Perifornical Lateral Hypothalamus Research Articles

Lesion of NPY Receptor-expressing Neurons in Perifornical Lateral Hypothalamus Attenuates Glucoprivic Feeding.

Glucoprivic feeding is one of several counterregulatory responses (CRRs) that facilitates restoration of euglycemia following acute glucose deficit (glucoprivation). Our previous work established that glucoprivic feeding requires ventrolateral medullary (VLM) catecholamine (CA) neurons that coexpress neuropeptide Y (NPY). However, the connections by which VLM CA/NPY neurons trigger increased feeding are uncertain. We have previously shown that glucoprivation, induced by an anti-glycolygic agent 2-deoxy-D-glucose (2DG), activates perifornical lateral hypothalamus (PeFLH) neurons and that expression of NPY in the VLM CA/NPY neurons is required for glucoprivic feeding. We therefore hypothesized that glucoprivic feeding and possibly other CRRs require NPY-sensitive PeFLH neurons. To test this, we used the ribosomal toxin conjugate NPY-saporin (NPY-SAP) to selectively lesion NPY receptor-expressing neurons in the PeFLH of male rats. We found that NPY-SAP destroyed a significant number of PeFLH neurons, including those expressing orexin, but not those expressing melanin-concentrating hormone. The PeFLH NPY-SAP lesions attenuated 2DG-induced feeding but did not affect 2DG-induced increase in locomotor activity, sympathoadrenal hyperglycemia, or corticosterone release. The 2DG-induced feeding response was also significantly attenuated in NPY-SAP-treated female rats. Interestingly, PeFLH NPY-SAP lesioned male rats had reduced body weights and decreased dark cycle feeding, but this effect was not seen in female rats. We conclude that a NPY projection to the PeFLH is necessary for glucoprivic feeding, but not locomotor activity, hyperglycemia, or corticosterone release, in both male and female rats.

From Molecule to Behavior: Hypocretin/orexin Revisited From a Sex-dependent Perspective.

The hypocretin/orexin (Hcrt/Orx) system in the perifornical lateral hypothalamus has been recognized as a critical node in a complex network of neuronal systems controlling both physiology and behavior in vertebrates. Our understanding of the Hcrt/Orx system and its array of functions and actions has grown exponentially in merely 2 decades. This review will examine the latest progress in discerning the roles played by the Hcrt/Orx system in regulating homeostatic functions and in executing instinctive and learned behaviors. Furthermore, the gaps that currently exist in our knowledge of sex-related differences in this field of study are discussed.

Patterns of neural activity in the mouse brain: Wakefulness vs. General anesthesia

In light of the general shift from rats to mice as the leading rodent model in neuroscience research we used c-Fos expression as a tool to survey brain regions in the mouse in which neural activity differs between the states of wakefulness and pentobarbital-induced general anesthesia. The aim was to complement prior surveys carried out in rats. In addition to a broad qualitative review, 28 specific regions of interest (ROIs) were evaluated quantitatively. Nearly all ROIs in the cerebral cortex showed suppressed activity during anesthesia. Subcortically, however, some ROIs showed suppression, some showed little change, and some showed increased activity. The overall picture was similar to the rat. Special attention was devoted to ROIs significantly activated during anesthesia, as such loci might actively drive the transition to anesthetic unconsciousness rather than responding passively to inhbitory agents distributed globally (the “wet blanket” hypothesis). Twelve such “anesthesia-on” ROIs were identified: the paraventricular hypothalamic nucleus, supraoptic nucleus, tuberomamillary nucleus, lateral habenular nucleus, dentate gyrus, nucleus raphe pallidus, central amygdaloid nucleus, perifornical lateral hypothalamus, ventro-lateral preoptic area, lateral septum, paraventricular thalamic nucleus and zona incerta. The same primary anti-FOS antibody was used in all mice, but two alternative reporter systems were employed: ABC-diaminobenzidine and the currently more popular AlexaFluor488. Fluorescence tagging revealed far fewer FOS-immunoreactive neurons, sounding an alert that the reporter system chosen can have major effects on results obtained.

Sleep well. Untangling the role of melatonin MT1 and MT2 receptors in sleep.

