Male rats with the testicular feminization mutation of the androgen receptor display elevated anxiety-related behavior and corticosterone response to mild stress

Testosterone influences the hypothalamic–pituitary–adrenal axis, anxiety-related behavior, and sensorimotor gating in rodents, but little is known about the role of the androgen receptor (AR) in mediating these influences. We compared levels of the stress hormone corticosterone at baseline and following exposure to a novel object in an open field in wild type (wt) male and female rats, and male rats with the testicular feminization mutation (Tfm) of the AR, which disables its function. Basal corticosterone was equivalent in all groups, but exposure to a novel object in an open field elicited a greater increase in corticosterone in Tfm males and wt females than in wt males. Tfm males also showed increased behavioral indices of anxiety compared to wt males and females in the test. Analysis of the immediate early gene c-Fos expression after exposure to a novel object revealed greater activation in Tfm males than wt males in some regions (medial preoptic area) and lesser activation in others (dentate gyrus, posterodorsal medial amygdala). No differences were found in a measure of sensorimotor gating (prepulse inhibition of the acoustic startle response), although Tfm males had an increased acoustic startle response compared to wt males and females. These findings demonstrate that ARs play a role in regulating anxiety-related behaviors, as well as corticosterone responses and neural activation following exposure to a mild stressor in rats.     

Mice with the testicular feminization mutation demonstrate a role for androgen receptors in the regulation of anxiety-related behaviors and the hypothalamic–pituitary–adrenal axis

Testosterone (T) appears to play a role in anxiety and sensorimotor gating in rodents, but whether T acts through the androgen receptor (AR) to influence these behaviors is less clear. We compared adult genetic male mice with the testicular feminization mutation (Tfm), which lack functional ARs, to wild type male littermates (wt males) on an assay of sensorimotor gating (prepulse inhibition of the acoustic startle response; PPI) and several tests thought to reflect anxiety: open field exposure, novel object exposure, elevated plus maze (EPM), and light/dark (LD) box. PPI was similar between groups, but indices of anxiety in the novel object and LD box tests were increased in Tfm males with no significant differences found in the open field or EPM. Since Tfm male mice have decreased circulating T, the same tests were conducted in mice that were gonadectomized (wt males) or sham-operated (Tfm males) as adults and supplemented with T or nothing (B). While T treatment reduced indices of anxiety in the novel object and LD box tests in wt males, it was ineffective in Tfm males. Increased indices of anxiety in Tfm males appear to be related to hyper-activation of the hypothalamic–pituitary–adrenal axis since levels of the stress hormone corticosterone were elevated in Tfm males compared to wt males at baseline and at several time points after exposure to a novel object. These findings demonstrate that ARs influence anxiety and stress responses in mice.     

Mice with the testicular feminization mutation demonstrate a role for androgen receptors in the regulation of anxiety-related behaviors and the hypothalamic-pituitary-adrenal axis

Testosterone (T) appears to play a role in anxiety and sensorimotor gating in rodents, but whether T acts through the androgen receptor (AR) to influence these behaviors is less clear. We compared adult genetic male mice with the testicular feminization mutation (Tfm), which lack functional ARs, to wild type male littermates (wt males) on an assay of sensorimotor gating (prepulse inhibition of the acoustic startle response; PPI) and several tests thought to reflect anxiety: open field exposure, novel object exposure, elevated plus maze (EPM), and light/dark (LD) box. PPI was similar between groups, but indices of anxiety in the novel object and LD box tests were increased in Tfm males with no significant differences found in the open field or EPM. Since Tfm male mice have decreased circulating T, the same tests were conducted in mice that were gonadectomized (wt males) or sham-operated (Tfm males) as adults and supplemented with T or nothing (B). While T treatment reduced indices of anxiety in the novel object and LD box tests in wt males, it was ineffective in Tfm males. Increased indices of anxiety in Tfm males appear to be related to hyper-activation of the hypothalamic–pituitary–adrenal axis since levels of the stress hormone corticosterone were elevated in Tfm males compared to wt males at baseline and at several time points after exposure to a novel object. These findings demonstrate that ARs influence anxiety and stress responses in mice.     

