Sex-specific modulation of cell proliferation by socially relevant stimuli in the adult green treefrog brain (Hyla cinerea).

Social experience plays an important role in regulating the neural, physiological and hormonal changes that accompany the expression of reproductive behavior in vertebrates. This suite of functions is sexually dimorphic, with different neural control areas preeminent in males and females. In anuran amphibians, social experience comes in the form of acoustic communication, which is central to their reproductive behavior. We sought to determine whether acoustic cues regulate cell proliferation in the brain of adult green treefrogs (Hyla cinerea). Our results show that both male and female treefrogs that heard a conspecific chorus during the breeding season exhibited increased brain cell proliferation compared to animals that heard random tones. Increased cell proliferation, as assessed by the number of 5-bromo-2'-deoxyuridine-immunoreactive (BrdU+) cells, were found near the ventricles of acoustically sensitive brain regions such as the preoptic area (POA) and the infundibular hypothalamus (IF). Sex differences emerged in the location of this socially modulated cell proliferation: increases occurred primarily in the male POA and the female IF. In addition, gonadal steroid hormones might have played a role in the social modulation of cell proliferation: by statistically control- ling for hormone level, we revealed that androgens might influence socially induced increases in BrdU+ cells in the male POA, but estrogen did not contribute to socially induced increases in the female IF. These results indicate that the reception of social cues increases cell proliferation in brain regions mediating sexual behavior and endocrine regulation, and moreover that social modulation of cell proliferation occurs in a sexually differentiated fashion.

Kisspeptin-like immunoreactive neuron distribution in the green anole (Anolis carolinensis).

Kisspeptins are a recently identified class of neuropeptides belonging to the RFamide peptide family. Despite growing evidence supporting kisspeptin as a key regulator of reproduction, data addressing whether kisspeptin is a conserved reproductive signal are lacking. We investigated the distribution of kisspeptin in adult green anole lizards (Anolis carolinensis) via immunohistochemistry. Additionally, we examined the possibility of a sexual dimorphism in kisspeptin expression. Kisspeptin immunoreactivity was observed rostrally in the preoptic area and caudally in an area lateral to the dorsal hypothalamic nucleus in both male and female anoles. These kisspeptin immunoreactive cells are associated with vesiculated fibers traveling through the paraventricular zone of the hypothalamus and preoptic area and extending into the rostral telencephalon. Preabsorption of the antiserum with gonadotropin inhibitory hormone (GnIH), a second RFamide peptide known to cross-react with the kisspeptin antiserum, eliminated staining of the caudal population of cells but retained staining in the rostral population, suggesting that kisspeptin is present in this area. Preabsorption with kisspeptin eliminated all immunoreactivity. These preabsorption results suggest that kisspeptin is restricted to a single population in the preoptic area in anoles. No sex differences were found in kisspeptin immunoreactive cell number. The presence of kisspeptin RFamide peptide in the green anole suggests that this reproductive regulatory signal is indeed evolutionarily conserved. Whether this reproductive signal functions similarly in regulating the reproductive axis of ectotherms requires further study.

Sex differences and androgen influences on midbrain auditory thresholds in the green treefrog, Hyla cinerea.

Reproductive hormones can modulate communication-evoked behavior by acting on neural systems associated with motivation; however, recent evidence suggests that modulation occurs at the sensory processing level as well. The anuran auditory midbrain processes communication stimuli, and is sensitive to steroid hormones. Using multiunit electrophysiology, we tested whether sex and circulating testosterone influence auditory sensitivity to pure tones and to the natural vocalization in the green treefrog, Hyla cinerea. Sex did not influence audiogram best frequencies although sexes did differ in the sensitivities at those frequencies with males more sensitive in the lower frequency range. Females were more sensitive than males in response to the natural vocalization, despite showing no difference in response to pure tones at frequencies found within the advertisement call. Thresholds to frequencies outside the range of the male advertisement call were higher in females. Additionally, circulating testosterone increased neural thresholds in females in a frequency-specific manner. These results demonstrate that sex differences are limited to frequency ranges that relate to the processing of natural vocalizations, and depend on the type of stimulus. The frequency-dependent and stimulus-dependent nature of sex and testosterone influences suggests that reproductive hormones influence the filtering properties of the auditory system.

Comparison of arginine vasotocin immunoreactivity differences in dominant and subordinate green anole lizards.

The neuropeptide arginine vasotocin (AVT) and its mammalian homologue arginine vasopressin (AVP) are believed to be involved in many social behaviors including territorial aggression. Testosterone (T) is also important for controlling territorial aggression, and it is believed to be involved in modulating AVT/AVP levels in the brain. In this study, male Anolis carolinensis were paired (n=11 pairs) in a neutral cage and were allowed to establish a dominant-subordinate relationship for 10 days (experimental groups) or housed in a neutral cage with or without a female (control groups; each n=4). On 10th day animals were sacrificed and their brain sections were processed for AVT immunohistochemistry and their serum was analyzed for testosterone levels. AVT immunoreactive (AVT-ir) cell numbers were counted in the anterior hypothalamus (AH), paraventricular nucleus (PN), posterior hypothalamus (PH), preoptic area (POA), and supra optic nuclei (SON). 2-way randomized block design was conducted to assess AVT-ir cell number differences between dominant and subordinate animals and Pearson's correlations were used to determine if a relationship existed between T levels and AVT-ir cell numbers. Dominant animals had more AVT-ir cells in the POA compared to subordinate animals, and subordinate animals had fewer AVT-ir cells in the POA compared to males housed either singly or with a female. There were no differences in AVT-ir cell numbers between dominant and subordinate animals in other areas. T levels were not correlated with the AVT-ir cell numbers in any area. Thus dominant animals have increased AVT-ir cell numbers compared to subordinate animals in a brain region known to be important in male sexual behavior. However, this difference is not related to differences in T.

