Chronic CNS oxytocin signaling preferentially induces fat loss in high-fat diet-fed rats by enhancing satiety responses and increasing lipid utilization.

Based largely on a number of short-term administration studies, growing evidence suggests that central oxytocin is important in the regulation of energy balance. The goal of the current work is to determine whether long-term third ventricular (3V) infusion of oxytocin into the central nervous system (CNS) is effective for obesity prevention and/or treatment in rat models. We found that chronic 3V oxytocin infusion between 21 and 26 days by osmotic minipumps both reduced weight gain associated with the progression of high-fat diet (HFD)-induced obesity and elicited a sustained reduction of fat mass with no decrease of lean mass in rats with established diet-induced obesity. We further demonstrated that these chronic oxytocin effects result from 1) maintenance of energy expenditure at preintervention levels despite ongoing weight loss, 2) a reduction in respiratory quotient, consistent with increased fat oxidation, and 3) an enhanced satiety response to cholecystokinin-8 and associated decrease of meal size. These weight-reducing effects persisted for approximately 10 days after termination of 3V oxytocin administration and occurred independently of whether sucrose was added to the HFD. We conclude that long-term 3V administration of oxytocin to rats can both prevent and treat diet-induced obesity.

Hindbrain oxytocin receptors contribute to the effects of circulating oxytocin on food intake in male rats.

Oxytocin (OT)-elicited hypophagia has been linked to neural activity in the nucleus of the solitary tract (NTS). Because plasma OT levels increase after a meal, we hypothesized that circulating OT acts at both peripheral and hindbrain OT receptors (OTRs) to limit food intake. To initially determine whether circulating OT inhibits food intake by acting at hindbrain OTRs, we pretreated rats with an OTR antagonist administered into the fourth ventricle (4V) followed by either central or systemic OT administration. Administration of the OTR antagonist into the 4V blocked anorexia induced by either 4V or i.p. injection of OT. However, blockade of peripheral OTRs also weakened the anorectic response to ip OT. Our data suggest a predominant role for hindbrain OTRs in the hypophagic response to peripheral OT administration. To elucidate central mechanisms of OT hypophagia, we tested whether OT activates NTS catecholaminergic neurons. OT (ip) increased the number of NTS cells expressing c-Fos, of which 10%-15% were catecholaminergic. Furthermore, electrophysiological studies in mice revealed that OT stimulated 47% (8 of 17) of NTS catecholamine neurons through a presynaptic mechanism. However, OT-elicited hypophagia did not appear to require activation of α1-adrenoceptors, and blockade of glucagon-like peptide-1 receptors similarly did not attenuate anorexia induced by OT. These findings demonstrate that OT elicits satiety through both central and peripheral OTRs and that although catecholamine neurons are a downstream target of OT signaling in the NTS, the hypophagic effect is mediated independently of α1-adrenoceptor signaling.

High-fat diet feeding causes rapid, non-apoptotic cleavage of caspase-3 in astrocytes.

Astrocytes respond to multiple forms of central nervous system (CNS) injury by entering a reactive state characterized by morphological changes and a specific pattern of altered protein expression. Termed astrogliosis, this response has been shown to strongly influence the injury response and functional recovery of CNS tissues. This pattern of CNS inflammation and injury associated with astrogliosis has recently been found to occur in the energy homeostasis centers of the hypothalamus during diet-induced obesity (DIO) in rodent models, but the characterization of the astrocyte response remains incomplete. Here, we report that astrocytes in the mediobasal hypothalamus respond robustly and rapidly to purified high-fat diet (HFD) feeding by cleaving caspase-3, a protease whose cleavage is often associated with apoptosis. Although obesity develops in HFD-fed rats by day 14, caspase-3 cleavage occurs by day 3, prior to the development of obesity, suggesting the possibility that it could play a causal role in the hypothalamic neuropathology and fat gain observed in DIO. Caspase-3 cleavage is not associated with an increase in the rate of apoptosis, as determined by TUNEL staining, suggesting it plays a non-apoptotic role analogous to the response to excitotoxic neuron injury. Our results indicate that astrocytes in the mediobasal hypothalamus respond rapidly and robustly to HFD feeding, activating caspase-3 in the absence of apoptosis, a process that has the potential to influence the course of DIO.