Aggression and arginine vasopressin immunoreactivity regulation by androgen receptor and estrogen receptor alpha

In the following study, we asked which steroid receptors regulate aggression and arginine vasopressin (AVP) immunoreactivity (– ir) in several limbic regions. Using spontaneous mutant and knockout mice, we generated a novel cross of mice whose offspring lacked estrogen receptor α (ERα), androgen receptor (AR) or both ERα and AR. The wild-type (WT) males and females were compared with ERα knockout (ERαKO) male, mutated AR (Tfm) male and ERαKO/Tfm (double knockout; DKO) male littermates. Animals were gonadectomized and treated with 17β-estradiol (E2) prior to resident-intruder aggression tests. WT and Tfm males showed aggression whereas WT females, ERαKO and DKO males did not. In the lateral septum, WT and Tfm male brains had significantly denser AVP-ir as compared with WT females and DKO males. ERαKO male brains were intermediate in the amount of AVP-ir present. In the medial amygdala, brains from all genotypes had equivalent AVP-ir, except DKO males, which had significantly less AVP-ir. Overall, the expression of aggressive behavior coincided with AVP-ir in WT, Tfm and DKO males. However, in ERαKO males and WT females, the amount of AVP-ir was not associated with resident-intruder aggression. In sum we have shown that E2 acts via ERα to regulate aggression in male mice. In contrast both ERα and AR contribute to AVP-ir in limbic brain regions.     

Knockout of 5-lipoxygenase results in age-dependent anxiety-like behavior in female mice

Background

The enzyme 5-lipoxygenase (5LO) has been implicated in a variety of neurological and psychiatric disorders including anxiety. Knockout of 5LO has previously been shown to alter anxiety-like behavior in mice at a young age but the effect of 5LO knockout on older animals has not been characterized.

Methodology/Principal Findings

Here we used the elevated plus maze behavioral paradigm to measure anxiety-like behavior in female mice lacking 5LO (5LO-KO) at three different ages. Adolescent 5LO-KO animals did not significantly differ from wild-type (WT) animals in anxiety-like behavior. However, adult and older mice exhibited increased anxiety-like behavior compared to WT controls.

Conclusions

These results indicate that 5LO plays a role in the development of the anxiety-like phenotype in an age-dependent manner in female mice. Future work should further investigate this interaction as 5LO may prove to be an important molecular target for the development of novel anxiolytic therapies.     

Testosterone suppresses the expression of regulatory enzymes of fatty acid synthesis and protects against hepatic steatosis in cholesterol-fed androgen deficient mice

Aims

Non-alcoholic fatty liver disease and its precursor hepatic steatosis is common in obesity and type-2 diabetes and is associated with cardiovascular disease (CVD). Men with type-2 diabetes and/or CVD have a high prevalence of testosterone deficiency. Testosterone replacement improves key cardiovascular risk factors. The effects of testosterone on hepatic steatosis are not fully understood.

Main methods

Testicular feminised (Tfm) mice, which have a non-functional androgen receptor (AR) and very low serum testosterone levels, were used to investigate testosterone effects on high-cholesterol diet-induced hepatic steatosis.

Key findings

Hepatic lipid deposition was increased in Tfm mice and orchidectomised wild-type littermates versus intact wild-type littermate controls with normal androgen physiology. Lipid deposition was reduced in Tfm mice receiving testosterone treatment compared to placebo. Oestrogen receptor blockade significantly, but only partially, reduced the beneficial effects of testosterone treatment on hepatic lipid accumulation. Expression of key regulatory enzymes of fatty acid synthesis, acetyl-CoA carboxylase alpha (ACACA) and fatty acid synthase (FASN) were elevated in placebo-treated Tfm mice versus placebo-treated littermates and Tfm mice receiving testosterone treatment. Tfm mice on normal diet had increased lipid accumulation compared to littermates but significantly less than cholesterol-fed Tfm mice and demonstrated increased gene expression of hormone sensitive lipase, stearyl-CoA desaturase-1 and peroxisome proliferator-activated receptor-gamma but FASN and ACACA were not altered.

