Rachel E. Cohen

Extensive early motor and non-motor behavioral deficits are followed by striatal neuronal loss in knock-in Huntington's disease mice.

Huntingtons disease is a neurodegenerative disorder, caused by an elongation of CAG repeats in the huntingtin gene. Mice with an insertion of an expanded polyglutamine repeat in the mouse huntingtin gene (knock-in mice) most closely model the disease because the mutation is expressed in the proper genomic and protein context. However, few knock-in mouse lines have been extensively characterized and available data suggest marked differences in the extent and time course of their behavioral and pathological phenotype. We have previously described behavioral anomalies in the open field as early as 1 month of age, followed by the appearance at 2 months of progressive huntingtin neuropathology, in a mouse carrying a portion of human exon 1 with approximately 140 CAG repeats inserted into the mouse huntingtin gene. Here we extend these observations by showing that early behavioral anomalies exist in a wide range of motor (climbing, vertical pole, rotarod, and running wheel performance) and non-motor functions (fear conditioning and anxiety) starting at 1-4 months of age, and are followed by progressive gliosis and decrease in dopamine and cyclic AMP-regulated phosphoprotein with molecular weight 32 kDa (DARPP32) (12 months) and a loss of striatal neurons at 2 years. At this age, mice also present striking spontaneous behavioral deficits in their home cage. The data show that this line of knock-in mice reproduces canonical characteristics of Huntingtons disease, preceded by deficits which may correspond to the protracted pre-manifest phase of the disease in humans. Accordingly, they provide a useful model to elucidate early mechanisms of pathophysiology and the progression to overt neurodegeneration.

Repeated Exposure to Methamphetamine Causes Long-Lasting Presynaptic Corticostriatal Depression that Is Renormalized with Drug Readministration

Addiction-associated behaviors such as drug craving and relapse are hypothesized to result from synaptic changes that persist long after withdrawal and are renormalized by drug reinstatement, although such chronic synaptic effects have not been identified. We report that exposure to the dopamine releaser methamphetamine for 10 days elicits a long-lasting (>4 month) depression at corticostriatal terminals that is reversed by methamphetamine readministration. Both methamphetamine-induced chronic presynaptic depression and the drugs selective renormalization in drug-experienced animals are independent of corresponding long-term changes in synaptic dopamine release but are due to alterations in D1 dopamine and cholinergic receptor systems. These mechanisms might provide a synaptic basis that underlies addiction and habit learning and their long-term maintenance.

Aromatase mRNA in the Brain of Adult Green Anole Lizards: Effects of Sex and Season

Neural testosterone metabolism, particularly the synthesis of oestradiol (E2) via the aromatase enzyme, is important for sexual behaviours in many vertebrates. In green anole lizards, E2 metabolised from testosterone facilitates female receptivity and increases sexual motivation in males. Testosterone treatment increases aromatase activity in the whole brain homogenates of gonadectomised male, but not female, anoles, which is an effect limited to the breeding season (BS). To investigate the potential for local effects of this enzyme in reproductive behaviour, we used in situ hybridisation for aromatase mRNA to examine expression during the BS and nonbreeding season (NBS) in areas of the brain that control male sexual behaviours [preoptic area (POA) and amygdala (AMY)], as well as one regulating female reproductive behaviours ventromedial hypothalamus (VMH). Males had a greater total number of aromatase‐expressing cells in the POA than females, and the density of aromatase‐expressing cells (number per unit volume) was greater in the VMH and AMY of females. This density was also higher during the BS than NBS in the POA. Expression of aromatase in the AMY appeared to be lateralised because trends were detected for the left side to have more total cells and more cells per unit volume than the right. These results suggest that, similar to other vertebrates, regional aromatisation of testosterone may be important for the control of sex‐specific reproductive behaviours.