The pharmacological potential of targeting selectively melatonin MT1 or MT2 receptors has not yet been exploited in medicine. Research using selective MT1/MT2 receptor ligands and MT1/MT2 receptor knockout mice has indicated that the activation of MT2 receptors selectively increases non-rapid eye movement (NREM) sleep whereas MT1 receptors seem mostly implicated in the regulation of REM sleep. Moreover, MT1 knockout mice show an increase in NREM sleep, while MT2 knockout a decrease, suggesting an opposite role of these two receptors. A recent paper in mice by Sharma et al (J Pineal Res, 2018, e12498) found that MT1 but not MT2 receptors are expressed on orexin neurons in the perifornical lateral hypothalamus (PFH). Moreover, after injecting melatonin or luzindole into the mouse PFH, the authors suggest that melatonin promotes NREM sleep because activates PFH MT1 receptors, which in turn inhibit orexin neurons that are important in promoting arousal and maintaining wakefulness. In this commentary, we have critically commented on some of these findings on the bases of previous literature. In addition, we highlighted the fact that no conclusions could be drawn on the melatonin receptor subtype mediating the effects of melatonin on sleep because the authors used the non-selective MT1/MT2 receptors antagonist luzindole. More solid research should further characterize the pharmacological function of these two melatonin receptors in sleep.

Melatonin promotes sleep in mice by inhibiting orexin neurons in the perifornical lateral hypothalamus.

Melatonin promotes sleep. However, the underlying mechanisms are unknown. Orexin neurons in the perifornical lateral hypothalamus (PFH) are pivotal for wake promotion. Does melatonin promote sleep by inhibiting orexin neurons? We used C57BL/6J mice and designed 4 experiments to address this question. Experiment 1 used double-labeled immunofluorescence and examined the presence of melatonin receptors on orexin neurons. Second, mice, implanted with bilateral guides targeted toward PFH and sleep-recording electrodes, were infused with melatonin (500pmole/50nL/side) at dark onset (onset of active period), and spontaneous bouts of sleep-wakefulness were examined. Third, mice, implanted with bilateral guides into the PFH, were infused with melatonin (500pmole/50nL/side) at dark onset and euthanized 2hours later, to examine the activation of orexin neurons using c-Fos expression in orexin neurons. Fourth, mice, implanted with PFH bilateral guides and sleep-recording electrodes, were infused with melatonin receptor antagonist, luzindole (10pmol/50nL/side), at light onset (onset of sleep period), and spontaneous bouts of sleep-wakefulness were examined. Our results suggest that orexin neurons express MT1, but not MT2 receptors. Melatonin infusion into the PFH, at dark onset, site-specifically and significantly increased NREM sleep (43.7%, P=.003) and reduced wakefulness (12.3%, P=.013). Local melatonin infusion at dark onset inhibited orexin neurons as evident by a significant reduction (66%, P=.0004) in the number of orexin neurons expressing c-Fos. Finally, luzindole infusion-induced blockade of melatonin receptors in PFH at sleep onset significantly increased wakefulness (44.1%, P=.015). Based on these results, we suggest that melatonin may act via the MT1 receptors to inhibit orexin neurons and promote sleep.

Relationship of the Chemokine, CXCL12, to Effects of Dietary Fat on Feeding-Related Behaviors and Hypothalamic Neuropeptide Systems.

