Effects of Mating and Social Exposure on Cell Proliferation in the Adult Male Prairie Vole (Microtus ochrogaster).

Microtus ochrogaster is a rodent with a monogamous reproductive strategy characterized by strong pair bond formation after 6 h of mating. Here, we determine whether mating-induced pair bonding increases cell proliferation in the subventricular zone (SVZ), rostral migratory stream (RMS), and dentate gyrus (DG) of the hippocampus in male voles. Males were assigned to one of the four groups: (1) control: males were placed alone in a clean cage; (2) social exposure to a female (SE m/f): males that could see, hear, and smell a sexually receptive female but where physical contact was not possible, because the animals were separated by an acrylic screen with small holes; (3) social exposure to a male (SE m/m): same as group 2 but males were exposed to another male without physical contact; and (4) social cohabitation with mating (SCM): males that mated freely with a receptive female for 6 h. This procedure leads to pair bond formation. Groups 2 and 3 were controls for social interaction. Male prairie voles were injected with 5-bromo-2'-deoxyuridine (BrdU) during the behavioral tests and were sacrificed 48 h later. Brains were processed to identify the new cells (BrdU-positive) and neuron precursor cells (neuroblasts). Our principal findings are that in the dorsal region of the SVZ, SCM and SE m/f and m/m increase the percentage of neuron precursor cells. In the anterior region of the RMS, SE m/f decreases the percentage of neuron precursor cells, and in the medial region SE m/f and m/m decrease the number of new cells and neuron precursor cells. In the infrapyramidal blade of the subgranular zone of the DG, SE m/m and SCM increase the number of new neuron precursor cells and SE m/m increases the percentage of these neurons. Our data suggests that social interaction, as well as sexual stimulation, leads to pair bonding in male voles modulating cell proliferation and differentiation to neuronal precursor cells at the SVZ, RMS, and DG.

Neurogenesis and sexual behavior.

Different conditions induce proliferation, migration and integration of new neurons in the adult brain. This process of neurogenesis is a clear example of long lasting plastic changes in the brain of different species. Sexual behavior is a motivated behavior that is crucial for the survival of the species, but an individual can spend all his life without displaying sexual behavior. In the present review, we briefly describe some of the effects of pheromones on neurogenesis. We review in detail studies describing the effects of sexual behavior in both males and females on proliferation, migration and integration of new cells and neurons. It will become evident that most of the studies have been done in rodents, assessing the effects of this behavior on neurogenesis within the dentate gyrus of the hippocampus and in the subventricular zone - rostral migratory stream - olfactory bulb system.

Mating and social exposure induces an opioid-dependent conditioned place preference in male but not in female prairie voles (Microtus ochrogaster).

In rodents, sexual stimulation induces a positive affective state that is evaluated by the conditioned place preference (CPP) test. Opioids are released during sexual behavior and modulate the rewarding properties of this behavior. Prairie voles (Microtus ochrogaster) are a socially monogamous species, in which copulation with cohabitation for 6h induces a pair bond. However, the mating-induced reward state that could contribute to the establishment of the long-term pair bond has not been evaluated in this species. The present study aimed to determine whether one ejaculation or cohabitation with mating for 6h is rewarding for voles. We also evaluated whether this state is opioid dependent. Our results demonstrate that mating with one ejaculation and social cohabitation with mating for 6h induce a CPP in males, while exposure to a sexually receptive female without mating did not induce CPP. In the female vole, mating until one ejaculation, social cohabitation with mating, or exposure to a male without physical interaction for 6h did not induce CPP. To evaluate whether the rewarding state in males is opioid dependent, the antagonist naloxone was injected i.p. The administration of naloxone blocked the rewarding state induced by one ejaculation and by social cohabitation with mating. Our results demonstrate that in the prairie vole, on the basis of the CPP in the testing conditions used here, the stimulation received with one ejaculation and the mating conditions that lead to pair bonding formation may be rewarding for males, and this reward state is opioid dependent.

Behavioral characterization of non-copulating male mice.

