Fructose-Induced Hypothalamic AMPK Activation Stimulates Hepatic PEPCK and Gluconeogenesis due to Increased Corticosterone Levels

Fructose consumption causes insulin resistance and favors hepatic gluconeogenesis through mechanisms that are not completely understood. Recent studies demonstrated that the activation of hypothalamic 5'-AMP-activated protein kinase (AMPK) controls dynamic fluctuations in hepatic glucose production. Thus, the present study was designed to investigate whether hypothalamic AMPK activation by fructose would mediate increased gluconeogenesis. Both ip and intracerebroventricular (icv) fructose treatment stimulated hypothalamic AMPK and acetyl-CoA carboxylase phosphorylation, in parallel with increased hepatic phosphoenolpyruvate carboxy kinase (PEPCK) and gluconeogenesis. An increase in AMPK phosphorylation by icv fructose was observed in the lateral hypothalamus as well as in the paraventricular nucleus and the arcuate nucleus. These effects were mimicked by icv 5-amino-imidazole-4-carboxamide-1-beta-D-ribofuranoside treatment. Hypothalamic AMPK inhibition with icv injection of compound C or with injection of a small interfering RNA targeted to AMPK alpha 2 in the mediobasal hypothalamus (MBH) suppressed the hepatic effects of ip fructose. We also found that fructose increased corticosterone levels through a mechanism that is dependent on hypothalamic AMPK activation. Concomitantly, fructose-stimulated gluconeogenesis, hepatic PEPCK expression, and glucocorticoid receptor binding to the PEPCK gene were suppressed by pharmacological glucocorticoid receptor blockage. Altogether the data presented herein support the hypothesis that fructose-induced hypothalamic AMPK activation stimulates hepatic gluconeogenesis by increasing corticosterone levels. (Endocrinology 153: 3633-3645, 2012)

Enhanced Expression of Heme Oxygenase-1 and Carbon Monoxide Excitatory Effects in Oxytocin and Vasopressin Neurones During Water Deprivation

A growing body of evidence indiates that carbon monoxide (CO) acts as a gas neurotransmitter within the central nervous system. Although CO has been shown to affect neurohypophyseal hormone release in response to osmotic stimuli, the precise sources, targets and mechanisms underlying the actions of CO within the magnocellular neurosecretory system remain largely unknown. In the present study, we combined immunohistochemistry and patch-clamp electrophysiology to study the cellular distribution of the CO-synthase enzyme heme oxygenase type 1 (HO-1), as well as the actions of CO on oxytocin (OT) and vasopressin (VP) magnocellular neurosecretory cells (MNCs), in euhydrated (EU) and 48-h water-deprived rats (48WD). Our results show the expression of HO-1 immunoreactivity both in OT and VP neurones, as well as in a small proportion of astrocytes, both in supraoptic (SON) and paraventricular (PVN) nuclei. HO-1 expression, and its colocalisation with OT and VP neurones within the SON and PVN, was significantly enhanced in 48WD rats. Inhibition of HO activity with chromium mesoporphyrin IX chloride (CrMP; 20 mu m) resulted in a slight membrane hyperpolarisation in SON neurones from EU rats, without significantly affecting their firing activity. In 48WD rats, on the other hand, CrMP resulted in a more robust membrane hyperpolarisation, significantly decreasing neuronal firing discharge. Taken together, our results indicate that magnocellular SON and PVN neurones express HO-1, and that CO acts as an excitatory gas neurotransmitter in this system. Moreover, we found that the expression and actions of CO were enhanced in water-deprived rats, suggesting that the state-dependent up-regulation of the HO-1/CO signalling pathway contributes to enhance MNCs firing activity during an osmotic challenge.

