Early postnatal hypothyroidism reduces juvenile play behavior, but prenatal hypothyroidism compensates for these effects.

Perinatal hypothyroidism causes long-lasting effects on behavior, including hyperactivity, cognitive delays/deficits, and a reduction in anxiety. Although there is some evidence that hypothyroidism during fetal development in humans has been associated with later autism spectrum disorder diagnosis or autism-like traits, the relationships between early thyroid hormones and social behaviors are largely unknown. Previously, we found that a moderate dose of the hypothyroid-inducing drug methimazole during embryonic and postnatal development dramatically increased juvenile play in male and female rats. The goal of the current study was to determine the extent to which thyroid hormones act in prenatal or postnatal development to organize later social behaviors. Subjects were exposed to methimazole in the drinking water during prenatal (embryonic day 12 to birth), postnatal (birth to postnatal day 23), or pre- and postnatal development; control animals received regular drinking water throughout the experiment. They were tested for play behavior as juveniles (P30-32). We found an interaction between pre- and postnatal methimazole administration such that postnatal hypothyroidism decreased some play behaviors, whereas sustained pre- and postnatal hypothyroidism restored play to control levels. The effects were similar in males and females. To our knowledge, this is the first report of an interaction between pre- and postnatal hypothyroidism on later behavior. The complexity of the timing of these effects may help explain why epidemiological studies have not consistently found a relationship between gestational hypothyroidism and later behavior.

Cooperative Group Learning in Undergraduate Neuroscience: Using Simulations to Complement Problem-Solving Assignments.

The incorporation of active learning improves student learning and persistence compared to traditional lecture-based teaching. However, there are numerous active learning strategies and the degree to which each one enhances learning relative to other techniques is largely unknown. I analyzed the effectiveness of the addition of simulations to cooperative group problem-solving assignments in an undergraduate 400-level neurobiology course. One section of the course carried out group problem-solving alone, whereas the other section used neuroscience simulations (Neuronify) as part of the problem-solving assignments. Overall, both groups of students learned course concepts effectively and did not differ in their performance on exams or specific exam questions related to the assignments. Students perceived that the assignments and simulations were helpful in their understanding of course material but did not overwhelmingly recommend including simulations in the future. Students using simulations were more likely to report gaining experience with experimental design, and this may be an effective way to build scientific reasoning in non-laboratory courses. However, student frustration with technology was the primary reason that students reported dissatisfaction with the simulations. Overall, cooperative group problem-solving with or without simulations is very effective at helping students learn neuroscience concepts.

Sex differences in juvenile play behavior differ among rat strains.

Juvenile male rats frequently play more than female rats, but the presence of sex differences is affected by testing conditions and may also depend on the strain of rat. In this experiment, we tested play and defensive behaviors in male and female Long-Evans, Sprague-Dawley, and Wistar rats. When observed with a cage mate during the juvenile period, Long-Evans rats played more than Wistar animals, but there were no sex differences in any strain. When tested with an unfamiliar sibling (not seen since weaning), both Long-Evans and Wistar rats played more than Sprague-Dawley animals, and Long-Evans females played more than males. We did not observe any sex or strain differences in defensive behaviors. Our data indicate that there are strain differences in play behavior, and sex differences in play depend on both strain and context. Variation among strains may reflect underlying differences in anxiety, novelty seeking, and circadian rhythms.

Perinatal hypothyroidism increases play behaviors in juvenile rats.

