Learning Deficits Induced by High-Calorie Feeding in the Rat are Associated With Impaired Brain Kynurenine Pathway Metabolism.

In addition to be a primary risk factor for type 2 diabetes and cardiovascular disease, obesity is associated with learning disabilities. Here we examined whether a dysregulation of the kynurenine pathway (KP) of tryptophan (Trp) metabolism might underlie the learning deficits exhibited by obese individuals. The KP is initiated by the enzymatic conversion of Trp into kynurenine (KYN) by indoleamine 2,3-dioxygenase (IDO). KYN is further converted to several signaling molecules including quinolinic acid (QA) which has a negative impact on learning. Wistar rats were fed either standard chow or made obese by exposure to a free choice high-fat high-sugar (fcHFHS) diet. Their learning capacities were evaluated using a combination of the novel object recognition and the novel object location tasks, and the concentrations of Trp and KYN-derived metabolites in several brain regions determined by ultra-performance liquid chromatography-tandem mass spectrometry. Male, but not female, obese rats exhibited reduced learning capacity characterized by impaired encoding along with increased hippocampal concentrations of QA, Xanthurenic acid (XA), Nicotinamide (Nam), and oxidized Nicotinamide Adenine Dinucleotide (NAD+). In contrast, no differences were detected in the serum levels of Trp or KP metabolites. Moreover, obesity enhanced the expression in the hippocampus and frontal cortex of kynurenine monooxygenase (KMO), an enzyme involved in the production of QA from kynurenine. QA stimulates the glutamatergic system and its increased production leads to cognitive impairment. These results suggest that the deleterious effects of obesity on cognition are sex dependent and that altered KP metabolism might contribute to obesity-associated learning disabilities.

Environmental Enrichment Differentially Activates Neural Circuits in FVB/N Mice, Inducing Social Interaction in Females but Agonistic Behavior in Males.

Environmental enrichment induces behavioral and structural modifications in rodents and influences the capability of mice to cope with stress. However, little is understood about hippocampal neurogenesis and the appearance of social/agonistic (aggressive) behavior upon activation of different neuronal circuits in FVB/N mice. Thus, in this study we hypothesized that environmental enrichment differentially regulates neurogenesis, neural circuit activation and social/agonistic behavior in male and female FVB/N mice. We explored the (1) neurogenic process as an indicative of neuroplasticity, (2) neuronal activation in the limbic system, and (3) social behavior using the resident-intruder test. On postnatal day 23 (PD23), mice were assigned to one of two groups: Standard Housing or Environmental Enrichment. At PD53, rodents underwent the resident-intruder test to evaluate social behaviors. Results revealed that environmental enrichment increased neurogenesis and social interaction in females. In males, environmental enrichment increased neurogenesis and agonistic behavior. Enriched male mice expressed higher levels of agonistic-related behavior than female mice housed under the same conditions. Neural circuit analysis showed lower activation in the amygdala of enriched males and higher activation in enriched females than their respective controls. Enriched females also showed higher activation in the frontal cortex without differences in male groups. Moreover, the insular cortex was less activated in females than in males. Thus, our results indicate that environmental enrichment has different effects on neuroplasticity and social/agonistic behavior in FVB/N mice, suggesting the relevance of sexual dimorphism in response to environmental stimuli.

Adult rats from undernourished dams show sex-dependent impaired expression in taste papillae and hypothalamus of genes responsible for sweet and fat detection and signalling.

AIM: Individuals undernourished in utero or during early life are at high risk of developing obesity and metabolic disorders and show an increased preference for consuming sugary and fatty food. This study aimed at determining whether impaired taste detection and signalling in the lingual epithelium and the brain might contribute to this altered pattern of food intake. METHODS: The preference for feeding fat and sweet food and the expression in circumvallate papillae and hypothalamus of genes coding for sweet and fat receptors and transducing pathways were evaluated in adult rats born to control or calorie-restricted dams. Expression in the hypothalamus and the brain's reward system of genes involved in the homeostatic and hedonic control of food intake was also determined. RESULTS: Male and female undernourished animals exhibited increased expression in taste papillae and hypothalamus of T1R1, T1R2, CD36, gustducin, TRMP5 and PLC-ß2 genes, all of which modulate sweet and fat detection and intracellular signalling. However, the severity of the effect was greater in females than in males. Moreover, male, but not female, undernourished rats consumed more standard and sweetened food than their control counterparts and presented increased hypothalamic AgRP and NPY mRNAs levels together with enhanced dopamine transporter and dopamine receptor D2 expression in the ventral tegmental area. CONCLUSIONS: Maternal undernutrition induces sex-specific changes in food preferences and gene expression in taste papillae, hypothalamus and brain reward regions. The gene expression alterations in the male offspring are in line with their preference for consuming sugary and fatty food.