Cannabinoid CB1 receptor restrains accentuated activity of hypothalamic corticotropin-releasing factor and brainstem tyrosine hydroxylase neurons in endotoxemia-induced hypophagia in rats

It is well known that endocannabinoids play an important role in the regulation of food intake and body weight. Endocannabinoids and cannabinoid receptors are found in the hypothalamus and brainstem, which are central areas involved in the control of food intake and energy expenditure. Activation of these areas is related to hypophagia observed during inflammatory stimulus. This study investigated the effects of cannabinoid (CB1) receptor blockade on lipopolysaccharide (LPS)-induced hypophagia. Male Wistar rats were pretreated with rimonabant (10 mg/kg, by gavage) or vehicle; 30 min later they received an injection of either LPS (100 mu g/kg, intraperitoneal) or saline. Food intake, body weight, corticosterone response, CRF and CART mRNA expression, Fos-CRF and Fos-alpha-MSH immunoreactivity in the hypothalamus and Fos-tyrosine hydroxylase (TH) immunoreactivity in the brainstem were evaluated. LPS administration decreased food intake and body weight gain and increased plasma corticosterone levels and CRF mRNA expression in the PVN. We also observed an increase in Fos-CRF and Fos-TH double-labeled neurons after LPS injection in vehicle-pretreated rats, with no changes in CART mRNA or Fos-alpha-MSH immunoreactive neurons in the ARC. In saline-treated animals, rimonabant pretreatment decreased food intake and body weight gain but did not modify hormone response or Fos expression in the hypothalamus and brainstem compared with vehicle-pretreated rats. Rimonabant pretreatment potentiated LPS-induced hypophagia, body weight loss and Fos-CRF and Fos-TH expressing neurons. Rimonabant did not modify corticosterone, CRF mRNA or Fos-alpha-MSH responses in rats treated with LPS. These data suggest that the endocannabinoid system, mediated by CB1 receptors, modulates hypothalamic and brainstem circuitry underlying the hypophagic effect during endotoxemia to prevent an exaggerated food intake decrease. This article is part of a Special Issue entitled 'Central Control of Food Intake'. (C) 2011 Elsevier Ltd. All rights reserved.

Pontomedullary and hypothalamic distribution of Fos-like immunoreactive neurons after acute exercise in rats

During exercise, intense brain activity orchestrates an increase in muscle tension. Additionally, there is an increase in cardiac output and ventilation to compensate the increased metabolic demand of muscle activity and to facilitate the removal of CO2 from and the delivery of O-2 to tissues. Here we tested the hypothesis that a subset of pontomedullary and hypothalamic neurons could be activated during dynamic acute exercise. Male Wistar rats (250-350 g) were divided into an exercise group (n = 12) that ran on a treadmill and a no-exercise group (n = 7). Immunohistochemistry of pontomedullary and hypothalamic sections to identify activation (c-Fos expression) of cardiorespiratory areas showed that the no-exercise rats exhibited minimal Fos expression. In contrast, there was intense activation of the nucleus of the solitary tract, the ventrolateral medulla (including the presumed central chemoreceptor neurons in the retrotrapezoid/parafacial region), the lateral parabrachial nucleus, the Kolliker-Fuse region, the perifornical region, which includes the perifornical area and the lateral hypothalamus, the dorsal medial hypothalamus, and the paraventricular nucleus of the hypothalamus after running exercise. Additionally, we observed Fos immunoreactivity in catecholaminergic neurons within the ventrolateral medulla (C1 region) without Fos expression in the A2, A5 and A7 neurons. In summary, we show for the first time that after acute exercise there is an intense activation of brain areas crucial for cardiorespiratory control. Possible involvement of the central command mechanism should be considered. Our results suggest whole brain-specific mobilization to correct and compensate the homeostatic changes produced by acute exercise. (c) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Chronic intermittent hypoxia alters glutamatergic control of sympathetic and respiratory activities in the commissural NTS of rats