Thyroid hormones play an instrumental role in the development of the central nervous system. During early development, the fetus is dependent on maternal thyroid hormone production due to the dysfunction of its own thyroid gland. Thus, maternal thyroid dysfunction has been shown to elicit significant abnormalities in neural development, neurochemistry, and behavior in offspring. Previous reports have suggested that human maternal hypothyroidism may increase the chances of having children with autism spectrum disorder and attention-deficit/hyperactivity disorder. However, very few studies have evaluated social behaviors in animal models of perinatal hypothyroidism. To evaluate the possibility that hypothyroidism during development influences the expression one of the most commonly observed non-reproductive social behaviors, juvenile play, we used the validated rat model of perinatal hypothyroidism by methimazole administration (MMI; 0.025% in drinking water) from GD12-PD23. Control animals had regular drinking water. During adolescence (PD33-35), we tested subjects for juvenile play behavior by introducing them to a same-sex, unfamiliar (since weaning) littermate for 30min. Play behaviors and other behaviors (sleep, social contact, locomotion) were then scored. MMI-treated subjects played more than twice as much as control animals, and the increase in some behaviors was particularly dramatic in males. Locomotor and other affiliative social behaviors were unaffected. These data suggest that perinatal hypothyroidism may alter the organization of the neural networks regulating play behaviors, but not other social behaviors. Moreover, this implicates perinatal hypothyroidism as a potential etiological factor in the development of neurobehavioral disorders, particularly those characterized by heightened social interactions and impulsivity.

Student-Designed Service-Learning Projects in an Undergraduate Neurobiology Course.

One of the challenges in teaching a service-learning course is obtaining student buy-in from all students in the course. To circumvent this problem, I have let students in my undergraduate Neurobiology course design their own service-learning projects at the beginning of the semester. Although this can be chaotic because it requires last-minute planning, I have made it successful through facilitating student communication in the classroom, requiring thorough project proposals, meeting with students regularly, and monitoring group progress through written reflection papers. Most of my students have strong opinions about the types of projects that they want to carry out, and many students have used connections that they have already made with local organizations. Almost all projects that students have designed to this point involve teaching basic concepts of neurobiology to children of various ages while simultaneously sparking their interest in science. Through taking ownership of the project and designing it such that it works well with their strengths, interests, and weekly schedule, students have become more engaged in service learning and view it as a valuable experience. Despite some class time being shifted away from more traditional assignments, students have performed equally well in the course, and they are more eager to talk with others about course concepts. Furthermore, the feedback that I have received from community partners has been excellent, and some students have maintained their work with the organizations.

Female juvenile play elicits Fos expression in dopaminergic neurons of the VTA.

Juveniles of many species engage in rough-and-tumble play behaviors, and these social encounters are important for the expression of typical social behaviors. Play is a highly motivated and rewarding behavior, which suggests that the mesocorticolimbic dopamine system is likely important for reinforcing the behavior. Indeed, systemic dopamine receptor antagonists decrease the expression of play behaviors, but the specific dopaminergic networks important for play are not known. In this study, we examined immediate-early gene expression in specific dopaminergic cell groups after juvenile male and female rats played or did not play. Subjects were housed with a same-sex sibling, and spontaneous play behavior (or lack thereof) was observed for 1 hr. Brains were harvested and immunohistochemistry was used to localize Fos and tyrosine hydroxylase. Cells expressing both proteins were counted in midbrain and forebrain dopaminergic cell groups. Females that played had more double-labeled cells in the ventral tegmental area (VTA) than females that did not play, but there was no effect of play on double-labeled cell counts in any brain region in males. Furthermore, many measures of play in females were positively correlated with the number of double-labeled cells in the VTA, including play duration and pin duration. Our results suggest that play in females likely induces dopamine release from mesocorticolimbic neurons to reinforce play behaviors. Our results also highlight a sex difference in the neural networks mediating play, thus emphasizing the importance of studying the neurobiology of play in both males and females.

Progesterone receptor expression in the brain of the socially monogamous and paternal male prairie vole.

Differences in the social organization and behavior of male mammals are attributable to species differences in neurochemistry, including differential expression of steroid hormone receptors. However, the distribution of progestin receptors (PR) in a socially monogamous and spontaneously parental male rodent has never been examined. Here we determined if PR exists and is regulated by testicular hormones in forebrain sites traditionally influencing socioreproductive behaviors in male prairie voles (Microtus ochrogaster). We hypothesized that PR expression in male prairie voles would differ from that described in other male rodents because PR activity inhibits parental behaviors and social memory in laboratory mice and rats. Adult male prairie voles received a sham surgery, were gonadectomized, or were gonadectomized and implanted with a testosterone-filled capsule. PR immunoreactivity (PRir) was measured four weeks later in areas of the hypothalamus and extended amygdala. A group of gonadally intact female prairie voles was included to reveal possible sex differences. We found considerable PRir in all sites examined. Castration reduced PRir in males' medial preoptic nucleus, anteroventral periventricular nucleus, ventromedial hypothalamus, and posterodorsal medial amygdala, and it was maintained in these sites by testosterone. This is the first study to examine PR expression in brain sites involved in socioreproductive behaviors in a socially monogamous and spontaneously paternal male rodent. Our results mostly reveal cross-species conservation in the distribution and hormone sensitivity of PR expression. Because PR interferes with aspects of sociality in other male rodents, PR may eventually be found to have different neurobiological actions in male prairie voles.