The intake of a high fat diet (HFD), in addition to stimulating orexigenic neuropeptides in the hypothalamus while promoting overeating and reducing locomotor behavior, is known to increase inflammatory mediators that modulate neuronal systems in the brain. To understand the involvement of chemokines in the effects of a HFD, we examined in rats whether HFD intake affects a specific chemokine, CXCL12, and its receptors, CXCR4 and CXCR7, in the hypothalamus together with the neuropeptides and whether CXCL12 itself acts similarly to a HFD in stimulating the neuropeptides and altering ingestion and locomotor behavior. Compared to low-fat chow, a HFD for 5 days significantly increased the expression of CXCL12 and its receptors, in both the paraventricular nucleus (PVN) where the neuropeptides enkephalin (ENK) and galanin were also stimulated and the perifornical lateral hypothalamus (PFLH) where orexin (OX) and melanin-concentrating hormone (MCH) were increased. In contrast, the HFD had no impact on expression of CXCL12 or its receptors in the arcuate nucleus (ARC) where the carbohydrate-related peptide, neuropeptide Y (NPY), was suppressed. Analysis of protein levels revealed a similar stimulatory effect of a HFD on CXCL12 levels in the PVN and PFLH, as well as in blood, and an increase in the number of CXCR4-positive cells in the PVN. In the ARC, in contrast, levels of CXCL12 and number of CXCR4-positive cells were too low to measure. When centrally administered, CXCL12 was found to have similar effects to a HFD. Injection of CXCL12 into the third cerebral ventricle immediately anterior to the hypothalamus significantly stimulated the ingestion of a HFD, reduced novelty-induced locomotor activity, and increased expression of ENK in the PVN where the CXCR4 receptors were dense. It had no impact, however, on NPY in the ARC or on OX and MCH in the PFLH where the CXCR4 receptors were not detected. These results, showing CXCL12 in the hypothalamus to be stimulated by a HFD and to mimic the effects of the HFD where its receptors are located, suggest that this chemokine system may have a role in mediating both the neuronal and behavioral effects induced by a fat-rich diet.

Prenatal fat exposure and hypothalamic PPAR β/δ: Possible relationship to increased neurogenesis of orexigenic peptide neurons

Gestational exposure to a fat-rich diet, while elevating maternal circulating fatty acids, increases in the offspring’s hypothalamus and amygdala the proliferation and density of neurons that express neuropeptides known to stimulate consummatory behavior. To understand the relationship between these phenomena, this study examined in the brain of postnatal offspring (day 15) the effect of prenatal fat exposure on the transcription factor, peroxisome proliferator-activated receptor (PPAR) β/δ, which is sensitive to fatty acids, and the relationship of PPAR β/δ to the orexigenic neuropeptides, orexin, melanin-concentrating hormone, and enkephalin. Prenatal exposure to a fat-rich diet compared to low-fat chow increased the density of cells immunoreactive for PPAR β/δ in the hypothalamic paraventricular nucleus (PVN), perifornical lateral hypothalamus (PFLH), and central nucleus of the amygdala (CeA), but not the hypothalamic arcuate nucleus or basolateral amygdaloid nucleus. It also increased co-labeling of PPAR β/δ with the cell proliferation marker, BrdU, or neuronal marker, NeuN, and the triple labeling of PPAR β/δ with BrdU plus NeuN, indicating an increase in proliferation and density of new PPAR β/δ neurons. Prenatal fat exposure stimulated the double-labeling of PPAR β/δ with orexin or melanin-concentrating hormone in the PFLH and enkephalin in the PVN and CeA and also triple-labeling of PPAR β/δ with BrdU and these neuropeptides, indicating that dietary fat increases the genesis of PPAR β/δ neurons that produce these peptides. These findings demonstrate a close anatomical relationship between PPAR β/δ and the increased proliferation and density of peptide-expressing neurons in the hypothalamus and amygdala of fat-exposed offspring.

5-Hydroxytryptamine 1A receptors in the dorsomedial hypothalamus connected to dorsal raphe nucleus inputs modulate defensive behaviours and mediate innate fear-induced antinociception

The dorsal raphe nucleus (DRN) is an important brainstem source of 5-hydroxytryptamine (5-HT), and 5-HT plays a key role in the regulation of panic attacks. The aim of the present study was to determine whether 5-HT1A receptor-containing neurons in the medial hypothalamus (MH) receive neural projections from DRN and to then determine the role of this neural substrate in defensive responses. The neurotracer biotinylated dextran amine (BDA) was iontophoretically microinjected into the DRN, and immunohistochemical approaches were then used to identify 5HT1A receptor-labelled neurons in the MH. Moreover, the effects of pre-treatment of the dorsomedial hypothalamus (DMH) with 8-OH-DPAT and WAY-100635, a 5-HT1A receptor agonist and antagonist, respectively, followed by local microinjections of bicuculline, a GABAA receptor antagonist, were investigated. We found that there are many projections from the DRN to the perifornical lateral hypothalamus (PeFLH) but also to DMH and ventromedial (VMH) nuclei, reaching 5HT1A receptor-labelled perikarya. DMH GABAA receptor blockade elicited defensive responses that were followed by antinociception. DMH treatment with 8-OH-DPAT decreased escape responses, which strongly suggests that the 5-HT1A receptor modulates the defensive responses. However, DMH treatment with WAY-100635 failed to alter bicuculline-induced defensive responses, suggesting that 5-HT exerts a phasic influence on 5-HT1A DMH neurons. The activation of the inhibitory 5-HT1A receptor had no effect on antinociception. However, blockade of the 5-HT1A receptor decreased fear-induced antinociception. The present data suggest that the ascending pathways from the DRN to the DMH modulate panic-like defensive behaviours and mediate antinociceptive phenomenon by recruiting 5-HT1A receptor in the MH.