Costa-Silva JH, Zoccal DB, Machado BH. Chronic intermittent hypoxia alters glutamatergic control of sympathetic and respiratory activities in the commissural NTS of rats. Am J Physiol Regul Integr Comp Physiol 302: R785-R793, 2012. First published December 28, 2011; doi:10.1152/ajpregu.00363.2011.-Sympathetic overactivity and altered respiratory control are commonly observed after chronic intermittent hypoxia (CIH) exposure. However, the central mechanisms underlying such neurovegetative dysfunctions remain unclear. Herein, we hypothesized that CIH (6% O-2 every 9 min, 8 h/day, 10 days) in juvenile rats alters glutamatergic transmission in the commissural nucleus tractus solitarius (cNTS), a pivotal site for integration of peripheral chemoreceptor inputs. Using an in situ working heart-brain stem preparation, we found that L-glutamate microinjections (1, 3, and 10 mM) into the cNTS of control rats (n = 8) evoked increases in thoracic sympathetic nerve (tSN) and central vagus nerve (cVN) activities combined with inhibition of phrenic nerve (PN) activity. Besides, the ionotropic glutamatergic receptor antagonism with kynurenic acid (KYN; 250 mM) in the cNTS of control group (n = 7) increased PN burst duration and frequency. In the CIH group (n = 10), the magnitude of L-glutamate-induced cVN excitation was smaller, and the PN inhibitory response was blunted (P < 0.05). In addition, KYN microinjections into the cNTS of CIH rats (n = 9) did not alter PN burst duration and produced smaller increases in its frequency compared with controls. Moreover, KYN microinjections into the cNTS attenuated the sympathoexcitatory response to peripheral chemoreflex activation in control but not in CIH rats (P < 0.05). These functional CIH-induced alterations were accompanied by a significant 10% increase of N-methyl-D-aspartate receptor 1 (NMDAR1) and glutamate receptor 2/3 (GluR2/3) receptor subunit density in the cNTS (n = 3-8, P < 0.05), evaluated by Western blot analysis. These data indicate that glutamatergic transmission is altered in the cNTS of CIH rats and may contribute to the sympathetic and respiratory changes observed in this experimental model.

Effect of the interaction between food state and the action of estrogen on oxytocinergic system activity

Increased plasma osmolality by food intake evokes augmentation of plasma oxytocin (OT). Ovarian steroids may also influence the balance of body fluids by acting on OT neurones. Our aim was to determine if estrogen influences the activity of OT neurones in paraventricular nucleus (PVN) and supraoptic nucleus (SON) under different osmotic situations. Ovariectomized rats (OVX) were treated with either estradiol (E-2) or vehicle and were divided into three groups: group I was fed ad libitum, group II underwent 48 h of fasting, and group III was refed after 48 h of fasting. On the day of the experiment, blood samples were collected to determine the plasma osmolality and OT. The animals were subsequently perfused, and OT/FOS immunofluorescence analysis was conducted on neurones in the PVN and the SON. When compared to animals which were fasted or fed ad libitum, the plasma osmolality of refed animals was higher, regardless of whether they were treated with vehicle or E-2. We observed neural activation of OT cells in vehicle-or E-2-treated OVX rats refed after 48 h of fasting, but not in animals fed ad libitum or in animals that only underwent 48 h of fasting. Finally, the percentage of neurones that co-expressed OT and FOS was lower in both the PVN and the SON of animals treated with E-2 and refed, when compared to vehicle-treated animals. These results suggest that E-2 may have an inhibitory effect on OT neurones and may modulate the secretion of OT in response to the increase of osmolality induced by refeeding. Journal of Endocrinology (2012) 212, 129-138

Glutamatergic neurotransmission in the hypothalamus PVN on heart rate variability in exercise trained rats

The paraventricular nucleus of hypothalamus (PVN) is a well known site of integration for autonomic and cardiovascular responses, and the glutamate neurotransmitter plays an important role. The aim of our study was to evaluate the cardiovascular parameters and autonomic modulation by means of spectral analysis after ionotropic glutamate receptor inhibition in the PVN in conscious sedentary (S) or swimming trained (ST) rats. After exercise training protocol, adult male Wistar rats, instrumented with guide cannulae to PVN and artery and vein catheters were submitted to mean arterial pressure (MAP) and heart rate (HR) recording. At baseline, physical training induced a resting bradycardia (S: 379 +/- 3, ST: 349 +/- 2 bpm, P<0.05) and promoted adaptations in HRV characterized by an increase of HF in normalized values and a decrease of LF in absolute and normalized units compared with the sedentary group. Microinjection of kynurenic acid (KYNA) in the PVN of sedentary and trained rats promoted decreases in MAP and HR, but the decrease in HR was smaller in the trained animals (Delta HRS: -48 +/- 7, ST: -28 +/- 4 bpm, P<0.05). Furthermore, the differences in baseline parameters of pulse interval, found between sedentary and trained animals, disappeared after KYNA microinjection in the PVN. Our data suggest that the cardiovascular and autonomic adaptations to the heart induced by exercise training may involve glutamatergic mechanisms in the PVN. (C) 2012 Elsevier B.V. All rights reserved.