L-amino acid decarboxylase- and tyrosine hydroxylase-immunoreactive cells in the extended olfactory amygdala and elsewhere in the adult prairie vole brain.

Neurons synthesizing dopamine (DA) are widely distributed in the brain and implicated in a tremendous number of physiological and behavioral functions, including socioreproductive behaviors in rodents. We have recently been investigating the possible involvement of sex- and species-specific TH-immunoreactive (TH-ir) cells in the male prairie vole (Microtus ochrogaster) principal bed nucleus of the stria terminalis (pBST) and posterodorsal medial amygdala (MeApd) in the chemosensory control of their monogamous pairbonding and parenting behaviors. These TH-ir cells are not immunoreactive for dopamine-beta-hydroxylase (DBH), suggesting they are not noradrenergic but possibly DAergic. A DAergic phenotype would require them to contain aromatic L-amino acid decarboxylase (AADC) and here we examined the existence of cells immunoreactive for both TH and AADC in the pBST and MeApd of adult virgin male and female prairie voles. We also investigated the presence of TH/AADC cells in the anteroventral periventricular nucleus (AVPV), medial preoptic area (MPO), arcuate nucleus (ARH), zona incerta (ZI), substantia nigra (SN) and ventral tegmental area (VTA). Among our findings were: (1) the pBST and MeApd each contained completely non-overlapping distributions of TH-ir and AADC-ir cells, (2) the AVPV contained surprisingly few AADC-ir cells and almost no TH-ir cells contained AADC-ir, (3) approximately 60% of the TH-ir cells in the MPO, ARH, and ZI also contained AADC-ir, (4) unexpectedly, only about half of TH-ir cells in the SN and VTA contained AADC-ir, and (5) notable populations of AADC-ir cells were found outside traditional monoamine-synthesizing regions, including some sites that do not contain AADC-ir cells in adult laboratory rats or cats (medial septum and cerebral cortex). In the absence of the chemical requirements to produce DA, monoenzymatic TH-ir cells in the virgin adult prairie vole pBST, MeApd, and elsewhere in their brain may instead produce L-DOPA as an end product and use it as a neurotransmitter or neuromodulator, similar to what has been observed for monoenzymatic TH-synthesizing cells in the laboratory rat brain.

Neuroanatomical projections of the species-specific tyrosine hydroxylase-immunoreactive cells of the male prairie vole bed nucleus of the stria terminalis and medial amygdala.

The principal nucleus of the bed nucleus of the stria terminalis (BSTpr) and posterodorsal part of the medial amygdalar nucleus (MEApd) are densely interconnected sites transmitting olfactory information to brain areas mediating sociosexual behaviors. In male prairie voles (Microtus ochrogaster), the BSTpr and MEApd contain hundreds of cells densely immunoreactive for tyrosine hydroxylase (TH). Such tremendous numbers of TH-immunoreactive (TH-ir) cells do not exist in other rodents examined, and studies from our laboratory suggest these cells may be part of a unique chemical network necessary for monogamous behaviors in prairie voles. To obtain information about how these TH-ir cells communicate with other sites involved in social behaviors, we first used biotinylated dextran amine (BDA) to determine sites that receive BSTpr efferents and also contain TH-ir fibers. Only in the medial preoptic area (MPO) and MEApd did we find considerable comingling of BDA-containing and TH-ir fibers. To examine if these sites receive input specifically from BSTpr TH-ir cells, the retrograde tracer Fluorogold was infused into the MPO or MEApd. Almost 80% of TH-ir projections to the MPO originated from the BSTpr or MEApd, involving about 40% of all TH-ir cells in these sites. In contrast, the MEApd received almost no input from TH-ir cells in the BSTpr, and received it primarily from the ventral tegmental area. Retrograde tracing from the BSTpr itself revealed substantial input from MEApd TH-ir cells. Thus, the male prairie vole brain contains a species-specific TH-ir network involving the BSTpr, MEApd, and MPO. By connecting brain sites involved in olfaction, sociality and motivation, this network may be essential for monogamous behaviors in this species.