Maternal DHA Supplementation during Pregnancy and Lactation in the Rat Protects the Offspring against High-Calorie Diet-Induced Hepatic Steatosis.

Maternal supplementation during pregnancy with docosahexaenoic acid (DHA) is internationally recommended to avoid postpartum maternal depression in the mother and improve cognitive and neurological outcomes in the offspring. This study was aimed at determining whether this nutritional intervention, in the rat, protects the offspring against the development of obesity and its associated metabolic disorders. Pregnant Wistar rats received an extract of fish oil enriched in DHA or saline (SAL) as placebo by mouth from the beginning of gestation to the end of lactation. At weaning, pups were fed standard chow or a free-choice, high-fat, high-sugar (fc-HFHS) diet. Compared to animals fed standard chow, rats exposed to the fc-HFHS diet exhibited increased body weight, liver weight, body fat and leptin in serum independently of saline or DHA maternal supplementation. Nevertheless, maternal DHA supplementation prevented both the glucose intolerance and the rise in serum insulin resulting from consumption of the fc-HFHS diet. In addition, animals from the DHA-fc-HFHS diet group showed decreased hepatic triglyceride accumulation compared to SAL-fc-HFHS rats. The beneficial effects on glucose homeostasis declined with age in male rats. Yet, the preventive action against hepatic steatosis was still present in 6-month-old animals of both sexes and was associated with decreased hepatic expression of lipogenic genes. The results of the present work show that maternal DHA supplementation during pregnancy programs a healthy phenotype into the offspring that was protective against the deleterious effects of an obesogenic diet.

Lack of interaction between prenatal stress and prenatal letrozole to induce same-sex preference in male rats.

Same-sex partner preference between males has been observed in all species in which this behavior has been studied. Disruption of brain estradiol synthesis during development has been proposed as one of the biological causes underlying this behavior in some mammals. In support of this possibility, perinatal administration of aromatase inhibitors (such as letrozole) to male rat pups, induces around half of them to have same-sex preference and female sexual behavior in adulthood. Another putative factor that modifies sex preference is prenatal stress. Several stress protocols, applied to the pregnant dam, cause some of the adult male progeny to have an increased male preference, a decreased preference for the female, and lordosis behavior. Interestingly, these effects of stress might be mediated by its inhibitory action on brain aromatase. The aim of the present study was to analyze a possible interaction between these two factors in male rats. Pregnant dams were exposed to one of the four treatments across gestation days 10-22 (G10-G22): 1) vehicle-treated non-stressed controls; 2) letrozole (0.56 µg/kg); 3) 30 min immobilization stress); 4) both letrozole and stress combined. The male offspring were tested in adulthood for partner preference in a three-chambered arena, where we also recorded the masculine and feminine sexual behaviors. One week later males were tested for masculine and feminine sexual behavior in cylindrical arenas where they interacted for 30 min with a receptive female and thereafter with a sexually active male for another 30 min. Letrozole, stress and their combination resulted in same-sex preference in 40, 31 and 50% of males, respectively, compared to 5% in the control group. In the sexual behavior tests, prenatal stress reduced the percentage of males displaying intromissions and ejaculation (impaired masculinization), while letrozole mainly increased lordosis (impaired defeminization). The males prenatally submitted to stress and treated with letrozole presented these behavioral features but did not differ from both treatments given independently. The results indicate that the changes induced by stress or the aromatase inhibition produced by letrozole only accounts for a shift in partner preference in around half of the males and that there was no interaction between these two factors.

Establishment of partner preference in male rats: Effect of prenatal letrozole and sexual experience.

Repeated testing for masculine sexual behavior influences female sex preference in males. Males perinatally treated with aromatase inhibitors show male preference, but also copulate with the receptive female. Such copulation modifies sex preference most likely because of its rewarding properties. In this study, we intended to equal the incentive value of both stimuli -in the sex preference test- by using receptive females with vaginal occlusion. Vehicle and letrozole-treated (0.56 µg/kg, gestation days 10-21) males were repeatedly tested for sex preference at 40, 55, 70, 85 and 100 days of age. These ages were selected because males of 40 days are unable to copulate, while by 100 days of age almost all males show the complete repertoire of masculine sexual behavior. At 40 days of age, males of all groups fail to show sex preference and none of them was able to copulate. In controls of 100 days of age all males showed female-sex preference and all intromitted the female. A large proportion (44%) of vehicle-treated males that could not copulate the female showed male preference. Twenty to 30% of the prenatally letrozole treated males also had same-sex preference even if they could copulate; and most of them (67%) had a male preference when copulation was precluded. These data support the idea that copulation is crucial for developing a female preference in control animals. The results suggest that brain changes produced by estrogens along early development and stimuli coming from the partner are essential for shaping sex preference.

Melatonin Influences Structural Plasticity in the Axons of Granule Cells in the Dentate Gyrus of Balb/C Mice.