Availability of a rich source of sodium during the perinatal period programs the fluid balance restoration pattern in adult offspring

Osmoregulatory mechanisms can be vulnerable to electrolyte and/or endocrine environmental changes during the perinatal period, differentially programming the developing offspring and affecting them even in adulthood. The aim of this study was to evaluate whether availability of hypertonic sodium solution during the perinatal period may induce a differential programming in adult offspring osmoregulatory mechanisms. With this aim, we studied water and sodium intake after Furosemide-sodium depletion in adult offspring exposed to hypertonic sodium solution from 1 week before mating until postnatal day 28 of the offspring, used as a perinatal manipulation model [PM-Na group]. In these animals, we also identified the cell population groups in brain nuclei activated by Furosemide-sodium depletion treatment, analyzing the spatial patterns of Fos and Fos-vasopressin immunoreactivity. In sodium depleted rats, sodium and water intake were significantly lower in the PM-Na group vs. animals without access to hypertonic sodium solution [PM-Ctrol group]. Interestingly, when comparing the volumes consumed of both solutions in each PM group, our data show the expected significant differences between both solutions ingested in the PM-Ctrol group, which makes an isotonic cocktail: however, in the PM-Na group there were no significant differences in the volumes of both solutions consumed after Furosemide-sodium depletion, and therefore the sodium concentration of total fluid ingested by this group was significantly higher than that in the PM-Ctrol group. With regard to brain Fos immunoreactivity, we observed that Furosemide-sodium depletion in the PM-Na group induced a higher number of activated cells in the subfornical organ, ventral subdivision of the paraventricular nucleus and vasopressinergic neurons of the supraoptic nucleus than in the PM-Ctrol animals. Moreover, along the brainstem, we found a decreased number of sodium depletion-activated cells within the nucleus of the solitary tract of the PM-Na group. Our data indicate that early sodium availability induces a long-term effect on fluid drinking and on the cell activity of brain nuclei involved in the control of hydromineral balance. These results also suggest that availability of a rich source of sodium during the perinatal period may provoke a larger anticipatory response in the offspring, activating the vasopressinergic system and reducing thirst after water and sodium depletion, as a result of central osmosensitive mechanism alterations. (C) 2011 Elsevier Inc. All rights reserved.

Brain Pathways Involved in the Modulatory Effects of Noradrenaline in Lateral Septal Area on Cardiovascular Responses

We have previously reported that stimulation of alpha-1 adrenoceptors by noradrenaline (NA) injected into the lateral septal area (LSA) of anaesthetized rats causes pressor and bradycardic responses that are mediated by acute vasopressin release into the circulation through activation of the paraventricular nucleus (PVN). Although the PVN is the final structure of this pathway, the LSA has no direct connections with the PVN, suggesting that other structures may connect these areas. To address this issue, the present study employed c-Fos immunohistochemistry to investigate changes caused by NA microinjection into the LSA in neuronal activation in brain structures related to systemic vasopressin release. NA microinjected in the LSA caused pressor and bradycardic responses, which were blocked by intraseptal administration of alpha-1 adrenoceptor antagonist (WB4101, 10 nmol/200 nL) or systemic V-1 receptor antagonist (dTyr(CH2)5(Me)AVP, 50 mu g/kg). NA also increased c-Fos immunoreactivity in the prelimbic cortex (PL), infralimbic cortex (IL), dorsomedial periaqueductal gray (dmPAG), bed nucleus of the stria terminalis (BNST), PVN, and medial amygdala (MeA). No differences in the diagonal band of Broca, cingulate cortex, and dorsolateral periaqueductal gray (dlPAG) were found. Systemic administration of the vasopressin receptor antagonist dTyr AVP (CH2)5(Me) did not change the increase in c-Fos expression induced by intra-septal NA. This latter effect, however, was prevented by local injection of the alpha-1 adrenoceptor antagonist WB4101. These results suggest that areas such as the PL, IL, dmPAG, BNST, MeA, and PVN could be part of a circuit responsible for vasopressin release after activation of alpha-1 adrenoceptors in the LSA.