Significance

An action of testosterone on hepatic lipid deposition which is independent of the classic AR is implicated. Testosterone may act in part via an effect on the key regulatory lipogenic enzymes to protect against hepatic steatosis.     

Novel androgen receptor full antagonists: Design,synthesis, and a docking study of glycerol and aminoglycerol derivatives that contain p-carborane cages

Based on the co-crystal structure of bicalutamide with a T877A-mutated androgen receptor (AR), glycerol and aminoglycerol derivatives were designed and synthesized as a novel type of carborane-containing AR modulators. The (R)-isomer of 6c, whose chirality is derived from the glycerol group, showed 20 times more potent cell inhibitory activity against LNCaP cell lines expressing T877A-mutated AR than the corresponding (S)-isomer. Docking studies of both isomers with AR suggested that (R)-6c is in closer spatial proximity to helix-12 of the AR than (S)-6c, which is the most important common motif in the secondary structure of AR for the expression of antagonistic activity.     

Function of androgen receptor in gene regulations

Most of the androgen actions are considered to be mediated by the androgen receptor (AR) of the target genes. The AR is composed of a fairly large molecule because of the long A/B domains of its N-terminal. However, the independent roles of the AR as well as those of the estrogen receptors largely remained unknown mainly due to the lack of the AR knockout (ARKO) mice line. We have succeeded in generating the ARKO mouse by means of a conditional targeting using the Cre/loxP system. The ARKO males grew healthily although they showed a typical feature of the testicular feminization mutation (Tfm) and the hormonal assay revealed significantly lower serum androgen and higher LH levels in comparison with those of the wild type (WT) males. The serum estrogen levels were, however, comparable between both the ARKO and the WT. Another hallmark of the ARKO males was a state of high bone turnover osteopenia, in which the acceleration in the bone resorption clearly exceeded the bone formation. Male-typical behaviors were disrupted in male ARKO mice. Aiming at a quick differentiation of an androgen-dependent polyQ disease such as Kennedy's disease, the authors also developed the Drosophila fly-eye model in which the wild type and the polyQ-expanded human AR (hAR) was induced in the eyes of Drosophila. When androgen was administered to the flies induced with the polyQ-expanded hAR, their optical nerves were devastated.     

Intrahippocampal administration of an androgen receptor antagonist, flutamide, can increase anxiety-like behavior in intact and DHT-replaced male rats

Testosterone (T) and its 5alpha-reduced metabolite, dihydrotestosterone (DHT), can decrease anxiety-like behavior; however, the mechanisms underlying these effects have not been established. First, we hypothesized that if T reduces anxiety-like behavior through actions of its 5alpha-reduced metabolite, DHT, then gonadectomy (GDX) would increase anxiety-like behavior, an effect which would be reversed by systemic administration of DHT. Second, we hypothesized that if T and DHT reduce anxiety-like behavior in part through actions at intracellular androgen receptors in the hippocampus, then administration of an androgen receptor antagonist, flutamide, directly to the hippocampus should increase anxiety-like behavior of intact and DHT-replaced, but not GDX, male rats. Inserts that were empty or contained flutamide were applied directly to the dorsal hippocampus of intact, GDX, or GDX and DHT-replaced rats 2 h prior to testing in the open field, elevated plus maze, or defensive freezing tasks. GDX rats exhibited significantly more anxiety-like behaviors than intact or DHT-replaced rats. Intact and DHT-replaced rats administered flutamide to the hippocampus showed significantly more anxiety-like behavior than did intact and DHT-replaced controls. However, flutamide alone did not increase anxiety-like behavior of GDX rats. Together, these findings suggest that androgens can decrease anxiety-like behavior of male rats in part through DHT's actions at androgen receptors in the hippocampus.     

Late onset of obesity in male androgen receptor-deficient (AR KO) mice

An androgen receptor (AR) null mutant mice line was generated by means of a Cre-lox P system. The male (AR(L-/Y)) (KO) mice exhibited typical features of testicular feminization mutant (Tfm) disease in external reproductive organs with growth retardation. The growth curve of the male AR KO mice was similar to that of the wild-type female littermates until the 10th week of age, but thereafter a drastic increase in the growth was observed with development of obesity. Clear increase in the wet weights of white adipose tissues, but not of brown adipose tissue, was found in the 30-week-old male AR KO mice. However, no significant alteration in serum lipid parameters and food intake was observed. Thus, the present results suggest that AR may serve as a negative regulator of adipose development in adult males.     