Hindbrain Catecholamine Neurons Activate Orexin Neurons During Systemic Glucoprivation in Male Rats.

Hindbrain catecholamine neurons are required for elicitation of feeding responses to glucose deficit, but the forebrain circuitry required for these responses is incompletely understood. Here we examined interactions of catecholamine and orexin neurons in eliciting glucoprivic feeding. Orexin neurons, located in the perifornical lateral hypothalamus (PeFLH), are heavily innervated by hindbrain catecholamine neurons, stimulate food intake, and increase arousal and behavioral activation. Orexin neurons may therefore contribute importantly to appetitive responses, such as food seeking, during glucoprivation. Retrograde tracing results showed that nearly all innervation of the PeFLH from the hindbrain originated from catecholamine neurons and some raphe nuclei. Results also suggested that many catecholamine neurons project collaterally to the PeFLH and paraventricular hypothalamic nucleus. Systemic administration of the antiglycolytic agent, 2-deoxy-D-glucose, increased food intake and c-Fos expression in orexin neurons. Both responses were eliminated by a lesion of catecholamine neurons innervating orexin neurons using the retrogradely transported immunotoxin, anti-dopamine-β-hydroxylase saporin, which is specifically internalized by dopamine-β-hydroxylase-expressing catecholamine neurons. Using designer receptors exclusively activated by designer drugs in transgenic rats expressing Cre recombinase under the control of tyrosine hydroxylase promoter, catecholamine neurons in cell groups A1 and C1 of the ventrolateral medulla were activated selectively by peripheral injection of clozapine-N-oxide. Clozapine-N-oxide injection increased food intake and c-Fos expression in PeFLH orexin neurons as well as in paraventricular hypothalamic nucleus neurons. In summary, catecholamine neurons are required for the activation of orexin neurons during glucoprivation. Activation of orexin neurons may contribute to appetitive responses required for glucoprivic feeding.

Appetitive associative learning recruits a distinct network with cortical, striatal, and hypothalamic regions

The amygdala, prefrontal cortex, striatum and other connected forebrain areas are important for reward-associated learning and subsequent behaviors. How these structurally and functionally dissociable regions are recruited during initial learning, however, is unclear. Recently, we showed amygdalar nuclei were differentially recruited across different stages of cue-food associations in a Pavlovian conditioning paradigm. Here, we systematically examined Fos induction in the forebrain, including areas associated with the amygdala, during early (day 1) and late (day 10) training sessions of cue-food conditioning. During training, rats in the conditioned group received tone-food pairings, while controls received presentations of the tone alone in the conditioning chamber followed by food delivery in their home cage. We found that a small subset of telencephalic and hypothalamic regions were differentially recruited during the early and late stages of training, suggesting evidence of learning-induced plasticity. Initial tone-food pairings recruited solely the amygdala, while late tone-food pairings came to induce Fos in distinct areas within the medial and lateral prefrontal cortex, the dorsal striatum, and the hypothalamus (lateral hypothalamus and paraventricular nucleus). Furthermore, within the perifornical lateral hypothalamus, tone-food pairings selectively recruited neurons that produce the orexigenic neuropeptide orexin/hypocretin. These data show a functional map of the forebrain areas recruited by appetitive associative learning and dependent on experience. These selectively activated regions include interconnected prefrontal, striatal, and hypothalamic regions that form a discrete but distributed network that is well placed to simultaneously inform cortical (cognitive) processing and behavioral (motivational) control during cue-food learning.

