The effects of delta-9-tetrahydrocannabinol administration in the regulation of female rat sociosexual behaviour.

By targeting the endocannabinoid system, delta-9-tetrahydrocannabinol (THC) modulates female motivated behaviours, influenced by sex hormones. Both medial preoptic nucleus (MPN) and ventromedial nucleus of the hypothalamus (VMN) are involved in the modulation of female sexual responses. The first triggers proceptivity, whereas the ventrolateral division of the latter (VMNvl) triggers receptivity. These nuclei are modulated by glutamate, which inhibits female receptivity, and GABA, which has a dichotomous action in female sexual motivation. Here, we evaluated the action of THC on the modulation of social and sexual behaviours, on signalling pathways of MPN and VMNvl and how sex hormones influence these parameters. Young ovariectomized female rats, given sex hormones (oestradiol benzoate, EB, and progesterone, P) and THC were used for behavioural testing and for immunofluorescence analyses of vesicular glutamate transporter 2 (VGlut2) and GAD (glutamic acid decarboxylase)67 expression. Results showed that females given EB + P exhibited a higher preference for male partner, as well as higher proceptivity and a higher receptivity than control or females given only EB. Females treated with THC presented similar responses in control or EB + P female rats and even more facilitated behavioural responses in EB females than the ones that did not receive THC. Immunofluorescence results in the MPN exhibited a decreased expression of GAD67 and VGlut2 in EB + THC-treated female rats. Within VMNvl of EB-primed rats no changes in the expression of both proteins were observed after THC exposure. This study demonstrates how the possible outcomes of endocannabinoid system instability within hypothalamic neuron connectivity can modify female rat sociosexual behaviour.

A Semi-Automatic Method for the Quantification of Astrocyte Number and Branching in Bulk Immunohistochemistry Images.

Immunohistochemical staining of cell and molecular targets in brain samples is a powerful tool that can provide valuable information on neurological mechanisms. However, post-processing of photomicrographs acquired after 3,3'-Diaminobenzidine (DAB) staining is particularly challenging due to the complexity associated with the size, samples number, analyzed targets, image quality, and even the subjectivity inherent to the analysis by different users. Conventionally, this analysis relies on the manual quantification of distinct parameters (e.g., the number and size of cells and the number and length of cell branching) in a large set of images. These represent extremely time-consuming and complex tasks, defaulting the processing of high amounts of information. Here we describe an improved semi-automatic method to quantify glial fibrillary acidic protein (GFAP)-labelled astrocytes in immunohistochemistry images of rat brains, at magnifications as low as 20×. This method is a straightforward adaptation of the Young & Morrison method, using ImageJ's plugin Skeletonize, coupled with intuitive data processing in datasheet-based software. It allows swifter and more efficient post-processing of brain tissue samples, regarding astrocyte size and number quantification, the total area occupied, as well as astrocyte branching and branch length (indicators of astrocyte activation), thus contributing to better understand the possible inflammatory response developed by astrocytes.

Unhealthy Diets Induce Distinct and Regional Effects on Intestinal Inflammatory Signalling Pathways and Long-Lasting Metabolic Dysfunction in Rats.

The intestinal epithelium is a principal site for environmental agents' detection. Several inflammation- and stress-related signalling pathways have been identified as key players in these processes. However, it is still unclear how the chronic intake of inadequate nutrients triggers inflammatory signalling pathways in different intestinal regions. We aimed to evaluate the impact of unhealthy dietary patterns, starting at a younger age, and the association with metabolic dysfunction, intestinal inflammatory response, and obesity in adulthood. A rat model was used to evaluate the effects of the consumption of sugary beverages (HSD) and a Western diet (WD), composed of ultra-processed foods. Both diets showed a positive correlation with adiposity index, but a positive correlation was found between the HSD diet and the levels of blood glucose and triglycerides, whereas the WD diet correlated positively with triglyceride levels. Moreover, a distinct inflammatory response was associated with either the WD or HSD diets. The WD induced an increase in TLR2, TLR4, and nuclear factor-kappa B (NF-κB) intestinal gene expression, with higher levels in the colon and overexpression of the inducible nitric oxide synthase. In turn, the HSD diet induced activation of the TLR2-mediated NF-κB signalling pathway in the small intestine. Altogether, these findings support the concept that early intake of unhealthy foods and nutrients are a main exogenous signal for disturbances of intestinal immune mechanisms and in a region-specific manner, ultimately leading to obesity-related disorders in later life.