Embryonic GABAB Receptor Blockade Alters Cell Migration,Adult Hypothalamic Structure,and Anxiety- and Depression-Like Behaviors Sex Specifically in Mice

Neurons of the paraventricular nucleus of the hypothalamus (PVN) regulate the hypothalamic- pituitary-adrenal (HPA) axis and the autonomic nervous system. Females lacking functional GABA_B receptors because of a genetic disruption of the R1 subunit have altered cellular characteristics in and around the PVN at birth. The genetic disruption precluded appropriate assessments of physiology or behavior in adulthood. The current study was conducted to test the long term impact of a temporally restricting pharmacological blockade of the GABA_B receptor to a 7-day critical period (E11–E17) during embryonic development. Experiments tested the role of GABA_B receptor signaling in fetal development of the PVN and later adult capacities for adult stress related behaviors and physiology. In organotypic slices containing fetal PVN, there was a female specific, 52% increase in cell movement speeds with GABA_B receptor antagonist treatment that was consistent with a sex-dependent lateral displacement of cells in vivo following 7 days of fetal exposure to GABA_B receptor antagonist. Anxiety-like and depression-like behaviors, open-field activity, and HPA mediated responses to restraint stress were measured in adult offspring of mothers treated with GABA_B receptor antagonist. Embryonic exposure to GABA_B receptor antagonist resulted in reduced HPA axis activation following restraint stress and reduced depression-like behaviors. There was also increased anxiety-like behavior selectively in females and hyperactivity in males. A sex dependent response to disruptions of GABA_B receptor signaling was identified for PVN formation and key aspects of physiology and behavior. These changes correspond to sex specific prevalence in similar human disorders, namely anxiety disorders and hyperactivity.     

Sex and exercise interact to alter the expression of anabolic androgenic steroid-induced anxiety-like behaviors in the mouse

Anabolic androgenic steroids (AAS) are taken by both sexes to enhance athletic performance and body image, nearly always in conjunction with an exercise regime. Although taken to improve physical attributes, chronic AAS use can promote negative behavior, including anxiety. Few studies have directly compared the impact of AAS use in males versus females or assessed the interaction of exercise and AAS. We show that AAS increase anxiety-like behaviors in female but not male mice and that voluntary exercise accentuates these sex-specific differences. We also show that levels of the anxiogenic peptide corticotrophin releasing factor (CRF) are significantly greater in males, but that AAS selectively increase CRF levels in females, thus abrogating this sex-specific difference. Exercise did not ameliorate AAS-induced anxiety or alter CRF levels in females. Exercise was anxiolytic in males, but this behavioral outcome did not correlate with CRF levels. Brain-derived neurotrophic factor (BDNF) has also been implicated in the expression of anxiety. As with CRF, levels of hippocampal BDNF mRNA were significantly greater in males than females. AAS and exercise were without effect on BDNF mRNA in females. In males, anxiolytic effects of exercise correlated with increased BDNF mRNA, however AAS-induced changes in BDNF mRNA and anxiety did not. In sum, we find that AAS elicit sex-specific differences in anxiety and that voluntary exercise accentuates these differences. In addition, our data suggest that these behavioral outcomes may reflect convergent actions of AAS and exercise on a sexually differentiated CRF signaling system within the extended amygdala.     

Differentiation between androgen and estrogen receptor mediated effects of testosterone on FSH using androgen receptor deficient (Tfm) and normal mice

The comparison of normal and androgen receptor (AR) deficient Tfm-mice allows distinction between AR mediated and estrogen receptor (ER) mediated effects of testosterone (T)--the latter after aromatization of T to estrogens--on serum and pituitary FSH. Normal male and female as well as Tfm mice were gonadectomized after 8 days and treated for 11 days with either T, estradiol (E2) or vehicle. Serum and pituitary FSH was determined by RIA for rat FSH. In Tfm mice T caused a suppression of serum FSH, indicating an ER mediated effect. Lower serum FSH levels after T in normal mice than Tfm mice indicate an additional AR mediated suppression. Lower serum FSH values in E2 treated Tfm than in T treated Tfm mice--where T acts only through ER--suggest two classes of estrophilic cells: one which aromatizes, thus being susceptible for both T and E2, and the other which does not aromatize. Only AR but not ER mediated T effects on pituitary FSH could be demonstrated.     

