Anders Agmo

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.

General arousal has been operationally defined as an enhanced motor activity and enhanced intensity of response to sensory stimuli. Even though the effects of gonadal hormones on mating behavior have been much studied, their potential effect on generalized arousal, as defined above, has never been evaluated. In the present study we employed a thoroughly validated assay of general arousal to determine the effects of estradiol (E) and testosterone (T) in gonadectomized female and male mice, respectively. The steroids were administered in three different ways: A fast-acting, water soluble preparation given intraperitoneally, an oil solution given subcutaneously, and an oil solution in a subcutaneous Silastic capsule. Motor activity and responses to sensory stimuli were recorded for 24h, 91h, and seven days following hormone administration, respectively. All measures of arousal varied according to the day/night cycle. The water soluble steroid preparation had no reliable effect. When the same doses of estradiol and testosterone were administered subcutaneously in an oil vehicle no effect of either treatment on arousal was observed. The subcutaneously implanted capsule containing estradiol or testosterone had a delayed effect on motor activity in females (four to seven days) but no effect in males. The long time required by the gonadal hormones for affecting arousal would be consistent with, but does not prove, a genomic action. The limited effects of E and T in our arousal assay suggest to us that the strongest actions of these hormones on arousal occur in the context of sequences of responses to sexually relevant stimuli.

Previous studies have shown that the opiate antagonist naloxone blocks the anxiolytic-like effects of benzodiazepines in several models of anxiety, including the elevated plus-maze. Although naloxone preferentially binds to the mu opioid receptor, its selectivity is rather low. The opioid receptor subtype important for anxiolytic-like actions of benzodiazepines in the plus-maze remains, therefore, unknown. In the present experiments, the ability of antagonists selective for subtypes of the opioid receptor to block the anxiolytic-like effects of chlordiazepoxide in the elevated plus-maze was evaluated in Swiss mice. Chlordiazepoxide, 5 mg/kg, increased the proportion as well as the number of open arms entries without modifying closed arms entries. Lower doses of the benzodiazepine were ineffective. The mu receptor antagonist beta-funaltrexamine, 10 and 20 mg/kg, the delta antagonist naltrindole, 10 mg/kg, and the kappa antagonist nor-binaltorphimine, 2.5 and 5 mg/kg, were then combin...

The locomotion-reducing effect of the GABAB agonist baclofen was compared with that of the GABAA agonists, aminopropanesulfonic acid (APSA) and THIP. It was found that baclofen was more potent than the other drugs. After intraventricular injection, baclofen induced almost complete immobility, whereas APSA did not affect locomotor activity. THIP had an intermediate effect. The GABA transaminase inhibitor gamma-acetylenic GABA (GAG) provoked a dose-dependent reduction of locomotion. Neither the effects of THIP nor those of GAG could be blocked by concurrent administration of bicuculline. The antagonist itself did not affect locomotor activity. It is concluded that the GABAA receptor is not important for the locomotion-reducing effects of GABAergic drugs.

D-ala2-met5-enkephalinamide (DALA) was found to be without effect on motility when injected in doses from 10 to 40 micrograms. When ICV injection of DALA was combined with IP injection of amphetamine, DALA markedly enhanced the stimulatory effect of amphetamine. The effects of DALA + amphetamine could be partially antagonized by naloxone. The locomotion-reducing effect of the dopamine antagonist pimozide was not affected by concurrent administration of DALA. These data suggest a complex interaction between opiates and dopamine. It is suggested that the effects of DALA are best explained assuming that the opiate inhibits GABAergic neurotransmission.