Hypothalamic peptides controlling alcohol intake: Differential effects on microstructure of drinking bouts

Different alcohol drinking patterns, involving either small and frequent drinking bouts or large and long-lasting bouts, are found to differentially affect the risk for developing alcohol-related diseases, suggesting that they have different underlying mechanisms. Such mechanisms may involve orexigenic peptides known to stimulate alcohol intake through their actions in the hypothalamic paraventricular nucleus (PVN). These include orexin (OX), which is expressed in the perifornical lateral hypothalamus, and galanin (GAL) and enkephalin (ENK), which are expressed within as well as outside the PVN. To investigate the possibility that these peptides affect different aspects of consumption, a microstructural analysis of ethanol drinking behavior was performed in male, Sprague–Dawley rats trained to drink 7% ethanol and implanted with guide shafts aimed at the PVN. While housed in specialized cages containing computerized intake monitors (BioDAQ Laboratory Intake Monitoring System, Research Diets Inc., New Brunswick, NJ) that measure bouts of ethanol drinking, these rats were given PVN injections of OX (0.9 nmol), GAL (1.0 nmol), or the ENK analog D-Ala2-met-enkephalinamide (DALA) (14.2 nmol), as compared to saline vehicle. Results revealed clear differences between the effects of these peptides. While all 3 stimulated ethanol intake, they had distinct effects on patterns of drinking, with OX increasing the number of drinking bouts, GAL increasing the size of the drinking bouts, and DALA increasing both the size and duration of the bouts. In contrast, these peptides had little impact on water or food intake. These results support the idea that different peptides can increase ethanol consumption by promoting distinct aspects of the ethanol drinking response. The stimulatory effect of OX on drinking frequency may be related to its neuronally stimulatory properties, while the stimulatory effect of GAL and ENK on bout size and duration may reflect a suppressive effect of these neuronally inhibitory peptides on the satiety-controlling PVN.

Differential Role of D1 and D2 Receptors in the Perifornical Lateral Hypothalamus in Controlling Ethanol Drinking and Food Intake: Possible Interaction with Local Orexin Neurons

The neurotransmitter dopamine (DA), acting in various mesolimbic brain regions, is well known for its role in promoting motivated behaviors, including ethanol (EtOH) drinking. Indirect evidence, however, suggests that DA in the perifornical lateral hypothalamus (PF/LH) has differential effects on EtOH consumption, depending on whether it acts on the DA 1 (D1) or DA 2 (D2) receptor subtype, and that these effects are mediated in part by local peptide systems, such as orexin/hypocretin (OX) and melanin-concentrating hormone (MCH), known to stimulate the consumption of EtOH. The present study in brain-cannulated Sprague-Dawley rats measured the effects of dopaminergic compounds in the PF/LH on drinking behavior in animals trained to consume 7% EtOH and also on local peptide mRNA expression using digoxigenin-labeled in situ hybridization in EtOH-naïve animals. Experiments 1 and 2 showed that the D1 agonist SKF81297 (10.8nmol/side) in the PF/LH significantly increased food intake, while tending to increase EtOH intake, and the D1 antagonist SCH23390 significantly decreased EtOH intake without affecting food intake. In contrast, the D2 agonist quinelorane (6.2nmol/side) in the PF/LH significantly reduced EtOH consumption, while the D2 antagonist sulpiride increased it. Experiments 3 and 4 revealed differential effects of PF/LH injection of the DA agonists on local OX mRNA, which was increased by the D1 agonist and decreased by the D2 agonist. These DA agonists had no impact on MCH expression. These results support a stimulatory role of the PF/LH D1 receptor in promoting the consumption of both EtOH and food, in contrast to a suppressive effect of the D2 receptor on EtOH drinking. They further suggest that these receptors affect consumption, in part, through local OX-expressing neurons. These findings provide new evidence for the function of PF/LH DA receptor subtypes in controlling EtOH and food intake.