Dysfunctional Behavioral Modulation of Corticostriatal Communication in the R6/2 Mouse Model of Huntington’s Disease

Background

In Huntington’s disease (HD), motor symptoms develop prior to the widespread loss of neurons in striatum and cerebral cortex. The aim of this study was to examine dysfunctional patterns of corticostriatal communication during spontaneously occurring behaviors in a transgenic mouse model of HD.

Methodology/Principal Findings

Local field potentials (LFPs) were recorded from two closely interconnected areas, motor cortex and dorsal striatum, in wild-type controls (WT, n = 14) and a widely used transgenic HD model (R6/2 mice, n = 12). All mice were between the ages of 7–9 weeks, a critical period of motor symptom development in R6/2s. Recordings were obtained while the mice were behaving freely in an open field. Specific LFP activity was extracted using timestamps for three increasingly demanding motor behaviors: 1) resting; 2) grooming; and 3) active exploration. Power spectral densities (PSD) were obtained for the cortical and striatal LFPs as well as coherence levels and relative phase across the frequency spectrum. In both brain regions, only R6/2s showed high frequency LFP oscillations during rest and grooming. As behavior increased from resting to exploring, corticostriatal synchrony at high frequencies declined in R6/2s, completely opposite to the WT pattern. R6/2s also exhibited nearly in-phase corticostriatal activity (cortex phase leads of ∼5°), while the WTs consistently showed cortical phase lags of ∼20° across all assessed behaviors, indicating a lead role for striatum.

Conclusions/Significance

Our results add to growing evidence for altered communication between cortex and striatum in HD and suggest more generally that increasingly demanding motor behaviors differentially modulate corticostriatal communication. Our data also suggest conduction delays in R6/2 corticostriatal transmission, leading to compensatory speeding of LFP activity, as evidenced by the presence of high frequency LFP oscillations.     

Sexually dimorphic androgen and estrogen receptor mRNA expression in the brain of túngara frogs

Sex steroid hormones are potent regulators of behavior and they exert their effects through influences on sensory, motor, and motivational systems. To elucidate where androgens and estrogens can act to regulate sex-typical behaviors in the túngara frog (Physalaemus pustulosus), we quantified expression of the androgen receptor (AR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) genes in the brains of male and females. To do so, we cloned túngara-specific sequences for AR, ERα, and ERβ, determined their distribution in the brain, and then quantified their expression in areas that are important in sexual communication. We found that AR, ERα, and ERβ were expressed in the pallium, limbic forebrain (preoptic area, hypothalamus, nucleus accumbens, amygdala, septum, striatum), parts of the thalamus, and the auditory midbrain (torus semicircularis). Males and females had a similar distribution of AR and ER expression, but expression levels differed in some brain regions. In the auditory midbrain, females had higher ERα and ERβ expression than males, whereas males had higher AR expression than females. In the forebrain, females had higher AR expression than males in the ventral hypothalamus and medial pallium (homolog to hippocampus), whereas males had higher ERα expression in the medial pallium. In the preoptic area, striatum, and septum, males and females had similar levels of AR and ER expression. Our results suggest that sex steroid hormones have sexually dimorphic effects on auditory processing, sexual motivation, and possibly memory and, therefore, have important implications for sexual communication in this system.     