Non-copulating (NC) males are those animals that do not mate in spite of repeated testing with sexually receptive females. They have been observed in several species including rats and mice. The present experiment was designed to perform a detailed behavioral characterization of NC male mice. Thus, we evaluated their sexual incentive motivation for a sexually receptive female or a sexually active male, olfactory preference for volatile and non-volatile odors from females or males, and olfactory discrimination between female and male volatile odors and food related odors (milk versus vinegar). We compared the activity of the accessory olfactory system (AOS) in copulating (C) and NC males in response to estrous bedding using the induction of Fos-immunoreactivity (Fos-IR) as a measure of neuronal activation. We also determined if estradiol or dopamine treatment could induce sexual behavior in NC males. Finally, we compared the testis weight and the number of penile spines in C, NC, and gonadectomized males. In the sexual incentive motivation test C males spend significantly more time in the female incentive zone than in the male incentive zone. On the other hand, NC males spend the same amount of time in both incentive zones. In tests of olfactory preference, NC males spent less time investigating estrous odors than C males. As well, NC males discriminate urine from conspecifics but they spend less time smelling these odors than C males. In addition, no increase in Fos expression is observed in NC males when they are exposed to odors from estrous females. Our data also suggest that the deficits observed in NC males are not due to lower circulating levels of gonadal hormones, because estradiol supplementation does not induce sexual behavior in these animals, and their testis weight and the number of penile spines are normal. The results suggest that NC males are not sexually motivated by the receptive females and their odors.

Sexual experience in female mice involves synaptophysin-related plasticity in the accessory olfactory bulb.

Sexually naïve female mice do not display high levels of sexual receptivity in their first sexual experience; they require around 4-5 sexual encounters to display the full receptive response, assessed by the lordosis reflex. In this study, we evaluated if repeated sexual stimulation with the same male is associated with changes in synaptic remodeling evaluated by synaptophysin (SYP) in brain structures involved in the control of sexual behavior such as the main and accessory olfactory bulbs (MOB and AOB, respectively), medial preoptic area (MPOA), ventromedial hypothalamus (VMH), and amygdala (AMG). Female mice were ovariectomized and hormonally primed to induce sexual receptivity. They were randomly distributed into three groups: a) sexually naïve (SN), with no prior sexual stimulation; b) sexually inexperienced (SI), with one prior mating session; and c) sexually experienced (SE), with six mating sessions. The SI group showed a significant decrease in SYP in the glomerular, mitral and granular layers of the AOB in comparison to SN and SE females. SYP expression increased in the SE group in comparison to SN and SI females in the glomerular and mitral cell layers of the AOB. No significant differences between groups were found in the other brain regions (MOB, MPOA, VMH or AMG). These changes in SYP expression in the AOB suggest that plastic modifications in this brain region can be associated with receptivity increase in sexual experience in female mice.

Sexual experience with a known male modulates c-Fos expression in response to mating and male pheromone exposure in female mice.

Sexually naïve female mice are not sexually receptive in their first mating opportunity. Four to five sexual encounters are needed to display high sexual receptivity as assessed by the lordosis reflex. The neuronal changes induced by sexual experience are not well understood. In this study, we evaluated if repeated sexual stimulation with the same male was associated with an increase in the neuronal activity evaluated by c-Fos expression in brain structures associated with the control of sexual behavior such as the accessory olfactory bulb (AOB), ventromedial hypothalamus (VMH), and the medial preoptic area (MPOA). Ovariectomized female mice were randomly distributed into three groups: sexually naïve (SN), with no prior sexual stimulation; sexually inexperienced (SI), with one prior mating session; and sexually experienced (SE), with six prior mating sessions. Females were primed with estradiol benzoate and progesterone once a week for 7 weeks. Neuronal activation in response to mating or soiled bedding was evaluated in the 7th week. Each group was subdivided into three subgroups: clean (exposure to clean bedding), male bedding (exposure to sawdust soiled with secretions from a male), or mating. Each female mated with her assigned male; in the exposure subgroup, soiled bedding was obtained from the male with whom she mated. Neuronal activity data showed that SE females had a higher c-Fos response in the VMH when they mated in comparison to females exposed to clean bedding. SI females that mated had a decrease c-Fos expression in the glomerular cell layer of the AOB, compared to females exposed to male bedding. The mitral cell layer showed a higher c-Fos response in SI females that mated in comparison to those exposed to male bedding. Comparisons between groups presented with the same stimulus indicate that SI females exposed to male bedding showed a decrease in c-Fos response in the mitral cell layer in comparison to SE and SN females. Correlation analysis demonstrated that the lordosis quotient from the last mating test correlated positively with the number of c-Fos-positive cells in the mitral cell layer in SE and SI groups. A similar correlation was found in the MPOA in SI females. Prior mating in female mice is required to increase sexual receptivity. Changes in the neuronal activity in the AOB and VMH may be involved in the neuronal plasticity induced by repeated sexual stimulation.