Prenatal Exposure to Nicotine Stimulates Neurogenesis of Orexigenic Peptide-Expressing Neurons in Hypothalamus and Amygdala

Animal and clinical studies show that gestational exposure to nicotine increases the propensity of offspring to consume nicotine, but the precise mechanism mediating this behavioral phenomenon is unclear. The present study in Sprague Dawley rats examined the possibility that the orexigenic peptide systems, enkephalin (ENK) and orexin (OX), which are stimulated by nicotine in adult animals and promote consummatory behavior, may be similarly responsive to nicotine's stimulatory effect in utero while having long-term behavioral consequences. The results demonstrated that nicotine exposure during gestation at low doses (0.75 or 1.5 mg/kg/d) significantly increased mRNA levels and density of neurons that express ENK in the hypothalamic paraventricular nucleus and central nucleus of the amygdala, OX, and another orexigenic peptide, melanin-concentrating hormone, in the perifornical lateral hypothalamus in preweanling offspring. These effects persisted in the absence of nicotine, at least until puberty. Colabeling of the cell proliferation marker BrdU with the neuronal marker NeuN and peptides revealed a marked stimulatory effect of prenatal nicotine on neurogenesis, but not gliogenesis, and also on the number of newly generated neurons expressing ENK, OX, or melanin-concentrating hormone. During adolescence, offspring also exhibited significant behavioral changes, increased consumption of nicotine and other substances of abuse, ethanol and a fat-rich diet, with no changes in chow and water intake or body weight. These findings reveal a marked sensitivity during gestation of the orexigenic peptide neurons to low nicotine doses that may increase the offspring's propensity to overconsume substances of abuse during adolescence.

Differential contribution of brain‐region‐specific melanocortin 5 receptor to physical activity levels in lean vs. obesity‐prone rats

The brain melanocortin (MC) system consists of peptides such as α‐MSH that act via MC receptors (MCR) to modulate energy balance. MC3R and MC4R are known to alter energy expenditure and food intake, but the role of MC5R in the brain remains poorly understood. We previously demonstrated that high‐activity rats bred for high‐aerobic capacity (HCR) show enhanced MC5R mRNA expression in the peri‐fornical lateral hypothalamus (PeFLH) compared to their low‐activity, low‐aerobic capacity counterparts (LCR). Here, we tested the hypothesis that hypothalamic MC5R expression is integral to the high‐activity phenotype. We measured physical activity (PA) after site‐directed microinjections in the PeFLH, using a specific agonist or antagonist of MC5R in a rat model of leanness vs. obesity (HCR and LCR). Intra‐PeFLH microinjections of the MC5R agonist significantly enhanced PA over a 3‐hour period, and this effect was greater in HCR relative to LCR. Moreoever, the MC5R antagonist significantly suppressed night‐time PA in lean HCR, but not obesity‐prone LCR. The MC5R agonist did not significantly alter food intake in either group. Taken together, these data implicate region‐specific MC5R in individual differences in PA; MC5R‐mediated PA may be a key trait distinguishing the lean phenotype. MC5R agonist was obtained from M. Bednarek (MedImmune Ltd, Cambridge, UK). Research support was from NIH R01NS055859 to CMN and AHA 11PRE7320029 to CS.

Opioids in the perifornical lateral hypothalamus suppress ethanol drinking

The opioid system is known to enhance motivated behaviors, including ethanol drinking and food ingestion, by acting in various reward-related brain regions, such as the nucleus accumbens, ventral tegmental area and medial hypothalamus. There is indirect evidence, however, suggesting that opioid peptides may act differently in the perifornical lateral hypothalamus (PF/LH), causing a suppression of consummatory behavior. Using brain-cannulated Sprague–Dawley rats trained to voluntarily drink 7% ethanol, the present study tested the hypothesis that opioids in the PF/LH can reduce the consumption of ethanol, with animals receiving PF/LH injections of the δ-opioid receptor agonist D-Ala2-met-enkephalinamide (DALA), the μ-receptor agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO), the κ-receptor agonist (±)-trans-U-50,488 methanesulfonate (U-50,488H), or the general opioid antagonist methylated naloxone (m-naloxone). The consumption of ethanol, lab chow, and water was monitored for 4 h after injection. The results showed that the three opioid receptor agonists injected into the PF/LH specifically and significantly reduced ethanol intake, while causing little change in chow or water intake, and the opposite effect, enhanced ethanol intake, was observed with the opioid antagonist. Of the three opioid agonists, the δ-agonist appears to produce the most consistent and long-lasting suppression of consumption. This effect was not observed with injections 2 mm dorsal to this area, focusing attention on the PF/LH as the main site of action. These results suggest that the opioid peptides have a specific role in the PF/LH of reducing ethanol drinking, which is distinct from their more commonly observed appetitive actions in other brain areas. The additional finding, that m-naloxone in the PF/LH stimulates ethanol intake in contrast to its generally suppressive effect in other regions, focuses attention on this hypothalamic area and its distinctive role in contributing to the variable effects sometimes observed with opioid antagonist therapy for alcoholism.