Melatonin, the main product synthesized by the pineal gland, acts as a regulator of the generation of new neurons in the dentate gyrus (DG). Newborn neurons buffer the deleterious effects of stress and are involved in learning and memory processes. Furthermore, melatonin, through the regulation of the cytoskeleton, favors dendrite maturation of newborn neurons. Moreover, newborn neurons send their axons via the mossy fiber tract to Cornu Ammonis 3 (CA3) region to form synapses with pyramidal neurons. Thus, axons of newborn cells contribute to the mossy fiber projection and their plasticity correlates with better performance in several behavioral tasks. Thus, in this study, we analyzed the impact of exogenous melatonin (8 mg/kg) administered daily for one- or six-months on the structural plasticity of infrapyramidal- and suprapyramidal mossy fiber projection of granule cells in the DG in male Balb/C mice. We analyzed the mossy fiber projection through the staining of calbindin, that is a calcium-binding protein localized in dendrites and axons. We first found an increase in the number of calbindin-positive cells in the granular cell layer in the DG (11%, 33%) after treatment. Futhermore, we found an increase in the volume of suprapyramidal (>135%, 59%) and infrapyramidal (>128%, 36%) mossy fiber projection of granule neurons in the DG after treatment. We also found an increase in the volume of CA3 region (>146%, 33%) after treatment, suggesting that melatonin modulates the structural plasticity of the mossy fiber projection to establish functional synapses in the hippocampus. Together, the data suggest that, in addition to the previously reported effects of melatonin on the generation of new neurons and its antidepressant like effects, melatonin also modulates the structural plasticity of axons in granule cells in the DG.

Prenatal administration of letrozole reduces SDN and SCN volume and cell number independent of partner preference in the male rat.

During development, the exposure to testosterone, and its conversion to estradiol by an enzyme complex termed aromatase, appears to be essential in adult male rats for the expression of typical male sexual behavior and female-sex preference. Some hypothalamic areas are the supposed neural bases of sexual preference/orientation; for example, male-oriented rams have a reduced volume of the sexually dimorphic nucleus (oSDN), while in homosexual men this nucleus does not differ from that of heterosexual men. In contrast, homosexual men showed a larger number of vasopressinergic cells in the suprachiasmatic nucleus (SCN). Interestingly, male rats perinatally treated with an aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), also showed bisexual preference and an increased number of vasopressinergic neurons in the SCN. However, this steroidal aromatase inhibitor has affinity for all three steroid receptors. Recently, we reported that the prenatal administration of the selective aromatase inhibitor, letrozole, produced a subpopulation of males with same-sex preference. The aim of this study was to compare the volume and number of cells of the SDN and SCN (the latter nucleus was immunohistochemically stained for vasopressin) between males treated with letrozole with same-sex preference, males treated with letrozole with female preference and control males with female preference. Results showed that all males prenatally treated with letrozole have a reduced volume and estimated cell number in the SDN and SCN, independent of their partner preference. These results indicate that the changes in these brain areas are not related to sexual preference, but rather to the effects of letrozole. The divergent results may be explained by species differences as well as by the critical windows during which the aromatase inhibitor was administered.

Male rats with same sex preference show high experimental anxiety and lack of anxiogenic-like effect of fluoxetine in the plus maze test.

Homosexual men show a 2-4 higher risk to suffer anxiety in comparison with heterosexuals. It is unknown if biological factors collaborate to increase such incidence. Fluoxetine produces differential brain activation in homosexuals as compared with heterosexuals, suggesting that it may produce a divergent behavioral effect dependant on sex-preference. The first aim was to evaluate experimental anxiety in male rats that show same-sex preference in the elevated plus maze (EPM). The second goal explored the putative differential effect of fluoxetine (10mg/kg) in male rats with female and same-sex preference in the EPM. To induce same-sex preference males were prenatally treated with letrozole (0.56µg/kg, 10-20 gestation days), while controls were males prenatally treated with letrozole that retain female-preference or which mothers received oil. In both groups we found animals with male preference, but the proportion was higher in males that prenatally received letrozole (10 vs. 27%). Males with same-sex preference spent less time and showed lower number of entries to the open arms of the EPM than males that prefer females, regardless of the prenatal treatment. In males with female preference, fluoxetine reduced the time spent and number of entries to the open arms that was absent in males with same-sex preference. These data suggest that biological factors contribute to the high levels of anxiety in subjects with same-sex preference and that fluoxetine in men may produce a divergent action depending on sexual orientation.

Prenatal letrozole produces a subpopulation of male rats with same-sex preference and arousal as well as female sexual behavior.