Social regulation of plasma estradiol concentration in a female anuran.

The behavior of an individual within a social aggregation profoundly influences behavior and physiology of other animals within the aggregation in such a way that these social interactions can enhance reproductive success, survival and fitness. This phenomenon is particularly important during the breeding season when males and female must synchronize their reproductive efforts. We examined whether exposure to conspecific social cues can elevate sex steroid levels, specifically estradiol and androgens, in female túngara frogs (Physalaemus pustulosus). We compared plasma estradiol and androgen concentrations in wild-caught females before and after exposure to either natural mate choruses or random tones. After exposure to mate choruses for 10 consecutive nights, estradiol concentrations were significantly elevated whereas there was no significant elevation in estradiol concentrations in the group of females exposed to random tones for 10 nights. Plasma androgen concentrations were not significantly changed after exposure to either natural mate choruses or random tones for 10 consecutive nights. Social modulation of estradiol concentrations may be important in maintaining a female's reproductive state while males are chorusing. To our knowledge, this is the first study to demonstrate social regulation of estradiol concentration in female anurans.

Social cues shift functional connectivity in the hypothalamus.

We determined how social stimuli that vary in behavioral relevance differentially activate functional networks in the frog hypothalamus. As measured by egr-1 mRNA levels, activity in three hypothalamic nuclei varied with acoustic stimulus, and these responses were correlated with egr-1 responses in different auditory regions regardless of stimulus. The correlations among hypothalamic nuclei, however, varied as a function of the behavioral relevance of the stimuli. Thus relevant social cues shift the functional connectivity within the hypothalamus, consistent with principles that underlie the simultaneous processing of sensory information in cognitive tasks.

Sex differences and hormone influences on tyrosine hydroxylase immunoreactive cells in the leopard frog.

We examined sex differences in tyrosine hydroxylase immunoreactive (TH-ir) cell populations in the preoptic area (POA), suprachiasmatic nucleus (SCN), posterior tuberculum (TP), and caudal hypothalamus (Hy) in the leopard frog (Rana pipiens), in addition to the effects of natural variation in sex steroid hormones on these same populations in both sexes. All four of these populations have been shown to be dopaminergic. Gonadal sex, androgens, and estrogen all influenced TH-ir cell numbers, but in a complicated pattern of interactions. After factoring out the effects of sex steroids by multiple regression, TH-ir cell numbers in all four areas differed between the sexes, with males having a greater number of TH-ir cells. The influence of androgens and estrogen differed by region and sex of the animals. Androgens were the main influence on TH-ir cell numbers in the POA and SCN. Plasma androgen concentrations were positively correlated with TH-ir cell numbers in both areas in males. In females, androgen concentration was negatively correlated with TH-ir cell numbers in the POA; there was no significant relationship in the SCN in females. In the more caudal populations, estrogen (E2) levels were positively correlated with TH-ir cell numbers in the TP of both males and females. In the caudal hypothalamus, E2 levels were positively correlated with TH-ir cell numbers in females, but there was no significant correlation in males. The results indicate that gonadal sex imposes a baseline sex difference in the four TH-ir (dopamine) populations, resulting in a higher number of such cells in males. Individual and sex-linked differences in gonadal steroid hormones lead to variation around this baseline condition, with androgens having a greater influence on rostral populations and estrogen on caudal populations. Last, an individual's gonadal sex determines the effect that androgens and estrogen have on each population.

Androgen effects on tyrosine hydroxylase cells in the northern leopard frog, Rana pipiens.

The interaction between gonadal steroids and dopamine neurons has been examined extensively in rodent model systems. However, there have been few investigations examining the functional relation between gonadal steroids and dopaminergic systems in nonmammalian taxa, and none in amphibians. We examined the effects of testosterone (T) and dihydrotestosterone (DHT) on changes in tyrosine hydroxylase immunoreactive (TH-ir) neuron number in the fore- and midbrain of male Rana pipiens, the Northern leopard frog, using a whole-mount immunohistochemical procedure. Gonadectomized males had significantly fewer TH-ir neurons in the medial preoptic area (POA), suprachiasmatic nucleus (SCN), and the caudal hypothalamus/posterior tubercular region (HY/TP) compared with T-implanted males. A follow-up study demonstrated that T- and DHT-implanted males had similar numbers of TH-ir neurons in these three regions compared with intact males and that all three of these groups possessed significantly more TH-ir neurons compared with gonadectomized males. These results suggest that circulating sex steroids have a significant impact on the activity of dopaminergic neurons in male R. pipiens. Although the function of these specific dopaminergic neurons is not yet known, the POA, SCN, and TP/DH are known to be integral brain regions underlying the neural control of reproductive behavior in frogs. These results suggest that dopamine may be important in controlling the behavior or neuroendocrine mechanisms of reproduction in these animals and that dopaminergic activity in these areas is regulated by gonadal steroids.