Uterine histopathological changes induced by acute administration of tamoxifen and its modulation by sex steroid hormones.

The endometrium is a particular sensitive target tissue for estradiol that is able to promptly modify its structure. Tamoxifen (TAM), a selective estrogen receptor modulator, was shown to promote a spectrum of uterine abnormalities, though the morphological and stereological effects of this drug in uterus is not clear. In this way, we have used an established model of ovariectomy followed by estradiol benzoate (EB) or TAM treatment and analyzed their effects in uterine histopathology and proliferation. Administration of EB promotes the unfolding and proliferation of the endometrium stroma, increasing uterine volume. No changes were found in uterine histomorphometric analysis upon TAM administration, except in the thickness of the luminal epithelium and endometrium layer. The latter may result from increased complexity and glandular volume density also observed in TAM treatment. In addition, EB induced PAX2 expression, an oncogene commonly found in epithelial tumors of the female genital tract, an effect that was weakened by previous TAM administration. Although treatments did not affect stroma cells proliferating index, in epithelial cells and, contrary to TAM, EB increased PCNA and not Ki67 expression. Collectively, our data suggest that the acute administration of TAM induces ERα-dependent atrophy of the uterine tissue and decreased the expression of proliferating cellular markers. On the contrary, if administered prior to EB, TAM is able to attenuate the action of the hormone in uterine morphology and biochemistry.

Effects of tamoxifen on neuronal morphology, connectivity and biochemistry of hypothalamic ventromedial neurons: Impact on the modulators of sexual behavior.

Tamoxifen (TAM) is a selective estrogen receptor modulator, widely used in the treatment and prevention of estrogen-dependent breast cancer. Although with great clinical results, women on TAM therapy still report several side effects, such as sexual dysfunction, which impairs quality of life. The anatomo-functional substrates of the human sexual behavior are still unknown; however, these same substrates are very well characterized in the rodent female sexual behavior, which has advantage of being a very simple reflexive response, dependent on the activation of estrogen receptors (ERs) in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl). In fact, in the female rodent, the sexual behavior is triggered by increasing circulation levels of estradiol that changes the nucleus neurochemistry and modulates its intricate neuronal network. Therefore, we considered of notice the examination of the possible neurochemical alterations and the synaptic plasticity impairment in VMNvl neurons of estradiol-primed female rats treated with TAM that may be in the basis of this neurological disorder. Accordingly, we used stereological and biochemical methods to study the action of TAM in axospinous and axodendritic synaptic plasticity and on ER expression. The administration of TAM changed the VMNvl neurochemistry by reducing ERα mRNA and increasing ERß mRNA expression. Furthermore, present results show that TAM induced neuronal atrophy and reduced synaptic connectivity, favoring electrical inactivity. These data suggest that these cellular and molecular changes may be a possible neuronal mechanism of TAM action in the disruption of the VMNvl network, leading to the development of behavioral disorders.

Unraveling the Hippocampal Molecular and Cellular Alterations behind Tramadol and Tapentadol Neurobehavioral Toxicity.

Tramadol and tapentadol are chemically related opioids prescribed for the analgesia of moderate to severe pain. Although safer than classical opioids, they are associated with neurotoxicity and behavioral dysfunction, which arise as a concern, considering their central action and growing misuse and abuse. The hippocampal formation is known to participate in memory and learning processes and has been documented to contribute to opioid dependence. Accordingly, the present study assessed molecular and cellular alterations in the hippocampal formation of Wistar rats intraperitoneally administered with 50 mg/kg tramadol or tapentadol for eight alternate days. Alterations were found in serum hydrogen peroxide, cysteine, homocysteine, and dopamine concentrations upon exposure to one or both opioids, as well as in hippocampal 8-hydroxydeoxyguanosine and gene expression levels of a panel of neurotoxicity, neuroinflammation, and neuromodulation biomarkers, assessed through quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical analysis of hippocampal formation sections showed increased glial fibrillary acidic protein (GFAP) and decreased cluster of differentiation 11b (CD11b) protein expression, suggesting opioid-induced astrogliosis and microgliosis. Collectively, the results emphasize the hippocampal neuromodulator effects of tramadol and tapentadol, with potential behavioral implications, underlining the need to prescribe and use both opioids cautiously.