Progesterone and medroxyprogesterone acetate differentially regulate alpha4 subunit expression of GABA(A) receptors in the CA1 hippocampus of female rats.

The Women's Health Initiative trials - in which more extreme adverse outcomes were observed in the medroxyprogesterone acetate (MPA) + conjugated equine estrogen (CEE) arm, as compared to the CEE only arm - suggest that the addition of MPA to estrogen treatment has undesirable consequences. An important question raised by these results is whether the adverse outcomes observed in the progestin arm can be attributed to effects that are unique to MPA or are common to all progestins. In this study we explored the potential for MPA and progesterone (P4) to differentially impact neuroendocrine function by comparing their effects on mRNA expression for the alpha4 subunit of GABA(a) receptors in the CA1 hippocampus of female rats. Prior research has shown that P4, acting through its reduced metabolite allopregnanolone (AP), can mediate alpha4 subunit expression, thereby altering GABA(A) receptor gated currents. By contrast, MPA competitively inhibits the enzymes necessary for the synthesis of AP. In this study, ovariectomized females were primed with estradiol benzoate and then treated with P4, MPA, or vehicle. Subjects were sacrificed 12 h or 24 h later and in situ hybridization was used to measure alpha4 mRNA in the CA1 hippocampus. At 12 h but not 24 h, alpha4 mRNA was reduced in the P4 group as compared to the MPA group, and as compared to the vehicle group. These results suggest that MPA, while progestational in terms of its effects in the uterus, is not a simple substitute for P4 in other systems. The relative impact of these two progestins on neuroendocrine function must be carefully explored.

Sex differences and effects of neonatal aromatase inhibition on masculine and feminine copulatory potentials in prairie voles.

Copulatory behaviors in most rodents are highly sexually dimorphic, even when circulating hormones are equated between the sexes. Prairie voles (Microtus ochrogaster) are monomorphic in their display of some social behaviors, including partner preferences and parenting, but differences between the sexes in their masculine and feminine copulatory behavior potentials have not been studied in detail. Furthermore, the role of neonatal aromatization of testosterone to estradiol on the development of prairie vole sexual behavior potentials or their brain is unknown. To address these issues, prairie vole pups were injected daily for the first week after birth with 0.5 mg of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) or oil. Masculine and feminine copulatory behaviors in response to testosterone or estradiol were later examined in both sexes. Males and females showed high mounting and thrusting in response to testosterone, but only males reliably showed ejaculatory behavior. Conversely, males never showed feminine copulatory behaviors in response to estradiol. Sex differences in these behaviors were not affected by neonatal ATD, but ATD-treated females received fewer mounts and thrusts than controls, possibly indicating reduced attractiveness to males. In other groups of subjects, neonatal ATD demasculinized males' tyrosine hydroxylase expression in the anteroventral periventricular preoptic area, and estrogen receptor alpha expression in the medial preoptic area. Thus, although sexual behavior in both sexes of prairie voles is highly masculinized, aromatase during neonatal life is necessary only for females' femininity. Furthermore, copulatory behavior potentials and at least some aspects of brain development in male prairie voles are dissociable by their requirement for neonatal aromatase.

Sexual dimorphism and steroid responsiveness of the posterodorsal medial amygdala in adult mice.

