors in addition to extending earlier observations on receptivity and proceptivity in the female mouse to the rat. Materials and Methods Subjects Male and female Wistar rats were obtained from Charles River WIGA (Sulzfeld, Germany). They were housed 2 per cage under a reversed light-dark cycle (12: 12 h, lights off 11: 00 h). Food (RM1, Special Diets Services, Witham, Essex, UK) and tap water were always available. Ambient temperature was maintained at 21 8 1 ° C and relative humidity was 55 8 10%. All females (250 g upon arrival to the animal facilities) were ovariectomized and some males (300 g upon arrival) were castrated under isofluorane anesthesia. All experimental procedures employed in the present experiment were approved by the National Animal Research Authority of Norway and were in agreement with the European Union council directive 86/609/EEC. Stereotaxic Surgery About 2 weeks after ovariectomy, some females received intracerebral infusions of either a shRNA encoded within an AAV vector directed against the ER gene (ER shRNA) or with a shRNA containing a nonsense base sequence (AAV control) as well as an independent EGFP (enhanced green fluorescent protein) expression cassette to detect neurons transduced by our viral vector. Hence, EGFP is used as a marker of correct injection localization. The vector AAV control was designed to suppress luciferase expression. The shRNAs employed here have been described in detail elsewhere [8] . Before infusion, the females were fixed in a stereotaxic frame under ketamine/xylazine anesthesia (100 and 10 mg/kg, respectively). The skull was exposed, and small holes were drilled at the appropriate places after removal of the fascia. Bilateral cannulae (30 gauge) were aimed at the VMN (coordi nates were: anteroposterior –2.80; mediolateral 8 0.6; dorsoventral –9.6; n = 13 for ER  shRNA as well as for AAV control), the MePDA (coordinates: anteroposterior –3.14; mediolateral 8 3.6; dorsoventral –8.2; n = 13 for ER  shRNA as well as for AAV control) or the cerebral cortex dorsal to the VMN (coordinates: anteroposterior –2.80; mediolateral 8 0.6; dorsoventral –2; n = 13).