Male rabbits were treated with the dopamine releasing drug amphetamine or the dopamine D1/D2 receptor antagonist cis(Z)-flupenthixol. Amphetamine, 1 to 4 mg/kg, had no effect on sexual behavior. Flupenthixol, 2 mg/kg, reduced the proportion of rabbits that ejaculated and the number of ejaculations per test. Lower doses were ineffective. Castrated males were treated with both drugs at two intervals after castration, 19-21 and 27-29 days postcastration, respectively. Amphetamine was without effect while flupenthixol, 1 mg/kg, reduced sexual behavior at the test 19-21 days postcastration. At the second test, sexual behavior was almost completely absent in control animals. Therefore, no further reduction could be observed after treatment with flupenthixol. Another group of animals was castrated and given androgen replacement. Testosterone decanoate was injected once weekly at a dose of 3 mg/kg. This treatment maintained a stable, low sexual activity. In these animals, amphetamine was again ineffective whereas flupenthixol, 1 mg/kg, inhibited sexual behavior. Gross motor function was evaluated in a water escape test. Amphetamine was inactive, and the effective dose of flupenthixol was 10 mg/kg. This dose is far above the dose required for inhibiting sexual behavior. In sum, facilitated dopaminergic transmission does not seem to affect on sexual behavior in the male rabbit, whereas reduced dopaminergic activity disrupts this behavior.

The effects of gamma-amino butyric acid (GABA)-ergic drugs on male rabbit sexual behaviour have been evaluated. The GABA(A) agonist 4,5,6,7-tetrahydroxixazolo-5,4c-pyridin-3-ol (THIP), the GABA(B) agonist R-baclofen and the GABA antagonists picrotoxin and bicuculline were used. Injection of THIP, 20 mg/kg, s.c. produced a complete suppression of sexual behaviour and R-baclofen, 2.5 mg/kg, s.c. a significant inhibition. Intraperitoneal injections produced effects at higher doses than did s.c. injections. The inhibition produced by R-baclofen was associated with strong motor effects as shown by the water escape test. It is probable, therefore, that the reduced sexual behaviour observed after treatment with this drug is a consequence of sedative or muscle relaxant effects. By contrast, the dose of THIP that inhibited sexual behaviour had no effect on the water escape test. These results show that the GABA(A) agonist inhibits sexual behaviour in the male rabbit independent of effects on the motor system. The GABA antagonists had marginal or no effects on sexual behaviour. When these data are compared to previous results in the rat, substantial differences are seen. As there are differences between the effects on rat and rabbit sexual behaviour by other types of drugs, it appears that drug action on sexual behaviour cannot be generalized from one species to another.

Rats emit ultrasonic vocalizations (USVs) of 22 kHz and 50 kHz before, during, and after copulation. The 50-kHz vocalizations can be subdivided into flat and frequency-modulated (FM) trill calls. In this study, the role of 50-kHz USVs in sexual incentive motivation in female rats was examined. USVs were recorded from sexually active males during the precopulatory phase. In the first two experiments, "full 10-min song," flat-call, and FM-trill-call stimuli were used. In Experiment 1, a combination of complex and trill calls was used as the FM-trill call, whereas a mixture of multistep and upward ramp calls was used in Experiment 2. The auditory stimuli were played back to sexually receptive female rats in a sexual incentive-motivation test. For comparison, the odor of an intact male rat was also used as incentive stimulus. The flat-call stimulus did not induce approach behavior in any experiment, whereas the FM trill showed a short-lived effect in Experiment 2. The females approached the "full song" during the first minute of stimulus exposure in Experiment 1, but not in Experiment 2. When the entire 10-min test period was analyzed, the females in Experiment 1 did not approach the full song more than background noise, though they did so in Experiment 2. The olfactory stimulus, to the contrary, had a clear incentive value in both experiments. In a third experiment, a devocalized male, an intact vocalizing male and a female rat were used as incentive stimuli. The females did not approach a vocalizing male more than they approached a silent male, showing that USVs do not contribute to the male rat's incentive value. Overall, the results of the present series of experiments show that the male rat's USVs do not have any consistent incentive value for the sexually receptive female rat. This sharply contrasts the strong and reliable effect of male odor.