TESTING ALTERNATIVE HYPOTHESES FOR EVOLUTIONARY DIVERSIFICATION IN AN AFRICAN SONGBIRD: RAINFOREST REFUGIA VERSUS ECOLOGICAL GRADIENTS

Geographic isolation in rainforest refugia and local adaptation to ecological gradients may both be important drivers of evolutionary diversification. However, their relative importance and the underlying mechanisms of these processes remain poorly understood because few empirical studies address both putative processes in a single system. A key question is to what extent is divergence in signals that are important in mate and species recognition driven by isolation in rainforest refugia or by divergent selection across ecological gradients? We studied the little greenbul, Andropadus virens, an African songbird, in Cameroon and Uganda, to determine whether refugial isolation or ecological gradients better explain existing song variation. We then tested whether song variation attributable to refugial or ecological divergence was biologically meaningful using reciprocal playback experiments to territorial males. We found that much of the existing song variation can be explained by both geographic isolation and ecological gradients, but that divergence across the gradient, and not geographic isolation, affects male response levels. These data suggest that ecologically divergent traits, independent of historical isolation during glacial cycles, can promote reproductive isolation. Our study provides further support for the importance of ecology in explaining patterns of evolutionary diversification in ecologically diverse regions of the planet.

Testosterone selectively affects aromatase and 5α-reductase activities in the green anole lizard brain

Testosterone (T) and its metabolites are important in the regulation of reproductive behavior in males of a variety of vertebrate species. Aromatase converts T to estradiol and 5alpha-reductase converts T to 5alpha-dihydrotestosterone (DHT). Male green anole reproduction depends on androgens, yet 5alpha-reductase in the brain is not sexually dimorphic and does not vary with season. In contrast, aromatase activity in the male brain is increased during the breeding compared to non-breeding season, and males have higher levels than females during the breeding season. Aromatase is important for female, but not male, sexual behaviors. The present experiment was conducted to determine whether 5alpha-reductase and aromatase are regulated by T. Enzyme activity was quantified in whole brain homogenates in both the breeding and non-breeding seasons in males and females that had been treated with either a T or blank implant. In males only, T increased 5alpha-reductase activity regardless of season and up-regulated aromatase during the breeding season specifically. Thus, regulation of both enzymes occurs in males, whereas females do not show parallel sensitivity to T. When considered with previous results, the data suggest that aromatase might influence a male function associated with the breeding season other than sexual behavior. 5alpha-Reductase can be mediated by T availability, but this regulation may not serve a sex- or season-specific purpose.

Relationships among reproductive morphology, behavior, and testosterone in a natural population of green anole lizards.

Laboratory studies of reproductive systems have long supported the idea that neural and/or muscular structures used frequently are often enhanced in size. However, field studies integrating behavioral, morphological, and hormonal data are needed to better understand relationships in natural environments. We examined a natural population of green anole lizards (Anolis carolinensis) to determine whether variation in reproductive morphology both within and between the sexes paralleled differences in courtship and copulatory behaviors and circulating testosterone levels. Display rate in males was positively correlated with the sizes of the cartilage supporting the dewlap (a throat fan used in courtship and aggression) and renal sex segments (portions of the kidney that function similarly to the mammalian prostate), but correlated negatively with seminiferous tubule size. Plasma testosterone in males was negatively correlated with display behavior and was not correlated with any measures of morphology. Females, which display rarely, exhibited no relationships between morphology and frequency of behavior. Comparisons between the sexes show that males have consistently larger courtship and copulation morphologies than females, even when accounting for sex differences in body size. The results not only support the idea of relationships between increased function and enhanced structures, but also show the complexity of mechanistic interactions associated with reproductive behavior in wild animals.

Aromatase and 5α-reductase type 2 mRNA in the green anole forebrain: an investigation of the effects of sex, season and testosterone manipulation.

Aromatase and 5α-reductase (5αR) catalyze the synthesis of testosterone (T) metabolites: estradiol and 5α-dihydrotestosterone, respectively. These enzymes are important in controlling sexual behaviors in male and female vertebrates. To investigate factors contributing to their regulation in reptiles, male and female green anole lizards were gonadectomized during the breeding and non-breeding seasons and treated with a T-filled or blank capsule. In situ hybridization was used to examine main effects of and interactions among sex, season, and T on expression of aromatase and one isozyme of 5αR (5αR2) in three brain regions that control reproductive behaviors: the preoptic area, ventromedial nucleus of the amygdala and ventromedial hypothalamus (VMH). Patterns of mRNA generally paralleled previous evaluations of intact animals. Although no main effects of T were detected, interactions were present in the VMH. Specifically, the density of 5αR2 expressing cells was greater in T-treated than control females in this region, regardless of season. Among breeding males, blank-treated males had a denser population of 5αR2 positive cells than T-treated males. Overall, T appears to have less of a role in the regulation of these enzymes than in other vertebrate groups, which is consistent with the primary role of T (rather than its metabolites) in regulation of reproductive behaviors in lizards. However, further investigation of protein and enzyme activity levels are needed before specific conclusions can be drawn.