Pharmacological studies on the anxiolytic effect of standardized Schisandra lignans extract on restraint-stressed mice

Fruits of Fructus Schisandrae were used as sedatives and hypnotics in traditional Chinese medicine for a long history. In this study, we investigated the effects of schisandra lignans extract (SLE) on anxiety disorder in restraint-stressed mice using light-dark (L-D) test. The influences of restraint stress on the levels of monoamines: noradrenaline (NE), dopamine (DA) and serotonin (5-HT) in cerebral cortex, as well as plasma corticosterone (CORT) were studied in mice. The HPLC fingerprint of SLE was recorded and the percentage composition of Schisandra lignans was determined as 82.63%. In L-D test, it was found out that 18 h of restraint stress significantly decreased the anxiolytic parameters (explorative behaviors, e.g. number of entries, time spent) in light area indicating high state of anxiety in stressed mice. In addition, restraint stress elevated NE, DA, and 5-HT levels in cerebral cortex of anxiety mice. Plasma CORT level was also increased. Oral administration of SLE (100 and 200 mg/kg/day, 8 days) emolliating the level of stress-induced anxiety by significantly increasing the anxiolytic parameters mentioned above. We also observed decreases in cerebral cortex monoamines levels, as well as plasma CORT level in stressed mice. These results suggested that SLE reversed stress-induced anxiety level, changes of cortex monoamine transmitters and plasma CORT. The anxiolytic effects of SLE might be related to its anti-stress activity by modulation of hyperactive HPA axis.     

Detection of spatially regulated gene expression by hybridization histochemistry.

Steady state measurements of kidney androgen-regulated protein (KAP) mRNA suggested that KAP gene expression was unusually sensitive to low hormone-receptor levels. Two of the criteria used to reach this conclusion involved relative insensitivity to treatment with a competitive antiandrogen and a partial androgen response of the gene in Tfm/Y androgen receptor (AR) deficient mice. These data may indicate the ability of the KAP gene to respond to an extremely low level of androgen-AR complex or that the effect of androgens is, at least in part, indirect. Hybridization in situ revealed that KAP mRNA expression was restricted to proximal tubule epithelial cells in the juxtamedullary region of castrated animals rather than throughout the cortex as in intact males. Examination of sections of kidneys from Tfm/Y mice before and after testosterone (T) treatment revealed that only the juxtamedullary tubules expressed KAP mRNA and that T increased the level of KAP mRNA in these cells. Treatment of Tfm/Y animals with other steroids showed that beta-estradiol treatment mimicked the effect of T while dihydrotestosterone (DHT) had no effect. The facts that DHT and T both stimulate cortical expression of KAP mRNA in normal animals but DHT has no effect on the juxtamedullary cells in the Tfm/Y variant may indicate that the action of T is due to an estrogenic metabolite. Castrated, hypophysectomized males exhibited no KAP gene expression, while in the presence of T, expression was observed throughout the cortex as in intact males. These results clearly indicate the involvement of pituitary hormones in KAP gene expression in the juxtamedullary tubules. These studies have shown that the regulation of KAP gene expression in the mouse kidney is much more complex than originally believed. Future studies will further investigate the roles of estrogen and specific pituitary hormones in KAP gene expression.     

Gestational Hypoxia Induces Sex-Differential Methylation of Crhr1 Linked to Anxiety-like Behavior

Stress during gestation increases vulnerability to disease and changes behavior in offspring. We previously reported that hypoxia and restraint during pregnancy sensitized the hypothalamic–pituitary–adrenal (HPA) axis and induced anxiety-like behavior in the adult offspring. Here, we report that gestational intermittent hypoxia (GIH) elicited a sex-dependent anxiety-like behavior in male P90 offspring and activation of corticotropin-releasing hormone (CRH) and CRH type-1 receptor (CRHR1) mRNA in the hypothalamic paraventricular nucleus (PVN) and in male E19 hypothalamus. These linked to demethylation at several specific sites of CpG island of Crhr1 promoter in P90 PVN and E19 embryo hypothalamus in GIH groups. Crhr1 DNA demethylation is more crucial in CpG island 1 than island 2 for activation of CRHR1 mRNA. DNMT3b is required for the Crhr1 DNA methylation than DNMT1 and DNMT3a in increased CRHR1 mRNA. We first address a novel hypothesis that GIH-induced male-sex-dependent demethylation at CpG sites of Crhr1 DNA in promoter triggers elevation of CRHR1 mRNA in PVN and anxiety-like behavior in adult offspring.