Obesity is associated with hypothalamic injury in rodents and humans.

Rodent models of obesity induced by consuming high-fat diet (HFD) are characterized by inflammation both in peripheral tissues and in hypothalamic areas critical for energy homeostasis. Here we report that unlike inflammation in peripheral tissues, which develops as a consequence of obesity, hypothalamic inflammatory signaling was evident in both rats and mice within 1 to 3 days of HFD onset, prior to substantial weight gain. Furthermore, both reactive gliosis and markers suggestive of neuron injury were evident in the hypothalamic arcuate nucleus of rats and mice within the first week of HFD feeding. Although these responses temporarily subsided, suggesting that neuroprotective mechanisms may initially limit the damage, with continued HFD feeding, inflammation and gliosis returned permanently to the mediobasal hypothalamus. Consistent with these data in rodents, we found evidence of increased gliosis in the mediobasal hypothalamus of obese humans, as assessed by MRI. These findings collectively suggest that, in both humans and rodent models, obesity is associated with neuronal injury in a brain area crucial for body weight control.

Deficits in substance P mRNA levels in the CeA are inversely associated with alcohol-motivated responding.

In the present study, in vitro and in vivo studies were conducted to determine the relationship between innate substance P (SP) levels and alcohol-motivated behavior in alcohol-preferring (P) and nonpreferring (NP) rat lines. In Experiment 1, in situ hybridization and quantitative autoradiography were used to detect and measure SP mRNA levels in discrete brain loci of the P and NP rats. The results indicated significantly lower SP mRNA levels in the central nucleus of the amygdala (CeA) of P compared with those of NP rats. Experiment 2 evaluated the effects of SP, microinfused into the CeA, on alcohol (10%, v/v) and sucrose (2%, w/v) motivated responding in the P rat. The results revealed that, when infused into the CeA (1-8 microg), SP reduced alcohol responding by 48-85% of control levels, with no effects on sucrose responding. Neuroanatomical control infusions (1-8 microg) into the caudate putamen (CPu) also failed to significantly alter alcohol- or sucrose-motivated behaviors. Given the selective reductions on alcohol (compared to sucrose) responding by direct intracranial infusion of SP, the data suggest that deficits in SP signaling within the CeA (an anxiety regulating locus) are inversely associated with alcohol-motivated behaviors. Activation of SP receptors in the CeA may reduce anxiety-like behavior in the P rat and contribute to reductions on alcohol responding. The SP system may be a suitable target for the development of drugs to reduce alcohol-drinking behavior in humans.

c-fos gene expression is increased in the paraventricular hypothalamic nucleus of Sprague-Dawley rats with visceral pain induced by acetic acid without detectable changes of corticotrophin-releasing factor mRNA: a quantitative approach with an image analysis system.

This study is the first of its kind to demonstrate that c-Fos immunoreactivity (ir) together with c-fos mRNA in their immediately adjacent tissue sections of a discrete brain region can be reliably measured. The c-fos gene expression in the paraventricular hypothalamic nucleus (PVN) of Sprague-Dawley rats for an animal model for visceral or somatovisceral pain induced by 2% acetic acid (AA) was used in this study. Specifically, c-fos mRNA signals were measured by quantitative autoradiography after in situ hybridization using c-fos oligodeoxynucleotide probe, and c-Fos-ir signals were represented by c-Fos immunostaining, as detected using c-Fos antibody in a regular immunohistochemistry. Signals from both c-Fos-ir and c-fos mRNA in the PVN were measured from their immediately adjacent cryostat sections. For the measurement of c-Fos-ir, it was carried out by reading 10 rectangles (1,000 microm(2)/rectangle) on each PVN section with c-Fos immunostaining. Specific signals were obtained from subtracting the nonspecific background signal from the total signals using a computer-assisted image analysis system. Results indicated that the AA treatment induced a significant increase of both c-Fos-ir and c-fos mRNA in the PVN. Interestingly, there was no increase of corticotrophin-releasing factor (CRF) mRNA expression in the PVN and central nucleus of the amygdala of Sprague-Dawley rats subjected to the AA treatment. In summary, this study has demonstrated that c-Fos-ir in the PVN with an anatomical resolution can be semiquantitatively measured after immunohistochemistry using an image analysis system, and that increased c-fos mRNA in the PVN 1 hr after the AA treatment is associated with no changes of the CRF mRNA expression.