Newly deposited maternal hormones can be detected in the yolks of oviductal eggs in the green anole lizard

Studies often examine egg yolks after oviposition with the goal of drawing conclusions about maternal allocation of gonadal steroid hormones and how it may affect offspring development. However, these hormones might originate from a few sources, including the ovary, blood plasma, or the embryo itself. The goal of this study was to investigate whether maternal steroids can enter oviductal eggs. In Experiment 1, gravid female green anole lizards were injected with 1 microCi 3H-T. Plasma, ovarian follicles (separated into yolking and non-yolking samples), and shelled oviductal eggs were collected at times ranging from 15 min to 24 hr after treatment. Main effects of tissue, time, and an interaction between them all existed on recovered 3H-hormone corrected for tissue mass. Of particular interest, there was a decrease in plasma with coincident increase in eggs. In Experiment 2, females were injected with doses ranging from 0.01 to 0.45 microCi of 3H-T per gram body weight. Across tissues, 3H-hormone levels corrected for mass were greater with increasing doses. Values also differed among tissues and an interaction was detected. Within each dose, plasma and non-yolking follicles generally had higher concentrations of 3H-hormone than did yolking follicles and oviductal eggs. However, at and after 6 hr, eggs had higher total radioactivity levels than both yolking and non-yolking follicles had (not corrected for mass). The results indicate that steroids can cross through relatively well-formed shells before oviposition, suggesting a way in which maternal hormones might influence developmental factors after yolk deposition.

The distribution of estrogen receptor β mRNA in male and female green anole lizards

Estrogens are critical for a variety of aspects of brain development and adult processes. These steroids act via receptors within specific tissues. Several estrogen receptors (ER) are thought to exist, including ERα and ERβ, which function via classical, genomic mechanisms. These two ERs are found in a variety of species and are critical to diverse functions, including reproductive behaviors. ERβ was discovered more recently than ERα, and very little work has been done on this receptor in reptiles. Currently no data are available on its distribution in the brain in this vertebrate group. Here, we have cloned ERβ in the green anole lizard, mapped its distribution using in situ hybridization, and quantified expression in three brain areas controlling reproductive behaviors-the preoptic area, ventromedial amygdala (AMY), and ventromedial hypothalamus (VMH). ERβ was detected in discrete areas throughout the anole brain, with high levels in limbic regions and motor nuclei in the brainstem. Females had a greater density of ERβ positive cells in the AMY and VMH than males. While the functional consequences of these differences are not clear, they may result in an increased ability to respond to local levels of estradiol. The present work documents that neural ERβ is distributed similarly in reptilian, rodent and avian species, suggesting that it may perform similar roles. However, more work is necessary to elucidate the function of ERβ in this group.

Distribution of Two Isozymes of 5α-Reductase in the Brains of Adult Male and Female Green Anole Lizards

The 5α-reductase (5αR) enzyme converts testosterone to 5α-dihydrotestosterone. This local metabolism within the brain is important for the full expression of male sexual behavior in many species, including green anole lizards. Two isozymes of 5αR exist and little is known about their specific distributions. We conducted in situ hybridization for both isozymes in intact male and female green anole brains during the breeding (BS) and non-breeding (NBS) seasons. 5αR1 mRNA was only detected in the brainstem, while 5αR2 was expressed in specific areas throughout the brain. As our primary interest was evaluating the potential role of 5αR in forebrain regulation of reproductive behavior, we quantified 5αR2 expression in the preoptic area, amygdala (AMY), and ventromedial hypothalamus (VMH). More 5αR2 cells were detected during the NBS than BS in the AMY, and the density of these cells was greater in females than males. In the VMH, the right side contained more 5αR2 cells than the left, an effect driven by a lateralized increase in the NBS. These data expand understanding of the distribution and potential roles of both isozymes in the adult brain, and differences in expression patterns between mammals and birds suggest that they may have been co-opted for different functions later in evolution.