The posterodorsal aspect of the medial amygdala (MePD) is sexually dimorphic in regional volume, rostrocaudal extent, and neuronal soma size in rats. These dimorphisms are maintained by circulating gonadal hormones, as castration of adult male rats reduces MePD measures, while testosterone treatment of females increases them. We now report that the MePD is also sexually dimorphic in volume, rostrocaudal extent, and somal area in BALB/c mice. Four weeks after castration of adult male mice, MePD regional volume and soma size are reduced, but rostrocaudal extent is not, compared to sham-castrated males. Treatment of adult ovariectomized females with an aromatized metabolite of testosterone, estradiol, for 8 weeks increased MePD volume and soma size, but not rostrocaudal extent. To probe the possible role of afferents in the steroid-induced plasticity of the MePD, we examined the effect of removing the olfactory bulbs in gonadally intact males and in estrogen-treated females. Bulbectomy had no effect on MePD morphology with one exception: among gonadally intact males, neuronal soma size was slightly smaller in the right MePD of bulbectomized males compared to males with intact bulbs. These results indicate that the sexual dimorphism and hormone responsiveness of the MePD that has been extensively studied in rats is also present in mice, which offers genetic tools for future research. We detected little or no evidence that olfactory bulb afferents play a role in maintaining MePD morphology in adult mice.

Sex and species differences in tyrosine hydroxylase-synthesizing cells of the rodent olfactory extended amygdala.

The bed nucleus of the stria terminalis (BST) and the medial amygdala (MeA) are anatomically connected sites necessary for chemosensory regulation of social behaviors in rodents. Prairie voles (Microtus ochrogaster) are a valuable model for studying the neural regulation of social behaviors because, unlike many other rodents, they are gregarious, pair bond after copulating, and are biparental. We herein describe sex and species differences in immunoreactivity for tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine synthesis, in the BST and MeA. Virgin male prairie voles had a large number of TH-immunoreactive cells in areas analogous to the rat principal nucleus of the BST (pBST) and the posterodorsal medial amygdala (MeAPd). Virgin female prairie voles had far fewer TH-immunoreactive cells in these sites ( approximately 17% of the number of cells as males in the pBST, approximately 35% of the number of cells in the MeAPd). A few TH-immunoreactive cells were found in the BST of male and female hamsters and meadow voles, but not in rats. The MeApd also contained a few TH-immunoreactive cells in male and female hamsters and male meadow voles, but not rats. Castration greatly reduced the number of TH-immunoreactive cells in the male prairie vole pBST and MeAPd, an effect that could be reversed with testosterone. Furthermore, treating ovariectomized females with testosterone substantially increased TH-immunoreactive cells in both sites. Therefore, a species-specific sex difference in TH expression is found in a chemosensory pathway in prairie voles. Expression of TH in these sites is influenced by circulating gonadal hormones in adults, which may be related to changes in their display of social behaviors across the reproductive cycle.

Medroxyprogesterone acetate acutely facilitates and sequentially inhibits sexual behavior in female rats.

Medroxyprogesterone acetate (MPA), a synthetic progestin commonly used in contraception and hormone replacement therapy, appears to inhibit libido in women, but little is known about the mechanisms through which it may exert this effect. We compared the acute and sequential actions of MPA and natural progesterone (P4) on sexual behavior in female rats to test the hypothesis that MPA inhibits sexual behavior, at least in part, by acting as a potent progesterone receptor (PR) agonist. Ovariectomized females were placed in one of three dose groups (high, mid, or low), and each subject was tested under three different conditions (MPA, P4, and vehicle). The order of progestin treatment was balanced among subjects, and within each dose group equimolar quantities of MPA and P4 were administered. During each trial, females were injected with estradiol benzoate (EB, 4 mug) followed by one of three progestin treatments (MPA, P4, or vehicle) at +44 h, and behavioral testing at +48 h. On the next day, all females were given a standard 500-microg injection of P4 at +68 h and were tested again for sexual behavior at +72 h. On the first day of behavioral testing, both MPA and P4 induced a pronounced rise in receptive and proceptive behavior at the mid and high doses, but at the lowest dose MPA had a much greater effect in comparison to P4. On the second day of behavioral testing, MPA attenuated the expression of proceptive and receptive behavior at both the mid and high doses, whereas P4 only attenuated the expression of lordosis and only did so at the highest dose. These findings illustrate that MPA and P4 have a similar impact on sexual behavior in female rats and suggest that the inhibitory effects of MPA may be attributable, at least in part, to its potent effects at the progesterone receptor.