Long-Term Tamoxifen Effects in the Cyclic Interaction of the Endocannabinoid and Endocrine System in the Rat Central Nervous System.

Steroid hormones can modulate the endocannabinoid system (ECS). Within the female reproductive tract, estrogen increases the expression of the cannabinoid receptors CB1 and CB2, and modifies the levels of anandamide (AEA), the major endocannabinoid, by altering the expression of both AEA synthesis (NAPE-PLD) and catabolic enzymes (FAAH). Here, we addressed the mechanisms involved in ECS fluctuations within the central nervous system and evaluated the effects of tamoxifen (TAM), a selective estrogen receptor modulator, in central AEA regulation. The current results suggest that the hypothalamic and pituitary AEA levels change differently according to the brain area and phase of the estrous cycle. In TAM-treated rats, there is a disruption of the cyclic fluctuation and reduction of the AEA levels in all brain areas. In the pituitary gland, NAPE-PLD expression increases in the metestrus phase, whereas throughout the rat cycle their expression remained constant, even upon TAM treatment. The fluctuations of pituitary AEA levels result from altered FAAH and NAPE-LPD expression. In contrast, no differences in FAAH or NAPE-PLD hypothalamic expression were observed. Overall, this study presents a broad view of the distribution and expression of ECS elements in the central nervous system and a way to suggest possible brain areas involved in the interaction of the endocannabinoid and neuroendocrine systems to induce several behavioral responses.

Structural, functional and behavioral impact of allergic rhinitis on olfactory pathway and prefrontal cortex.

BACKGROUND: Allergic rhinitis (AR) has been identified as a cause of olfactory dysfunction. Beyond the classic symptoms, AR has been associated with altered sleep patterns, a decline in cognitive performance and higher likelihood of depression and anxiety. The olfactory pathway has been postulated to be a possible link between nasal inflammation and central nervous system (CNS) modifications. Thus, we aimed to investigate the structural, functional and behavioral changes in the olfactory pathway and related areas in an animal model of AR. METHODS: AR was induced in adult Wistar rats by ovalbumin sensitization and challenge. Following olfactory and behavioral tests we investigated the synaptic structure of the olfactory bulb (OB), anterior olfactory nuclei (AON), piriform cortex and prefrontal cortex (PFC), by immunofluorescence detection of synaptophysin (Syn) and glutamatergic, GABAergic and dopaminergic neuronal markers. RESULTS: We detected a significant decrease in Syn in the glomerular layer (GL) of OB and in the PFC of the AR group. Additionally, the optical density of GAD67 and VGLUT2 was reduced in the OB, AON and PFC, compared to controls. The behavioral tests demonstrated olfactory dysfunction and reduced male aggressiveness in AR rats, but we did not find any difference in the cognition and anxiety-like behavior. CONCLUSIONS: We confirmed olfactory dysfunction in a rat model of AR and we identified modifications in synaptic activity by reduction of Syn optical density in the GL of the OB and in the PFC. This was accompanied by structural changes in glutamatergic and GABAergic activity in essential components of the olfactory pathway and PFC.

Effects of chronic tamoxifen treatment in female rat sexual behaviour.