Ultrasonic vocalizations (USVs) are emitted in response to a sexual partner before, during, and after copulation. These vocalizations are the so-called 50-kHz USVs and can be subdivided into flat and frequency-modulated (FM) 50-kHz trill calls. In the present series of experiments, the potential unconditioned and conditioned incentive properties of female 50-kHz USVs for male rats were examined. USVs were recorded from sexually receptive females during the precopulatory phase. A complete 10-min song, or single flat or FM trill calls were selected as auditory stimuli for Experiments 1 and 2. As FM trill calls, a multistep call was used in Experiments 1 and 2a, and an upward ramp call was used in Experiment 2b. The auditory stimuli were played back with a loudspeaker to naïve and sexually experienced male rats in a sexual incentive motivation test. The odor of a sexually receptive female rat was also used as an incentive stimulus for comparison. In a third experiment, a devocalized female, a sham female and a male rat were used as incentive stimuli. It was found that the auditory stimuli did not induce approach behavior in naïve and sexually experienced male rats, but the olfactory stimulus did. In addition, the males spent equal amounts of time in the vicinity of devocalized and vocalizing females. These data show that 50-kHz USVs neither are unconditioned nor conditioned incentives for male rats.

The present experiment was designed to determine whether infusions of naloxone into specific brain sites can block sexual reinforcement as evaluated with the conditioned place preference procedure. Methylnaloxonium (5 micrograms/cannula) was infused bilaterally either into the medial preoptic area (MPOA) or into the nucleus accumbens (NAC) of sexually experienced male rats. We chose the MPOA because it is important for sexual behavior, and several opioid peptides have been shown to modify sexual behavior when infused there. The NAC appears to be a critical structure for drug-induced reward. Methylnaloxonium blocked place preference produced by ejaculation after infusion into the MPOA without affecting sexual behavior. Infusion of the antagonist into the NAC did not reduce the reinforcing properties of ejaculation. These data suggest that the MPOA may be a site where sexual reward is produced.

Sexual arousal affects cognitive processing, which depends on the coordinated functioning among cortical areas. The aim of this research was to determine whether previous observation of videos with sexual content affects the degree of cortical electroencephalographic (EEG) coupling during performance of an executive task. Cortical EEG correlations were calculated in three groups of heterosexual men under three conditions: at rest; during observation of a video with neutral, aggressive, or erotic content; and while performing the Tower of Hanoi task (TOH). Based on self-reports, it was shown that the erotic video induced general and sexual arousal, while the aggressive video affected valence and general arousal. Task performance was similar in all three groups. During performance of TOH, only the erotic group showed a decreased correlation between prefrontal areas with an increased correlation between parietal and prefrontotemporal areas, specifically in the slow bands. It is likely that these changes in the degree of cortical coupling could be associated with the cognitive strategies or functional adaptations that participants require to adequately solve the task during a state of sexual arousal. These data could contribute to improving our understanding of the central nervous mechanisms that underlie the effect of sexual arousal on the cognitive processes involved in tasks like TOH.

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.

Female paracopulatory behavior is believed to incite the male to start copulation and to allow the female control over the pace of sexual interaction. It is also supposed to represent female sexual motivation. However, there is little direct experimental evidence for these presumed functions. In the present study, we determined the temporal relationship between female paracopulatory behavior, male pursuit of the female, and male copulatory acts. Groups of 4 intact female and 3 male Wistar rats were housed in a seminatural environment for 8 days. Sociosexual behavior during the entire period of behavioral estrus was analyzed. All episodes of paracopulatory behavior were identified, and the male behavior occurring up to 10s before or after each episode was determined. Most paracopulatory behaviors were unrelated to male copulatory acts or male pursuit. To the contrary, 96% of copulatory acts occurred within 5s of female paracopulatory behavior and 83% within 5s of male pursuit of the female. Sexual interaction was initiated by a male as often as it was initiated by a female. The duration of an episode of paracopulatory behavior or male pursuit was an important determinant of the likelihood that a copulatory act would follow. Copulatory acts were a consequence of a subtle interaction between the male and the female, with the behavior of both being equally important. No support was obtained for the notion that sexual interactions are mainly controlled by the female.

