Ana C. Ribeiro

Concepts and mechanisms of generalized central nervous system arousal.

A concept of generalized arousal of the CNS is presented and given an operational definition that leads to quantitative physical measures. Because this primitive arousal function underlies all motivated behavioral responses, cognitive functions, and emotional expression, disorders of generalized arousal can be associated with a large number of problems in medicine and public health, including vegetative states, attentional disorders, depression, occupational hazards, and problems with sleep and anesthesia. Some of its known mechanisms are briefly reviewed, at the levels of neuroanatomy, neurophysiology, and functional genomics. Generalized arousal contributes to the excitement and the activation of behaviors during specific arousal states. Data are summarized for four genomic/neurochemical systems through which changes in generalized arousal could affect sexual arousal, two of which heighten, and the other two of which reduce arousal.

The role of the estrogen receptor α 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.

Relationships among estrogen receptor, oxytocin and vasopressin gene expression and social interaction in male mice.

The incidence of social disorders such as autism and schizophrenia is significantly higher in males, and the presentation more severe, than in females. This suggests the possible contribution of sex hormones to the development of these psychiatric disorders. There is also evidence that these disorders are highly heritable. To contribute toward our understanding of the mechanisms underlying social behaviors, particularly social interaction, we assessed the relationship of social interaction with gene expression for two neuropeptides, oxytocin (OT) and arginine vasopressin (AVP), using adult male mice. Social interaction was positively correlated with: oxytocin receptor (OTR) and vasopressin receptor (V1aR) mRNA expression in the medial amygdala; and OT and AVP mRNA expression in the paraventricular nucleus of the hypothalamus (PVN). When mice representing extremes of social interaction were compared, all of these mRNAs were more highly expressed in high social interaction mice than in low social interaction mice. OTR and V1aR mRNAs were highly correlated with estrogen receptor α (ERα) mRNA in the medial amygdala, and OT and AVP mRNAs with estrogen receptor β (ERβ) mRNA in the PVN, indicating that OT and AVP systems are tightly regulated by estrogen receptors. A significant difference in the level of ERα mRNA in the medial amygdala between high and low social interaction mice was also observed. These results support the hypothesis that variations of estrogen receptor levels are associated with differences in social interaction through the OT and AVP systems, by upregulating gene expression for those peptides and their receptors.

Contrasting Effects of Leptin on Food Anticipatory and Total Locomotor Activity

Obese, leptin deficient obob mice have profoundly decreased activity and increased food seeking behavior. The decreased activity has been attributed to obesity. In mice, we tested the hypothesis that leptin increases total locomotor activity but inhibits food anticipatory activity. We also sought to determine if leptin induced increases in total locomotor activity are independent of changes in body weight and obesity. We studied obob mice and also created a novel transgenic mouse where leptin is over-expressed in a tetracycline-off system and can be abruptly and non-invasively suppressed by doxycycline within few hours. The studies were performed using two independent behavioral assays: home cage activity (HCA) and running wheel activity (RWA). Systemic administration of leptin (150 ng/hr) to obob mice produced a 122%±30% (mean ± SEM) increase (p≤0.01) in locomotor activity within 2 days In addition, cerebroventricular administration of leptin (5 ng/hr) also produced an early and progressive increase in total locomotor activity beginning on the 1st day (+28±8%; p≤0.05) and increasing to +69±23% on day 3 without a decrease in body weight during this time. The increase in activity was restricted to the dark phase. Conversely, in a tet-off transgenic obob mouse line, acute leptin suppression reduced spontaneous locomotor activity. To further define activities that are leptin regulated, we assayed food anticipatory activity (FAA) and found that it was markedly augmented in obob mice compared to wild type mice (+38±6.7 in obob vs +20±6.3% in wild type at peak; mean ± SEM; p≤0.001) and abolished by leptin. Although melanocortin-3 receptors (MC3R) reportedly mediate FAA, we found augmented FAA and preserved inhibitory effects of leptin on FAA in MC3R−/−obob mice. In summary, this study demonstrates that total activity and FAA are regulated independently by leptin. Leptin, acting in the central nervous system and at physiologic levels, produces early increases in locomotor activity before substantial weight loss. In contrast, leptin suppresses augmented food anticipatory activity in obob mice.