The medial preoptic (MPN) and the ventromedial hypothalamic nuclei (VMN) modulate the estrogen receptor (ER)-dependent female sexual behavior, a response that is inhibited by tamoxifen (TAM), a modulator of the steroid receptor activation. With the objective to assess TAM action in the brain areas involved in the modulation sexual cues, an animal model on long-term TAM therapy to intact female rats, was used to mimic the 5-year prophylactic TAM therapy offered to women at higher risk of breast cancer. After three months treatment, female sexual behavior with a stud male rat was evaluated. Upon sacrifice, the brains were removed and the MPN and the ventrolateral division of the VMN were screened for the effects of TAM in the expression of ERα, ERß and progesterone receptor. Results show that TAM inhibited the receptive component of the female sexual behavior. Even though TAM decreased estrogen and progesterone levels to values similar to the ones of estrous and diestrus rats, the biochemical data failed to demonstrate such possible causation for the behavioral response. In fact, TAM administration induced a constant low level of ovarian hormones that changed the pattern of ER and PR expression as well as receptor co-expression in the brain areas regulating the behavioral response, dissimilar to the ones seen in the cycle phases with the same low hormone levels. Nevertheless, present data suggests that by affecting ER- and/or PR-dependent mechanisms, TAM may modulate the hypothalamus, a region known to participate in several social behaviors.

Gut Microbiome Composition and Metabolic Status Are Differently Affected by Early Exposure to Unhealthy Diets in a Rat Model.

Childhood is a critical stage of development during which diet can have profound influence on the microbiota-host interactions, leading to potentially lifelong impacts. This study aimed to investigate whether the consumption of cafeteria diet (CAFD) and sugary drinks during early rat life alters the structure of the gut microbial community and the metabolic activity. Four-week-old male Wistar rats (n = 27) were fed a standard chow diet with ad libitum access to water (CD) or to sucrose solution (HSD), and a third group was fed with CAFD and a sucrose solution for 14 weeks. HSD and CAFD consumption induced alterations in Firmicutes to Bacteroidetes ratio, Proteobacteria, and Verrucomicrobia. HSD increased the abundance of Barnesiella, whereas CAFD induced a depletion of Saccharibacteria. CAFD increased total white adipose tissue (WAT) weight (p < 0.0005) compared to CD. When CAFD was compared to HSD, a significant difference was found only for retroperitoneal WAT (p < 0.0005). Unhealthy diet-fed groups presented higher glucose (p < 0.0005), total cholesterol and creatinine serum levels (p < 0.005) compared to the CD rats. Early-life consumption of HSD, and of CAFD even more so, can have long-lasting negative effects on metabolic function. The gut microbiota communities were distinctively perturbed by diet composition.

Evaluation of progressive hepatic histopathology in long-term tamoxifen therapy.

Tamoxifen (TAM) therapy is the better treatment for breast cancer and the drug use the prophylaxis of this disease in young premenopausal women. Yet, the effects associated with this therapy are unknown. To better understand the extension of this problem, we developed an animal model to mimic this therapy, aiming to evaluate its potential biochemical and histopathological changes in the liver. Young cycling female rats were treated with TAM for one, two and three months and toxicological biomarkers and liver histomorphometry were evaluated. Starting at two months, TAM-treatment prevented the normal age-dependent increase in body weight, without inducing changes in food intake. Serum levels of cholesterol and of the metabolic enzymes creatine kinase and aspartate aminotransferase were reduced in all TAM treatment periods. Serum levels of the metabolic enzymes alkaline phosphatase and lactate dehydrogenase were increased after the first month but returned to control levels upon 3 months of drug exposition. Moderate microvesicular steatosis, classified only at the first month of TAM treatment, was reduced afterwards. Our model showed an adaptive response of liver upon 3 months of treatment, suggesting that at the stated conditions, TAM will not promote hepatotoxicity. In this way, the present model may be useful in the study of possible key endocrine effects of TAM use and the search for better clinical outcomes.

Dynamics of progesterone and estrogen receptor alpha in the ventromedial hypothalamus.