Corticotropin-releasing factor mRNA and substance P receptor binding in the paraventricular hypothalamic nucleus, central nucleus of the amygdala, and locus coeruleus of Sprague-Dawley rats following restraint-induced stress.

The central mechanism of stress is poorly understood. This study was designed to examine how corticotropin-releasing factor (CRF) neurons, together with substance P (SP) receptors in the paraventricular hypothalamic nucleus (PVN), central nucleus of the amygdala (CeA), and locus coeruleus (LC), are affected by stress. Sprague-Dawley rats were restrained for 2 h. Animals were sacrificed by decapitation immediately after the 2-h restraint (the 0-h group) and 4, 24, or 48 h after restraint. Tissue sections were cut and collected on two sets of slides. Tissue sections of the first set were processed for studying CRF mRNA using 33P-labeled 60-mer oligonucleotide probe. Immediately adjacent tissue sections were processed for studying SP receptor-binding capacity using 125I-SP ligand. Quantitative results showed that CRF mRNAs in the PVN were significantly up-regulated at the 4- and 24-h stages, and they seemed not to be regulated by SP receptors. In addition, SP receptors in the CeA were up-regulated at the 24- and 48-h stages, whereas SP receptors were down-regulated in the LC at the same stages. In concert with the literature indicating SP antagonist's antidepressive effects, up-regulated SP receptors in the CeA might contribute to the development of stress-related depression.

Innate differences in neuropeptide Y (NPY) mRNA expression in discrete brain regions between alcohol-preferring (P) and -nonpreferring (NP) rats: a significantly low level of NPY mRNA in dentate gyrus of the hippocampus and absence of NPY mRNA in the medial habenular nucleus of P rats.

The neuropeptide Y (NPY) gene in rat chromosome 4 has been shown to play an important role in alcohol-seeking behavior. NPY knockout mice drink more alcohol than wild-type mice, implicating a link between NPY deficiency and high alcohol intake. This is supported by recent studies showing that intracerebroventricular injections of NPY reduce alcohol intake in both alcohol-preferring (P) and high alcohol-drinking rats. However, it is unknown which anatomical NPY systems are involved in alcohol preference. This study was designed to investigate whether there are innate differences in NPY mRNA in cerebral cortical areas, dentate gyrus (DG) of the hippocampus and medial habenular nucleus (MHb) between P and alcohol-nonpreferring (NP) rats, as these discrete brain regions are rich in NPY mRNA. [(33)P]-labeled 28-mer oligodeoxynucleotide probe was applied for the in situ hybridization study to detect the NPY mRNA, measured using quantitative autoradiography. This study revealed an absence of NPY mRNA in the MHb of P rats. We found that NPY mRNA was significantly lower in the DG of P rats than NP rats. This innate difference of NPY mRNA expression in the DG between P and NP rats is region specific. For example, in most of the cerebral cortical areas examined, an innate difference was not seen. Our study suggests that lower NPY gene expression in the DG and MHb of P rats may be factors contributing to some of the phenotypic differences observed between the P and NP lines of rats.

Corticotropin-releasing factor gene expression is down-regulated in the central nucleus of the amygdala of alcohol-preferring rats which exhibit high anxiety: a comparison between rat lines selectively bred for high and low alcohol preference.

The role of amygdaloid corticotropin-releasing factor (CRF) in alcoholism is not clear. Alcohol-preferring (P) rats and high alcohol-drinking (HAD) rats are selectively bred for high alcohol preference, and have been considered suitable animal models for studying alcoholism. The CRF neurons in the central nucleus of the amygdala (CeA) of P rats and HAD rats were studied in comparison with those of their respective counterparts, namely, alcohol-nonpreferring (NP) rats and low alcohol-drinking (LAD) rats. Specifically, CRF-immunoreactivity (ir) in the CeA and paraventricular hypothalamic nucleus (PVN) was assessed using radioimmunohistochemical (RIH) assay in alcohol-naive P/NP rats, and HAD/LAD rats. Furthermore, CRF mRNA was examined using in situ hybridization in the CeA of P/NP rats. Anxiety levels were also evaluated using an elevated plus maze. Results of the present study showed that CRF-ir was significantly lower in the CeA of P rats than NP rats. Moreover, CRF mRNA in the CeA was also much lower in P rats than NP rats. Such differences were not seen in the PVN. Interestingly, those P rats exhibited higher anxiety than NP rats. In contrary, there were no innate differences of CRF-ir in both the CeA and PVN between HAD and LAD rats whose anxiety levels were similar. This study is consistent with the literature showing CRF knockout (KO) induces alcohol drinking, and central administrations of CRF reduce alcohol intake. Collectively, the present study suggests that reduced CRF gene expression in the CeA of P rats is associated with their alcohol preference and anxiety.