Neuronal NOS Inhibitor and Conventional Antidepressant Drugs Attenuate Stress-induced Fos Expression in Overlapping Brain Regions

Recent evidence indicates that the administration of inhibitors of neuronal nitric oxide synthase (nNOS) induces antidepressant-like effects in animal models such as the forced swimming test (FST). However, the neural circuits involved in these effects are not yet known. Therefore, this study investigated the expression of Fos protein, a marker of neuronal activity, in the brain of rats submitted to FST and treated with the preferential nNOS inhibitor, 7-nitroindazole (7-NI), or with classical antidepressant drugs (Venlafaxine and Fluoxetine). Male Wistar rats were submitted to a forced swimming pretest (PT) and, immediately after, started receiving a sequence of three ip injections (0, 5, and 23 h after PT) of Fluoxetine (10 mg/kg), Venlafaxine (10 mg/kg), 7-NI (30 mg/kg) or respective vehicles. One hour after the last drug injection the animals were submitted to the test session, when immobility time was recorded. After the FST they were sacrificed and had their brains removed and processed for Fos immunohistochemistry. Independent group of non-stressed animals received the same drug treatments, or no treatment (naive). 7-NI, Venlafaxine or Fluoxetine reduced immobility time in the FST, an antidepressant-like effect. None of the treatments induce significant changes in Fos expression per se. However, swimming stress induced significant increases in Fos expression in the following brain regions: medial prefrontal cortex, nucleus accumbens, locus coeruleus, raphe nuclei, striatum, hypothalamic nucleus, periaqueductal grey, amygdala, habenula, paraventricular nucleus of hypothalamus, and bed nucleus of stria terminalis. This effect was attenuated by 7-NI, Venlafaxine or Fluoxetine. These results show that 7-NI produces similar behavioral and neuronal activation effects to those of typical antidepressants, suggesting that these drugs share common neurobiological substrates.

Glial cell activity is maintained during prolonged inflammatory challenge in rats

We evaluated the expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), ionized calcium binding adaptor protein-1 (Iba-1), and ferritin in rats after single or repeated lipopolysaccharide (LPS) treatment, which is known to induce endotoxin tolerance and glial activation. Male Wistar rats (200-250 g) received ip injections of LPS (100 µg/kg) or saline for 6 days: 6 saline (N = 5), 5 saline + 1 LPS (N = 6) and 6 LPS (N = 6). After the sixth injection, the rats were perfused and the brains were collected for immunohistochemistry. After a single LPS dose, the number of GFAP-positive cells increased in the hypothalamic arcuate nucleus (ARC; 1 LPS: 35.6 ± 1.4 vs control: 23.1 ± 2.5) and hippocampus (1 LPS: 165.0 ± 3.0 vs control: 137.5 ± 2.5), and interestingly, 6 LPS injections further increased GFAP expression in these regions (ARC = 52.5 ± 4.3; hippocampus = 182.2 ± 4.1). We found a higher GS expression only in the hippocampus of the 6 LPS injections group (56.6 ± 0.8 vs 46.7 ± 1.9). Ferritin-positive cells increased similarly in the hippocampus of rats treated with a single (49.2 ± 1.7 vs 28.1 ± 1.9) or repeated (47.6 ± 1.1 vs 28.1 ± 1.9) LPS dose. Single LPS enhanced Iba-1 in the paraventricular nucleus (PVN: 92.8 ± 4.1 vs 65.2 ± 2.2) and hippocampus (99.4 ± 4.4 vs 73.8 ± 2.1), but had no effect in the retrochiasmatic nucleus (RCA) and ARC. Interestingly, 6 LPS increased the Iba-1 expression in these hypothalamic and hippocampal regions (RCA: 57.8 ± 4.6 vs 36.6 ± 2.2; ARC: 62.4 ± 6.0 vs 37.0 ± 2.2; PVN: 100.7 ± 4.4 vs 65.2 ± 2.2; hippocampus: 123.0 ± 3.8 vs 73.8 ± 2.1). The results suggest that repeated LPS treatment stimulates the expression of glial activation markers, protecting neuronal activity during prolonged inflammatory challenges.