Intrasexual competition for access to a female mate is believed to be unusual in wild male rats, which suggests that female choosiness could be important. Even if competition is unusual, males still have to inevitably approach one partner first for copulation. In females, it has been shown that females spend longer time with one male compared to the others when tested in a multiple partner paradigm. The male mate preference was investigated in this study. In addition, the role of ultrasonic vocalizations (USVs) and female odors in the male's initial choice to approach one female instead of another was studied in this experiment. Male rats could choose between three different sexually receptive females. The experiment started with a 15-minute period with inaccessible females followed by a 15-minute period with accessible females in which the males could copulate with the females of his choice. The results showed that male rats spent more time with the female of 1st entry over the...

The role of opioid systems in the anticonflict effect of chlordiazepoxide, diazepam and pentobarbital was evaluated with a modified Vogel procedure. First, morphine, ineffective by itself, was combined with subeffective or marginally effective doses of the benzodiazepines in order to detect possible potentiation. However, the combined treatment reduced licking in the Vogel procedure as well as in a licking test where no shock was administered. Several doses of the benzodiazepines and pentobarbital were then administered in combination with several doses of the opiate antagonist naloxone. A dose-dependent inhibition of anticonflict effect was obtained. In an additional experiment, it was shown that naloxone blocked the effects of diazepam in the elevated plus-maze procedure. Motor deficiencies, as evaluated with a rotarod test, produced by the benzodiazepines and pentobarbital could not be antagonized by naloxone. It is concluded that opioids are important for the anticonflict but not for the motor effects of these drugs. An analysis of published studies concerning the interaction of opioids and benzodiazepines in several procedures supposed to reflect anxiolytic effects shows that the inhibition obtained with naloxone is reliable and not procedure specific. The mechanisms by which opiate antagonists produce this inhibition of anticonflict activity are not known. It is tentatively suggested that opioid activation associated with stress may be a necessary component of anxiolysis.

Female rats were allowed to pace sexual interactions in a bilevel chamber, where a sexually vigorous male was tethered to the bottom level. Exploratory behaviors (sniffing, rearing), locomotor activity (expressed as number of level changes and periods of inactivity) as well as items of sexual motivation (latency to descend to the male's level, approaches towards the male and genital exploration) were recorded. In addition, sexual receptivity was evaluated in a non-paced situation. A test for motor impairment was also performed. The GABA transaminase inhibitor gamma-acetylene GABA reduced exploratory behaviors at doses much lower than those needed to reduce receptivity. The GABA reuptake inhibitor SKF 100330A did not affect any behavior category at doses of 15 and 30 mg/kg, but had a sedative action at 60 mg/kg. This was shown as impaired motor coordination and an almost total absence of activity in the bilevel chamber. Receptivity was not impaired, however. The mixed GABAA/ GABAB agonist progabide reduced exploratory behaviors and receptivity without producing motor impairment at a dose of 400 mg/kg. The GABAA agonist THIP impaired motor coordination and reduced receptivity and exploratory behaviors at a dose of 10 mg/kg. A larger dose, 20 mg/kg, had a strong sedative action. Only a small proportion of the animals descended to the males level. The GABAB agonist baclofen reduced receptivity at a dose that had no effect on motor coordination or exploratory behaviors. None of the drugs had a specific effect on sexual motivation. Whenever behaviors reflecting motivation were reduced, there were also other behavioral effects indicative of sedation. These data show that GABA receptor agonists, particularly the GABAB agonist baclofen, reduce sexual receptivity at doses that have only slight effect on motor functions or exploratory behaviors. In contrast, non-specific enhancement of GABAergic activity by a transaminase or reuptake inhibitor have effects on motor functions and exploratory behaviors at doses much lower than those needed to reduce receptivity.