Cyclic fluctuations of estradiol and progesterone in females influence neuronal activity in the ventrolateral division of the ventromedial hypothalamic nucleus (VMNvl), through the activation of progesterone receptors (PRs) and estrogen receptors (ERs). The expression of ER and PR in the VMNvl is influenced by their cognate ligands and is a central upstream trigger in the pathway of VMNvl-dependent modulation of endocrine responses. By studying the role played by estradiol and progesterone in PR and ERa expression in the VMNvl along the estrous cycle and how the two receptors interact in the same neuron, we aim to evaluate the synergistic action of both ovarian hormones in the regulation of VMNvl activity. In animals at all phases of the estrous cycle, the number of VMN neurons expressing PR or ERa was estimated by stereological methods, and the percentage, and rostro-caudal distribution, of neurons simultaneously expressing both receptors was determined. The highest number of PR-immunoreactive neurons was seen at proestrus, and of ERa-immunoreactive neurons was seen at proestrus and metestrus. The ERa/PR co-localization is increased at caudal levels. Approximately half the neurons expressing PR co-express ERa, a proportion that stays constant along the estrous cycle. The percentage of ERa neurons co-expressing PR changes from 60% at proestrus to 40% at metestrus. Fluctuations in circulating ovarian hormone levels promote coordinated changes in PR and ERa expression and co-localization. This may be an important mechanism in the regulation of input relayed by the VMNvl, allowing a precise modulation of endocrine responses.

The integrity of the nucleus of the lateral olfactory tract is essential for the normal functioning of the olfactory system.

The nucleus of the lateral olfactory tract (nLOT) is a relatively small component of the cortical pallial amygdala, with peculiar neurogenic, neurochemical and connectivity patterns. Although it has been suggested that it might be involved in non-pheromonal olfactory-guided behaviors, particularly feeding, the functional implications of the nLOT have never been investigated. In view of this fact, we have tackled this subject by performing a series of behavioral tests and by quantifying biological and biochemical parameters in sexually naïve adult male rats that were submitted to bilateral excitotoxic lesions of the nLOT. nLOT-lesioned rats had severe olfactory deficits with inability to detect and discriminate between odors. Additionally, they did not display innate behavioral responses to biologically relevant chemosignals. Specifically, nLOT-lesioned rats did not show avoidance towards predator odors or aggressive behaviors towards intruders, and had severely impaired sexual behavior. In fact, nLOT lesions abolished preference for odors of receptive females, reduced chemoinvestigatory behavior and eliminated mounting behavior. nLOT-lesioned rats had normal circulating levels of testosterone, did not display anxiety- or depressive-like behaviors, and had unimpaired cognitive functions and fear acquisition and memory. Altogether, our results suggest that the nLOT integrity is required for the normal functioning of the olfactory system.

Regulation of NPY and α-MSH expression by estradiol in the arcuate nucleus of Wistar female rats: a stereological study.

OBJECTIVES: Feeding behavior in both animals and humans is modulated by estrogens, as shown by the increased adiposity observed in women and rats upon the drop of estradiol levels at menopause. Estradiol action on food intake is mediated through its cognate receptors within several hypothalamic nuclei, namely the arcuate nucleus (ARN). The ARN contains two neuronal populations expressing peptides that exert opposing effects on the central control of feeding: the orexigenic neuropeptide Y (NPY) and the anorexigenic α-melanocyte-stimulating hormone (α-MSH). METHODS: To understand the role played by estradiol in the modulation of food intake, we have used an animal model of cyclic 17ß-estradiol benzoate (EB) administration and stereological methods to estimate the total number of neurons immunoreactive for NPY and α-MSH in the ARN of ovariectomized rats. RESULTS: Present results show that the experimentally induced EB cyclicity prompted a decrease in food consumption and in body weight. Data also show that ovariectomy induced an increase in NPY expression and a decrease in α-MSH expression in the ARN that were reverted by EB administration. Conversely, EB blocked the expression of NPY and increased the synthesis of α-MSH in ARN neurons, without affecting the overall sum of NPY and α-MSH neurons. DISCUSSION: These results suggest that estradiol affects food intake and, consequently, body weight gain, through an overriding mechanism superimposed in the physiological balance between both peptides in the ARN of female rats.

Induction and subcellular redistribution of progesterone receptor A and B by tamoxifen in the hypothalamic ventromedial neurons of young adult female Wistar rats.

The ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) is a brain center for estrogen-dependent triggering of female sexual behavior upon progesterone receptor (PR) activation. We examined the agonistic and antagonistic actions of tamoxifen in this nucleus by analyzing its effects on the total number of PR-immunoreactive neurons, PR mRNA and protein levels, and subcellular location of PRs in ovariectomized Wistar rats. The results show that tamoxifen has no agonistic action in the number of PR-immunoreactive neurons, but increases PR expression and labeling in the nucleus and cytoplasm of VMNvl neurons that constitutively express PRs. As an antagonist, tamoxifen partially inhibited the estradiol-dependent increase in the number of PR-immunoreactive neurons and in PR mRNA and protein levels, without interfering with the subcellular location of the protein. We suggest that tamoxifen influence on PR expression in the VMNvl critically depends on the presence or absence of estradiol.

Changes in the female arcuate nucleus morphology and neurochemistry after chronic ethanol consumption and long-term withdrawal.

Ethanol is a macronutrient whose intake is a form of ingestive behavior, sharing physiological mechanisms with food intake. Chronic ethanol consumption is detrimental to the brain, inducing gender-dependent neuronal damage. The hypothalamic arcuate nucleus (ARN) is a modulator of food intake that expresses feeding-regulatory neuropeptides, such as alpha melanocyte-stimulating hormone (α-MSH) and neuropeptide Y (NPY). Despite its involvement in pathways associated with eating disorders and ethanol abuse, the impact of ethanol consumption and withdrawal in the ARN structure and neurochemistry in females is unknown. We used female rat models of 20% ethanol consumption for six months and of subsequent ethanol withdrawal for two months. Food intake and body weights were measured. ARN morphology was stereologically analyzed to estimate its volume, total number of neurons and total number of neurons expressing NPY, α-MSH, tyrosine hydroxylase (TH) and estrogen receptor alpha (ERα). Ethanol decreased energy intake and body weights. However, it did not change the ARN morphology or the expression of NPY, α-MSH and TH, while increasing ERα expression. Withdrawal induced a significant volume and neuron loss that was accompanied by an increase in NPY expression without affecting α-MSH and TH expression. These findings indicate that the female ARN is more vulnerable to withdrawal than to excess alcohol. The data also support the hypothesis that the same pathways that regulate the expression of NPY and α-MSH in long-term ethanol intake may regulate food intake. The present model of long-term ethanol intake and withdrawal induces new physiological conditions with adaptive responses.

Estrogen modulates the sexually dimorphic synaptic connectivity of the ventromedial nucleus.

Neurons in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) display a remarkable estrogen-dependent functional and structural plasticity, which is likely to be mediated, in part at least, by neuronal afferents. The present study was designed to determine whether the number of synapses per neuron and the size of individual synapses in the VMNvl vary across the estrus cycle and, also, whether they differ between the sexes. To accomplish this, the VMNvl of adult female rats at proestrus or diestrus day 1 and of age-matched male rats was analyzed using electron microscopy. We found that a single VMNvl neuron receives around 7,000 synapses during diestrus and approximately 10,000 during proestrus. This estrus cycle-related variation is accounted for by increases in the number of all types of synapses. In males, the number of synapses received by each VMNvl neuron is similar to that of diestrus rats (approximately 7,500). However, in males the number of axodendritic and axospinous synapses is smaller than in proestrus rats, whereas the number of axosomatic synapses is higher than in diestrus rats. In addition, we found that the size of the postsynaptic densities of axospinous and axosomatic synapses is consistently larger in males than in females. Our results show that the synaptic organization of the VMNvl is sexually dimorphic, with females having more dendritic synapses and males more somatic synapses. They also show that the synaptic plasticity induced by estrogen in the VMNvl is characterized by changes in the number, but not the size, of the synapses.

Estrogen receptors α and ß have different roles in the induction and trafficking of progesterone receptors in hypothalamic ventromedial neurons.