Reduced neuropeptide Y mRNA expression in the central nucleus of amygdala of alcohol preferring (P) rats: its potential involvement in alcohol preference and anxiety.

Levels of neuropeptide Y (NPY) mRNA expression in discrete brain regions of alcohol preferring (P) rats and alcohol nonpreferring (NP) rats were examined using in situ hybridization. NPY mRNA expression was significantly lower in the central nucleus of amygdala (CeA) of P rats than NP rats, whereas no differences were found in the medial or basolateral amygdaloid nuclei. This study suggests that reduced NPY gene expression in the CeA may contribute to differences in alcohol preference and other behavioral differences observed between P and NP rats.

Angiotensin II receptor binding in the locus ceruleus of spontaneously hypertensive rats and Wistar-Kyoto rats: a quantitative autoradiographic study with references to hypertension and cardiac/testicular hypertrophy.

The locus ceruleus (LC) contains a high density of angiotensin II (AII) receptors. The role of AII receptors at the LC in genetic hypertension and organ function is unclear. Spontaneously hypertensive (SHR) rats and Wistar-Kyoto (WKY) rats were studied, and blood pressure of animals was measured using the tail-cuff method. Animals were decapitated and the heart weight (HW) and testicular weight (TW) of animals measured. AII receptor binding was carried out by incubating the LC tissue sections with 200 pM [(125)I]-AII receptor ligand, and measured using quantitative autoradiography. Results showed that the HW/BW ratio was significantly higher in SHR rats than WKY rats. However, the TW/BW ratio was higher in SHR rats than WKY rats only at two hypertensive stages, whereas AII receptor binding capacity in the LC was also statistically higher in SHR rats than WKY rats. Results indicated that cardiac and testicular hypertrophies were related to higher AII receptor binding in the LC of SHR rats, when compared with WKY rats. Interestingly, the literature shows that there is an LC-testes axis. In conclusion, this study indicated that AII receptors in the LC are associated with genetic hypertension, and testicular weight could be a reasonable index for essential hypertension.

Calcitonin gene-related peptide (CGRP) levels and alcohol.

Calcitonin gene-related peptide (CGRP) when administered into the brain exerts stress-like effects such as increased pain sensitivity, anorexia, and potentiation of fear-related behaviours. Since alcohol consumption may be related to alcohol's anxiolytic properties, the present study sought to determine if brain CGRP levels were correlated with genetic differences in preference for drinking alcohol and/or affected by alcohol exposure/withdrawal. CGRP-like immunoreactivity (CGRP-LI) was measured by radioimmunoassay (RIA) in amygdala, hippocampus, frontal cortex, hypothalamus, and caudate. In the first experiment, CGRP-LI was compared in alcohol-naive rats [preferring (P) and non-preferring (NP)], lower concentrations were found in the hippocampus (U = 153.5; d.f. = 1,28; p < 0.014) and frontal cortex (U = 183.0; d.f. = 1,28; p < 0.0001) of the P rats. In a second experiment, a group of outbred Wistar rats were exposed to alcohol in vapour chambers, or control conditions. At 7 wk of alcohol exposure there were no differences in exposed rats as compared to controls. However, at 4 wk following ethanol withdrawal, higher concentrations of CGRP-LI were found in the hippocampus (U = 26.5; d.f. = 1,20 p < 0.05), hypothalamus (U = 17.5; d.f. = 1,20; p < 0.009), and caudate-putamen (U = 17.0; d.f. = 1,20; p < 0.009) of the previously exposed animals. These studies suggest that CGRP may modulate alcohol preference and additionally, that exposure/withdrawal from ethanol produces long-lasting effects on CGRP-LI.