Fluoxetine exposure during pregnancy and lactation: effects on acute stress response and behavior in the novelty-suppressed feeding are age and gender-dependent in rats

Fluoxetine (FLX) is commonly used to treat anxiety and depressive disorders in pregnant women. Since FLX crosses the placenta and is excreted in milk, maternal treatment with this antidepressant may expose the fetus and neonate to increased levels of serotonin (5-HT). Long-term behavioral abnormalities have been reported in rodents exposed to higher levels of 5-HT during neurodevelopment. In this study we evaluated if maternal exposure to FLX during pregnancy and lactation would result in behavioral and/or stress response disruption in adolescent and adult rats. Our results indicate that exposure to FLX influenced restraint stress-induced Fos expression in the amygdala in a gender and age-specific manner. In male animals, a decreased expression was observed in the basolateral amygdala at adolescence and adulthood; whereas at adulthood, a decrease was also observed in the medial amygdala. A lack of FLX exposure effect was observed in females and also in the paraventricular nucleus of both genders. Regarding the behavioral evaluation, FLX exposure did not induce anhedonia in the sucrose preference test but decreased the latency to feed of both male and female adolescent rats evaluated in the novelty-suppressed feeding test. In conclusion, FLX exposure during pregnancy and lactation decreases acute amygdalar stress response to a psychological stressor in males (adolescents and adults) as well as influences the behavior of adolescents (males and females) in a model that evaluates anxiety and/or depressive-like behavior. Even though FLX seems to be a developmental neurotoxicant, the translation of these findings to human safe assessment remains to be determined since it is recognized that not treating a pregnant or lactating woman may also impact negatively the development of the descendants.

The Effect of Chronic Estrogen Administration on Same-Sex Social Behavior and Central Oxytocin Levels in Prairie Voles (Microtus ochrogaster)

The purpose of our study is to investigate the effects of chronic estrogen administration on same-sex interactions during exposure to a social stressor and on oxytocin (OT) levels in prairie voles (Microtus orchrogaster). Estrogen and OT are two hormones known to be involved with social behavior and stress. Estogen is involved in the transcription of OT and its receptor. Because of this, it is generally thought that estrogen upregulates OT, but evidence to support this assumption is weak. While estrogen has been shown to either increase or decrease stress, OT has been shown to have stress-dampening properties. The goal of our experiment is to determine how estrogen affects OT levels as well as behavior in a social stressor in the voles. In addition, estrogen is required for many opposite-sex interactions, but little is known about its influence on same-sex interactions. We hypothesized that prairie voles receiving chronic estrogen injections would show an increase in OT levels in the brain and alter behavior in response to a social stressor called the resident-intruder test. To test this hypothesis, 73 female prairie voles were ovariectomized and then administered daily injections of estrogen (0.05 ¿g in peanut oil, s.c.) or vehicle for 8 days. On the final day of injections, half of the voles were given the resident-intruder test, a stressful 5 min interaction with a same-sex stranger. Their behavior was video-recorded. These animals were then sacrificed either 10 minutes or 60 minutes after the conclusion of the test. Half of the animals (no stress group) were not given the resident-intruder test. After sacrifice, trunk blood and brains were collected from the animals. Videos of the resident-intruder tests were analyzed for pro-social and aggressive behavior. Density of OT-activated neurons in the brain was measured via pixel count using immunohistochemistry. No differences were found in pro-social behavior (focal sniffing, p = 0.242; focal initiated sniffing p = 0.142; focal initiated sniffing/focal sniffing, p = 0.884) or aggressive behavior (total time fighting, p= 0.763; number of fights, p= 0.148; number of strikes, p = 0.714). No differences were found in activation of OT neurons in the brain, neither in the anterior paraventricular nucleus (PVN) (pixel count p= 0.358; % area that contains pixelated neurons p = 0.443) nor in the medial PVN (pixel count p= 0.999; % area that contains pixelated neurons p = 0.916). These results suggest that estrogen most likely does not directly upregulate OT and that estrogen does not alter behavior in stressful social interactions with a same-sex stranger. Estrogen may prepare the animal to respond to OT, instead of increasing the production of the peptide itself, suggesting that we need to shift the framework in which we consider estrogen and OT interactions.