Pregnancy affects FOS rhythms in brain regions regulating sleep/wake state and body temperature in rats

Circadian rhythms in behavior and physiology change substantially as female mammals undergo the transition from a non-pregnant to a pregnant state. Here, we examined the possibility that site-specific changes in brain regions known to regulate the sleep/wake cycle and body temperature might reflect altered rhythms in these overt functions. Specifically, we compared daily patterns of immunoreactive FOS in early pregnant and diestrous rats in the medial septum (MS), vertical and horizontal diagonal bands of Broca (VDB and HDB), perifornical lateral hypothalamus (LH), and ventrolateral, medial, and median preoptic areas (VLPO, MPA, and MnPO, respectively). In the pregnant animals, FOS expression was reduced and the daily rhythms of expression were lost or attenuated in the MS, VDB, and LH, areas known to support wakefulness, and in the MPA, a brain region that may coordinate sleep/wake patterns with temperature changes. However, despite the well-documented differences in sleep patterns between diestrous and pregnant rats, reproductive state did not affect FOS expression in the VLPO or MnPO, two brain regions in which FOS expression usually correlates with sleep. These data indicate that plasticity in sleep/wake patterns during early pregnancy may be driven by a reduction in wakefulness-promotion by the brain, rather than by an increase in sleep drive.

Region-specific differences in brain melanocortin receptors in rats of the lean phenotype

The brain melanocortin (MC) system is one of numerous overlapping systems regulating energy balance; it consists of peptides including α-melanocyte-stimulating hormone that act through melanocortin receptors (MCRs). Mutations and polymorphisms in MC3R and MC4R have been identified as one of the most common genetic contributors to obesity in human studies. Brain MC3R and MC4R are known to modulate energy expenditure (EE) and food intake, but much less is known regarding brain MC5R. To test the hypothesis that brain MC modulates physical activity (PA) and EE, we compared brain MCR profiles in rats that consistently show high versus low levels of 'spontaneous' daily PA. Compared with low-activity rats, high-activity rats show enhanced mRNA expression of MCRs in the brain, specifically of MC3R in the paraventricular nucleus (PVN), and MC4R and MC5R in the perifornical lateral hypothalamus. Next, we microinjected the MCR agonist melanotan II into the PVN region and measured PA and EE. Intra-PVN melanotan II induced a dose-dependent increase in PA and this effect was greater in high-activity rats compared with low-activity rats. These results indicate region-specific brain MCR expression in the heightened PA seen in association with high endurance capacity and identify promising targets in the brain MC system that may contribute to interindividual variability in energy balance.

Prenatal ethanol exposure stimulates neurogenesis in hypothalamic and limbic peptide systems: Possible mechanism for offspring ethanol overconsumption

Exposure to ethanol during the prenatal period contributes to increased alcohol consumption and preference in rodents and increased risk for alcoholism in humans. With studies in adult animals showing the orexigenic peptides, enkephalin (ENK), galanin (GAL) and orexin (OX), to stimulate ethanol consumption, the question addressed here is whether prenatal ethanol alters the development in utero of specific neurons that express these peptides. With reports describing suppressive effects of high doses of ethanol, we examined the offspring of dams gavaged from embryonic day 9 to parturition with a control solution or lower ethanol doses, 1 and 3g/kg/day, known to promote ethanol consumption in the offspring. To understand underlying mechanisms, measurements were taken in postnatal offspring of the expression of ENK in the hypothalamic paraventricular nucleus (PVN) and nucleus accumbens (NAc), GAL in the PVN, and OX in the perifornical lateral hypothalamus (PFLH) using real-time qPCR and in situ hybridization, and also of the cell proliferation marker, 5-bromo-2-deoxyuridine (BrdU), and its double-labeling with either neuronal nuclei (NeuN), a marker of mature neurons, or the peptides. On postnatal day 15 (P15), after two weeks without ethanol, the offspring showed increased expression of ENK in the PVN and NAc core but not shell, GAL in the PVN, and OX in the PFLH. In these same areas, prenatal ethanol compared to control increased the density at birth (P0) of neurons expressing these peptides and at P0 and P15 of neurons double-labeling BrdU and NeuN, indicating increased neurogenesis. These BrdU-positive neurons were found to express ENK, GAL and OX, indicating that prenatal ethanol promotes neurogenesis in these specific peptide systems. There were no changes in gliogenesis or apoptosis. This increase in neurogenesis and density of peptide-expressing neurons suggests the involvement of these hypothalamic and accumbal peptide systems in mediating the increased alcohol consumption observed in prenatal ethanol-exposed offspring.