The First Mating Experience Induces New Neurons in the Olfactory Bulb in Male Mice.

In rodents, neurogenesis in the olfactory bulbs (OBs) is enhanced by exposure to olfactory enriched environments including sexually relevant odors. In the present study we evaluated whether sexual stimulation in male mice increases the number of newly generated cells that reach the OB and whether these cells differentiate into neurons. To this end, we used sexually naive male C57BL mice randomly assigned to one of three groups: (1) control, in which animals were left alone in their home cages; (2) exposure, in which animals were exposed to a receptive female precluding any physical contact; and (3) mating, in which males copulated with females. Males were given three injections of the DNA synthesis marker 5-bromo-2'-deoxyuridine (BrdU) 2 h before, at the end and 2 h after the test. Fifteen days after BrdU administration, brains were removed and processed to identify new cells and evaluate if they had differentiated into neurons in the granular (GR), mitral (MI) and glomerular (GL) cell layers of the main and accessory OB (MOB and AOB, respectively). We found an increase in the percentage of new cells that differentiate into neurons in the GL cell layer of the MOB of males from the mating group compared with those from the exposure and control groups. No differences were found in the number of new cells or percentage of new neurons in the rest of the analyzed regions. In male mice, the first sexual experience increases the percentage of new cells that differentiate into neurons in the GL cell layer of the MOB.

Neuronal activity of aromatase enzyme in non-copulating male rats.

There are apparently normal male rats that fail to initiate copulation; these animals are called non-copulating (NC) males. Several research groups have demonstrated that conversion of testosterone to oestradiol (aromatisation) in specific brain areas known to be involved in the control of masculine sexual behaviour is fundamental in the control of masculine sexual behaviour. The aim of the present study was to test the hypothesis that the concentration of aromatase activity (AA) in the brain is lower in NC males than in copulating males (C). We quantified AA in several brain nuclei and also evaluated whether NC rats have altered concentrations of testosterone in their plasma. We found that AA was reduced in the medial preoptic nuclei (MPN) of NC male rats vs C males. In addition, NC and C male rats had similar plasma levels of testosterone. These data suggest that reduced levels of AA in the MPN could be a crucial factor associated with lack of male coital behaviour in rats.

Comparative analysis of immunoreactive cells for androgen receptors and oestrogen receptor alpha in copulating and non-copulating male rats.

In some species, including gerbils, guinea pigs, mice, rams and rats, some apparently normal males fail to mate. These kinds of animals have been named 'noncopulating (NC)'. The cause of this behavioural deficit is unknown. The present study aimed to determine whether NC male rats have alterations in the amount of androgen (AR) and oestrogen receptor alpha (ERalpha) in a neuronal circuit important for the control of male sexual behaviour; the vomeronasal projection pathway. We evaluated the number of AR and ERalpha immunoreactive (AR-IR and ERalpha-IR) cells in the accessory olfactory bulb (AOB), the bed nucleus of the stria terminalis (BNST), the anterior-dorsal medial amygdala (MeAD), the posterior dorsal amygdala (MePD) and the medial preoptic area (MPOA). The results demonstrate that the number of AR-IR cells in NC males was significantly higher compared to copulating (C) males in the MePD, but no significant differences were found in any of the other structures analysed. ERalpha-IR cells were more abundant in NC than in C males in the MeAD and the MePD. However, in the MPOA the number of ERalpha-IR cells was significantly reduced in NC males. No significant differences were found in the AOB or in the BNST. A similar pattern of results was observed when regions within these structures that are activated by Fos expression, on mating or exposure to sexually relevant cues were analysed. The differences in the number of AR and ER in particular brain areas could be associated with alterations in sexual behaviour as well as partner and olfactory preference for receptive females seen in NC male rats.