Progesterone receptor (PR) activation in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) is essential for promoting female sexual behavior. Estrogen receptor (ER) α, in contrast to ERß, has been implicated in the induction of PRs. The simultaneous activation of ERα and ERß, although not increasing the number of PR-immunoreactive neurons in the VMNvl, facilitates lordosis, which suggests that ERß and/or the ERα-ERß interaction might play a role in PR dynamics and/or PR expression by individual neurons. To address this question, we used western blot and immunohistochemical studies to determine the amounts and subcellular distributions of both PR isoforms in VMNvl neurons of ovariectomized rats injected with estradiol benzoate or with specific agonists of ERα and ERß, alone or in association. The present data show that ERα activation does not change PR expression in individual neurons, but increases the number of PRs in the VMNvl, because it increases the number of neurons expressing PRs. Conversely, ERß activation does not change the total number of PRs in the VMNvl, but increases the labeling intensity of the perikaryal cytoplasm, which suggests that it promotes the transport of PRs from neurites into cell bodies. In addition, the simultaneous activation of ERα and ERß increases the expression of PRs by individual neurons and, consequently, increases the total number of PRs in the VMNvl. Our findings reveal that individual and simultaneous activation of ERα and ERß have different effects on the levels and subcellular location of PRs in VMNvl neurons.

Timed hypocaloric food restriction alters the synthesis and expression of vasopressin and vasoactive intestinal peptide in the suprachiasmatic nucleus.

In mammals, the main circadian pacemaker is located in the suprachiasmatic nucleus (SCN) and its most potent synchronizer is the daily variation of the intensity of light. However, other nonphotic cues, such as timed food restriction, can induce changes in the circadian rhythms, leading also to the appearance of a food-entrained oscillator. The present study was designed to establish if the alterations of the circadian rhythms induced by timed hypocaloric food restriction are accompanied by structural changes in the SCN. Two groups of adult rats, both maintained on 12-h light/12-h dark cycles, were used; in one group, animals had permanent free access to food, whereas in the other they were subjected to a restricted hypocaloric early morning feeding during 7 months. Using stereological techniques and in situ hybridization, we have examined the structure of the SCN and the synthesis and expression of vasopressin (AVP) and vasoactive intestinal peptide (VIP). The volume of the SCN and the total number of neurons did not vary between the two groups. However, the total number of AVP- and VIP-immunoreactive neurons and the AVP and VIP mRNA levels were significantly decreased in timed hypocaloric food-restricted animals. The results indicate that timed hypocaloric food restriction has led to changes of AVP and VIP content of the neurons. They furthermore suggest the existence of a coupling between the food-entrainable oscillator and the light-entrainable pacemaker.

Role of plasma membrane estrogen receptors in mediating the estrogen induction of progesterone receptors in hypothalamic ventromedial neurons.

Progesterone is well known for its role in the modulation of sexual behavior. In the ventromedial nucleus (VMN), a part of the mediobasal hypothalamus that regulates sexual behavior in female rodents, estrogens induce the expression of progesterone receptors (PRs). This effect is known to be dependent on the activation of nuclear estrogen receptors (ERs). However, recent studies have documented estrogen activation of genomic transcription triggered by protein-protein phosphorylation cascades initiated at membrane receptors. The aim of this study was to examine if membrane-initiated estradiol (E2 ) stimulation is able to induce PR expression in the VMN or, at least, to modulate nuclear ER action. To achieve this goal, 2-month-old ovariectomized Wistar rats were injected bilaterally, in the vicinity of VMN, with free E2 and with E2 conjugated with bovine serum albumin (E2 BSA), alone or in sequence, by using a two-pulse injection paradigm. Stereological methods and western blot analysis were used to estimate the total number of PR-immunoreactive neurons in the VMN and the PR protein content of the VMN, respectively. The results showed that the administration of E2 BSA alone increases the number of PR-immunoreactive neurons and the expression level of PR protein to values similar to those resulting from E2 administration. They also showed that the sequential administration of E2 and E2 BSA potentiates the effects resulting from the injection of E2 or E2 BSA alone. These data provide the first evidence that membrane-initiated E2 stimulation is able to induce and to potentiate the genomic activation of PR expression in the VMN.