Neurochemical Heterogeneity of Rats Predicted by Different Measures to be High Ethanol Consumers

Alcoholism is a heterogeneous disease, with subjects possibly differing both in the best measure that predicts their excess consumption and in their most effective pharmacotherapy. Two different measures, high novelty-induced activity and high-fat-induced triglycerides (TGs), are known to identify subgroups of animals prone to consuming higher amounts of ethanol (EtOH). The question investigated here is whether these subgroups are, in fact, similar in their neurochemical phenotype that may contribute to their overconsumption. EtOH-naïve, Sprague-Dawley rats were subgrouped based on the 2 predictor measures of activity or TG levels, and then quantitative real-time polymerase chain reaction and digoxigenin-labeled in situ hybridization were used to measure their expression of hypothalamic peptides that affect EtOH intake. In additional subgroups subsequently trained to drink 9% EtOH, the opioid antagonist and alcoholism medication, naltrexone, was tested at a low dose (0.02 mg/kg, s.c.) to determine the rats' sensitivity to its effects. The 2 measures, while both effective in predicting amount of EtOH intake, were found to identify distinctive subgroups. Rats with high compared to low activity exhibited significantly greater expression of galanin and enkephalin in the paraventricular nucleus (PVN) and of orexin in the perifornical lateral hypothalamus (PFLH), but no difference in melanin-concentrating hormone in PFLH or neuropeptide Y in arcuate nucleus. This contrasts with rats having high TG, which exhibited greater expression only of PVN galanin, along with reduced PFLH orexin. The high-activity rats with elevated enkephalin, but not high-TG rats, were also unusually sensitive to naltrexone, which significantly reduced their alcohol intake. In addition to revealing differences in endogenous peptides and drug responsiveness in predicted high EtOH drinkers, this study demonstrates that these disturbances differ markedly between the 2 at-risk subgroups. This indicates that simple tests may be effective in identifying subjects most responsive to a specific pharmacotherapy.

Role of brain melanocortin receptor 5 in physical activity in rats

The brain melanocortin (MC) system is one of several systems that regulate energy balance; it consists of peptides such as α‐MSH that act via MC receptors (MCR). Brain MC3R and MC4R are known to modulate energy expenditure and food intake; little is known about the role of MC5R in the brain, however. We previously demonstrated that, compared to low‐activity rats, high‐activity rats show enhanced MC5R mRNA expression in the peri‐fornical lateral hypothalamus (PeFLH) but not in the paraventricular nucleus (PVN). To test the hypothesis that hypothalamic MC5R expression is related to enhanced physical activity (PA), we stereotaxically implanted guide cannulae in male Sprague‐Dawley rats, then measured the ability of a novel MC5R‐specific agonist in the PVN and PeFLH to increase PA. Intra‐PeFLH microinjections significantly enhanced PA in a dose‐dependent manner, while no such effect was seen in the PVN. The MC5R agonist (compared to vehicle) did not significantly suppress food intake in either of the hypothalamic regions, however. Thus, specific, regional activation of brain MC receptors could affect different aspects of energy balance, particularly PA. We will now investigate if these site‐ and receptor subtype‐specific MC differences may underlie individual differences in daily PA. MC5R agonist was obtained from M. Bednarek (MedImmune Ltd, Cambridge, UK). Research support was from NIH R01NS055859 to CMN and AHA11PRE7320029 to CS.