Disruption of the sexual differentiation process during critical periods in male rodents produces changes in partner preference and sexual behavior. In this study we used prenatal (gestation days 10-22) letrozole (0.31 and 0.56 µg/kg) to inhibit aromatase and alter normal sexual differentiation of males. These animals and control rats (injected with vehicle) were used when adults to study: a) sexual preference (where the experimental male could choose to interact with a receptive female or a sexually experienced male); b) masculine and feminine sexual behaviors (tested in cylindrical arenas); c) non-contact erections when exposed to a female or a male and, d) serum sex steroids and gonadotropin levels. The results showed that 30% of the males treated with letrozole (0.56 µg/kg) had same-sex preference, 33% displayed lordosis and 63% showed non-contact erections in the presence of a sexually experienced male. However, 44% of these males also exhibited complete masculine sexual behavior towards receptive females. None of the control males displayed lordosis when mounted by another male and very few (12%) showed non-contact erections when exposed to a sexually experienced male. Similar low percentages were found in those males prenatally treated with the low letrozole dose (0.31 µg/kg). No difference was found in the serum levels of testosterone, estradiol, LH and FSH between control and letrozole-treated males regardless of their sexual preference. These results indicate that prenatal selective inhibition of aromatization produces feminization of sexual partner preference, arousal and sexual behavior but does not affect masculine sexual behavior.

Perinatal administration of aromatase inhibitors in rodents as animal models of human male homosexuality: similarities and differences.

In this chapter we briefly review the evidence supporting the existence of biological influences on sexual orientation. We focus on basic research studies that have affected the estrogen synthesis during the critical periods of brain sexual differentiation in male rat offspring with the use of aromatase inhibitors, such as 1,4,6-androstatriene-3,17 (ATD) and letrozole. The results after prenatal and/or postnatal treatment with ATD reveal that these animals, when adults, show female sexual responses, such as lordosis or proceptive behaviors, but retain their ability to display male sexual activity with a receptive female. Interestingly, the preference and sexual behavior of these rats vary depending upon the circadian rhythm.Recently, we have established that the treatment with low doses of letrozole during the second half of pregnancy produces male rat offspring, that when adults spend more time in the company of a sexually active male than with a receptive female in a preference test. In addition, they display female sexual behavior when forced to interact with a sexually experienced male and some typical male sexual behavior when faced with a sexually receptive female. Interestingly, these males displayed both sexual behavior patterns spontaneously, i.e., in absence of exogenous steroid hormone treatment. Most of these features correspond with those found in human male homosexuals; however, the "bisexual" behavior shown by the letrozole-treated rats may be related to a particular human population. All these data, taken together, permit to propose letrozole prenatal treatment as a suitable animal model to study human male homosexuality and reinforce the hypothesis that human sexual orientation is underlied by changes in the endocrine milieu during early development.

Sex- and endocrine-stage-differences in middle-aged rats in an animal model of OCD.

Various clinical studies suggest that many features of OCD are influenced by sex, age and fluctuations in hormonal levels. Animal models have confirmed these differences, and suggest they are mediated by the serotonergic system. We compared the perseveration behavior in a T-maze after the administration of the 5-HT1A agonist, 8-OH-DPAT (2.0 mg/kg) and the preventive action of subchronic fluoxetine (10 mg/kg, 3 times) in middle-aged (11-14 months) males and female rats in two endocrine states: irregular cycles (tested in diestrus) or persistent diestrus. After 8-OH-DPAT, females with persistent diestrus presented higher perseveration scores than males and females with irregular cycles. Fluoxetine produced an anticompulsive-like effect only in females with persistent diestrus. Females in persistent diestrus showed higher estradiol levels than those in irregular cycles or males. In all groups 8-OH-DPAT increased ambulation and fluoxetine did not modify this action. In males, the combined administration of fluoxetine and 8-OH-DPAT impaired motor coordination. Data are discussed on the basis of estradiol levels and sex differences.

Sex differences in the burying behavior test in middle-aged rats: effects of diazepam.

The full behavioral profile displayed during the burying behavior test was studied in middle aged (11-14 months) males, females with irregular estrous cycles, and females in persistent diestrus, with and without diazepam (0.5-2.0mg/kg). Ambulation and motor coordination were also tested to discern behavioral changes from general motor alterations. Without diazepam treatment, middle-aged males showed longer burying behavior latencies, more prod explorations and less freezing than both groups of females. Untreated middle aged males also showed less cumulative burying and more immobility compared to females with irregular cycles. None of the parameters showed any difference between the female groups. Diazepam (0.5 and 1.0mg/kg) increased burying behavior latency in females, but had no effect on any parameter in middle aged males. However, a higher dose (2.0mg/kg) of diazepam increased immobility, freezing and the number of prod shocks and decreased prod explorations and groomings, but impaired motor coordination in males. In contrast with young males and females, diazepam at any dose reduced cumulative burying. Data are discussed on the bases of (1) sex and age differences in burying behavior and on (2) the anxiolytic-like action of diazepam and its side effects.