Deficits in odor-guided behaviors in the transgenic 3xTg-AD female mouse model of Alzheimer׳s disease.

Alzheimer׳s disease (AD) is characterized by a number of alterations including those in cognition and olfaction. An early symptom of AD is decreased olfactory ability, which may affect odor-guided behaviors. To test this possibility we evaluated alterations in sexual incentive motivation, sexual olfactory preference, sexual olfactory discrimination, nursing-relevant olfactory preference and olfactory discrimination in female mice. We tested 3xTg-AD (a triple transgenic model, which is a "knock in" of PS1M146V, APPSwe, and tauP300L) and wild type (WT) female mice when receptive (estrous) and non-receptive (anestrous). Subjects were divided into three groups of different ages: (1) 4-5 months, (2) 10-11 months, and (3) 16-18 months. In the sexual incentive motivation task, the receptive 3xTg-AD females showed no preference for a sexually active male at any age studied, in contrast to the WT females. In the sexual olfactory preference test, the receptive WT females were able to identify sexually active male secretions at all ages, but the oldest (16-18 months old) 3xTg-AD females could not. In addition, the oldest 3xTg-AD females showed no preference for nursing-relevant odors in dam secretions and were unable to discriminate between cinnamon and strawberry odors, indicating olfactory alterations. Thus, the present study suggests that the olfactory deficits in this mouse model are associated with changes in sexual incentive motivation and discrimination of food-related odors.

Repeated paced mating promotes the arrival of more newborn neurons in the main and accessory olfactory bulbs of adult female rats.

We have previously shown that the first-paced mating encounter increases the number of newborn cells in the granule cell layer (Gra; also known as internal cell layer, ICL) of the accessory olfactory bulb (AOB) in the adult female rat (Corona et al., 2011). In the present study we evaluated if repetition of the stimulus (paced mating) could increase the arrival of more newborn neurons in the olfactory bulb generated during the first session of paced sexual contact. Sexually naive female rats were bilaterally ovariectomized, hormonally supplemented with estradiol (E2) and progesterone (P) and randomly assigned to one of four groups: (1) without sexual contact, (2) one session of paced mating, (3) four sessions of paced mating, and (4) four sessions of non-paced mating. We also included a group of gonadally intact females. On the first day of the experiment, all females were i.p. injected with the marker of DNA synthesis bromodeoxyuridine and were killed 16 days later. Blood was collected at sacrifice to determine the plasma levels of E2 and P. The number of newborn neurons that arrived at the ICL of the AOB and the Gra of the main olfactory bulb (MOB) increased, relative to all other groups, only in the group that repeatedly mated under pacing conditions. No differences were found in E2 and P levels between supplemented groups indicating that our results are not influenced by changes in hormone concentrations. We suggest that repeated paced mating promotes the arrival of more newborn neurons in the AOB and MOB.

Steroid hormone action in the brain: cross-talk between signalling pathways.

Ovarian steroid hormones, oestradiol and progesterone, modulate neuroendocrine functions in the central nervous system, resulting in alterations in physiology and behaviour. The classical model of steroid hormone action assumes that these neural effects are predominantly mediated via their intracellular receptors functioning as 'ligand-dependent' transcription factors in the steroid-sensitive neurones regulating genes and genomic networks with profound behavioural consequences. Studies from our laboratory demonstrate that, in addition to their cognate ligands, intracellular steroid receptors can be activated in a 'ligand-independent' manner by the neurotransmitter dopamine, which alters the dynamic equilibrium between neuronal phosphatases and kinases. A high degree of cross-talk between membrane-initiated signalling pathways and the classical intracellular signalling pathways mediates hormone-dependent behaviour in mammals. The molecular mechanisms, by which a multitude of signals converge with steroid receptors to delineate a genomic level of cross-talk in brain and behaviour are discussed.