Estrogen-Induced Sexual Incentive Motivation, Proceptivity and Receptivity Depend on a Functional Estrogen Receptor α 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 α (ERα) 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 ERα 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 ERα in the MePDA lacked these effects. Likewise, the inactive control AAV vector failed to modify any behavior. Thus, the ERα 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 ERα 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.

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.

Estradiol modulates behavioral arousal and induces changes in gene expression profiles in brain regions involved in the control of vigilance.

Estrogens reduce lipocalin‐type prostaglandin D synthase (L‐PGDS) expression in a region‐dependent manner in the mouse preoptic area (POA). This result linked sex hormones with sleep–wake cycle regulation. In turn, the somnogenic effects of prostaglandin D2 have been shown to be mediated via increases in adenosine, and a select group of sleep‐active ventrolateral preoptic nucleus (VLPO) neurons are directly activated by adenosine 2A (A2A) agonists. We hypothesized that increased arousal after estrogen administration is mediated by a reduction of L‐PGDS and lowered A2A receptor expression in the POA. To test this hypothesis, running wheel activity (RWA) of ovariectomized female mice treated with oil or different doses of estradiol benzoate (EB) was studied, followed by quantitative reverse‐transcriptase polymerase chain reaction to determine mRNA expression of genes related to sleep and arousal in brain region extracts from oil‐treated and EB‐treated mice. RWA was increased in estrogen‐treated mice, and these effects followed an inverted‐U dose–response curve. The most effective dose (1.25 μg EB/capsule) increased RWA more than 2.5‐fold, as compared with control animals, and EB doses that were higher or lower were less effective. Increases in RWA were accompanied by decreased L‐PGDS mRNA in the POA and decreased A2A receptor mRNA in the POA and VLPO. Given that EB‐treated animals have higher motor activity and lower levels of L‐PGDS and A2A receptor mRNAs in sleep‐active areas, these correlational findings support the hypothesis that EB may increase behavioral arousal by decreasing the levels of well‐known sleep‐inducing molecules within the preoptic region.

Relations between mechanisms of CNS arousal and mechanisms of stress

In the centennial year of the birth of Hans Selye, this review compares his classical concepts of stress with a modern approach to mechanisms of CNS arousal. Relations between the two concepts are described. Neuroanatomical, neurophysiological, and functional genomic mechanisms underlying CNS arousal are briefly reviewed. Controls over stress responses and arousal are compared to particular concepts of control system engineering. Understanding these two systems is of crucial importance because their dysregulation is associated with large numbers of disease states.

Two forces for arousal: Pitting hunger versus circadian influences and identifying neurons responsible for changes in behavioral arousal

The mechanisms underlying CNS arousal in response to homeostatic pressures are not known. In this study, we pitted two forces for CNS arousal against each other (circadian influences vs. restricted food availability) and measured the neuronal activation that occurs in a behaviorally defined group of animals that exhibited increased arousal in anticipation of feeding restricted to their normal sleeping time. The number of c-FOS+ neurons was significantly increased only in the ventromedial nucleus of the hypothalamus (VMH) in these mice, compared with control animals whose feeding was restricted to their normal active and feeding time (P < 0.01). Because the activation of VMH neurons coincides with the earliest signs of behavioral arousal preceding a change in meal time, we infer that VMH activation is involved in the increased arousal in anticipation of food.

Genetic Mechanisms in Neural and Hormonal Controls over Female Reproductive Behaviors

Building on well-established mechanisms that produce the primary female mating behavior, lordosis, research is extending into mechanisms for sexual arousal. Genes and neurochemcal pathways supporting sexual arousal are reviewed, and four neurochemical/biophysical routes by which generalized arousal could influence sexual arousal are charted.