Assessment of Alcohol Reward in Prairie Voles (Microtus ochrogaster)

The purpose of our study was to assess whether prairie voles find alcohol rewarding. Prairie voles have recently become a species of interest for alcohol studies, which have traditionally used other rodent model species including several different strains of mice and rats. The prairie vole is one of only two known rodent species that readily administers high levels of unsweetened alcohol, implicating it as a potentially effective animal model for studying alcohol abuse. However, voluntary consumption does not necessarily imply that prairie voles find it rewarding. Therefore the purpose of our study was to investigate if alcohol has rewarding properties for prairie voles using three different approaches: place conditioning, flavor conditioning, and immunohistochemistry. Furthermore, we sought to characterize their reward profile and compare it to other commonly used rodent models ¿ C57BL/6 mice, DBA/2J mice, and Sprague-Dawley rats. Place and flavor conditioning are behavioral methods that rely on the learned association between a stimulus and the effects of a drug; the drug of interest in these studies is alcohol. To assess whether prairie voles will demonstrate a conditioned preference for alcohol-paired stimuli, seven place conditioning studies were run that investigated a range of different doses, individual conditioning session durations, and trial durations. Video analysis revealed no difference in the amount of time spent on the alcohol-paired floor, suggesting no conditioned place preference for alcohol. Two flavor conditioning tests were conducted to assess whether voles would demonstrate a preference for an alcohol-paired flavored saccharin solution. Voles demonstrated reduced consumption of the alcohol-paired flavored saccharin solution, regardless of dose or flavor, when alcohol administration occurred after conditioning sessions (p=<0.001). When alcohol was administered before conditioning sessions, no difference in consumption of the alcohol-paired and saline-paired flavored saccharin solutions was seen (p=0.545). Previous studies that have documented similar behavior have hypothesized that this is an example of an anticipatory contrast effect. This theory proposes that prairie voles reduce their intake of a hedonic solution (flavored saccharin solution) in anticipation of later drug administration (alcohol). However, conditioning-based behavioral methods of studying alcohol reward are highly sensitive to the parameters of the conditioned stimulus, thus it is possible that voles will not show preference for alcohol-related stimuli, even if they do find alcohol rewarding. Immunohistochemical analysis supplemented this behavioral data by allowing us to identify specific neural regions that were directly activated in response to the acute administration of alcohol. No difference in the number of activated c-Fos neurons in the Nucleus Accumbens (NAc) core or shell was seen (p=0.3364; p=0.6698) in animals that received an acute injection of alcohol or saline. There was a significant increase in the number of activated c-Fos neurons in the Paraventricular Nucleus of the Hypothalamus (PVN) in alcohol-treated animals compared to saline-treated animals (p=0.0034). There was no difference in the pixel count of activated c-Fos neurons or in the % area activated in the Arcuate Nucleus between alcohol and saline-treated animals (p=0.4523; p=0.3304). In conclusion, the place conditioning studies that were conducted in this thesis suggest that prairie voles do not demonstrate preference or aversion towards alcohol-paired stimuli. The flavor conditioning studies suggest that prairie voles do not demonstrate aversion but rather avoidance of the alcohol-paired flavor in anticipation of future alcohol administration. The preliminary immunohistochemical data collected is inconclusive but cannot rule out the possibility of neuronal activation patterns indicative of reward. Taken together, our data indicate that prairie voles hav

Galanin regulates prolactin release and lactotroph proliferation

The neuropeptide galanin is predominantly expressed by the lactotrophs (the prolactin secreting cell type) in the rodent anterior pituitary and in the median eminence and paraventricular nucleus of the hypothalamus. Prolactin and galanin colocalize in the same secretory granule, the expression of both proteins is extremely sensitive to the estrogen status of the animal. The administration of estradiol-17β induces pituitary hyperplasia followed by adenoma formation and causes a 3,000-fold increase in the galanin mRNA content of the lactotroph. To further study the role of galanin in prolactin release and lactotroph growth we now report the generation of mice carrying a loss-of-function mutation of the endogenous galanin gene. There is no evidence of embryonic lethality and the mutant mice grow normally. The specific endocrine abnormalities identified to date, relate to the expression of prolactin. Pituitary prolactin message levels and protein content of adult female mutant mice are reduced by 30–40% compared with wild-type controls. Mutant females fail to lactate and pups die of starvation/dehydration unless fostered onto wild-type mothers. Prolactin secretion in mutant females is markedly reduced at 7 days postpartum compared with wild-type controls with an associated failure in mammary gland maturation. There is an almost complete abrogation of the proliferative response of the lactotroph to high doses of estrogen, with a failure to up-regulate prolactin release, STAT5 expression or to increase pituitary cell number. These data further support the hypothesis that galanin acts as a paracrine regulator of prolactin expression and as a growth factor to the lactotroph.