Dorsomedial Nucleus Research Articles

Transsynaptic connections between the hypothalamus and adipose tissue: relationship to reproduction

Neurophysiological mechanisms that control energy balance are reciprocally linked to those that control reproduction. Neuromorphological studies using retrograde tracing methods revealed that nerve cells within the central (CNS) and autonomic (ANS) nervous systems in different species, including the pig, are transsynaptically connected to different fat tissue depots. In the pig, neurons localised in the paraventricular nucleus, supraoptic nucleus and arcuate nucleus were infected with pseudorabies virus (PRV) 9 days after injections into both the perirenal and subcutaneous adipose tissue depots. Infected neurons were in the ventromedial nucleus, dorsomedial nucleus and preoptic area after injection of PRV into perirenal adipose tissue, while infected cells in the lateral hypothalamic area projected only to the subcutaneous adipose tissue depot. Additionally, numerous centres of the ANS innervate adipose tissue depots in the pig. Fast blue stained (FB+) neurons, which projected to the subcutaneous adipose tissue overlaying the thoracolumbar area were located in the thoraco-lumbar region of the sympathetic chain ganglia (SChG). However, neurons supplying perirenal and mesentery adipose tissue depots were found in both the SChG and prevertebral ganglia. The vast majority of labelled neurons, in both the CNS and ANS, which innervated adipose tissue depots, expressed leptin receptor (OBR) immunoreactivity. The purpose of this brief review is to establish evidence for a multisynaptic circuit of neurons, which innervate adipose tissue in the pig and demonstrate that hypothalamic nuclei and sympathetic ganglion neurons involved in reproductive processes are transsynaptically connected to different adipose tissue depots.

Hypothalamic-Pituitary and Adipose Tissue Responses to the Effect of Resistin in Sheep: The Integration of Leptin and Resistin Signaling Involving a Suppressor of Cytokine Signaling 3 and the Long Form of the Leptin Receptor.

We hypothesized that resistin is engaged in the development of leptin central insensitivity/resistance in sheep, which is a unique animal model to explore reversible leptin resistance. Thirty Polish Longwool ewes, which were ovariectomized with estrogen replacement, were used. Treatments consisted of the intravenous injection of control (saline) or recombinant bovine resistin (rbresistin): control (Control; n = 10), a low dose of rbresistin (R1; 1.0 μg/kg body weight (BW); n = 10), and a high dose of rbresistin (R2; 10.0 μg/kg BW; n = 10). The studies were performed during short-day (SD) and long-day (LD) photoperiods. Leptin and resistin concentrations were determined. Expression levels of a suppressor of cytokine signaling (SOCS)-3 and the long form of the leptin receptor (LeptRb) were determined in selected brain regions, including in the anterior pituitary (AP), hypothalamic arcuate nucleus (ARC), preoptic area (POA), and ventro- and dorsomedial nuclei (VMH/DMH). The results indicate that resistin induced a consistent decrease in LeptRb (except in POA) and an increase in SOCS-3 expression during the LD photoperiod in all selected brain regions. In conclusion, the results demonstrate that the action of resistin appears to be strongly associated with photoperiod-driven changes in the leptin signaling pathway, which may underlie the phenomenon of central leptin resistance.

Systemic Glucose Administration Alters Water Diffusion and Microvascular Blood Flow in Mouse Hypothalamic Nuclei - An fMRI Study.

The hypothalamus is the principal regulator of global energy balance, enclosing additionally essential neuronal centers for glucose-sensing and osmoregulation. Disturbances in these tightly regulated neuronal networks are thought to underlie the development of severe pandemic syndromes, including obesity and diabetes. In this work, we investigate in vivo the response of individual hypothalamic nuclei to the i.p. administration of glucose or vehicle solutions, using two groups of adult male C57BL6/J fasted mice and a combination of non-invasive T2∗-weighted and diffusion-weighted functional magnetic resonance imaging (fMRI) approaches. MRI parameters were assessed in both groups of animals before, during and in a post-stimulus phase, following the administration of glucose or vehicle solutions. Hypothalamic nuclei depicted different patterns of activation characterized by: (i) generalized glucose-induced increases of neuronal activation and perfusion-markers in the lateral hypothalamus, arcuate and dorsomedial nuclei, (ii) cellular shrinking events and decreases in microvascular blood flow in the dorsomedial, ventromedial and lateral hypothalamus, following the administration of vehicle solutions and (iii) increased neuronal activity markers and decreased microperfusion parameters in the ARC nuclei of vehicle-administered animals. Immunohistochemical studies performed after the post-stimulus phase confirmed the presence of c-Fos immunoreactive neurons in the arcuate nucleus (ARC) from both animal groups, with significantly higher numbers in the glucose-treated animals. Together, our results reveal that fMRI methods are able to detect in vivo diverse patterns of glucose or vehicle-induced effects in the different hypothalamic nuclei.

Hindbrain lactoprivic regulation of hypothalamic neuron transactivation and gluco-regulatory neurotransmitter expression: Impact of antecedent insulin-induced hypoglycemia

Hindbrain energy state shapes hypothalamic control of glucostasis. Dorsal vagal complex (DVC) L-lactate deficiency is a potent glucose-stimulatory signal that triggers neuronal transcriptional activation in key hypothalamic metabolic loci. The energy gauge AMPK is activated in DVC metabolic-sensory A2 noradrenergic neurons by hypoglycemia-associated lactoprivation, but sensor reactivity is diminished by antecedent hypoglycemia (AH). Current research addressed the premise that AH alters hindbrain lactoprivic regulation of hypothalamic metabolic transmitter function. AH did not modify reductions in A2 dopamine-beta-hydroxylase and monocarboxylate-2 (MCT2) protein expression elicited by caudal fourth ventricular delivery of the MCT inhibitor alpha-cyano-4-hydroxycinnamic acid (4CIN), but attenuated 4CIN activation of A2 AMPK. 4CIN constraint of hypothalamic norepinephrine (NE) activity was averted by AH in a site-specific manner. 4CIN induction of Fos immunolabeling in hypothalamic arcuate (ARH), ventromedial (VMN), dorsomedial (DMN) and paraventricular (PVN) nuclei and lateral hypothalamic area (LHA) was avoided by AH. AH affected reactivity of select hypothalamic metabolic neurotransmitter/enzyme marker proteins, e.g. ARH neuropeptide Y, VMN glutamate decarboxylase, DMN RFamide-related peptide-1 and -3, and LHA orexin-A profiles to 4CIN, but did not alleviate drug inhibition of ARH proopiomelanocortin. AH prevented 4CIN augmentation of circulating glucagon, but did not alter hyperglycemic or hypocorticosteronemic responses to that treatment. Results identify hindbrain lactate deficiency as a stimulus for glucagon secretion, and imply that habituation of this critical counter-regulatory hormone to recurring hypoglycemia may involve one or more hypothalamic neurotransmitters characterized here by acclimation to this critical sensory stimulus.

Glucose-Dependent Insulinotropic Polypeptide Receptor-Expressing Cells in the Hypothalamus Regulate Food Intake

SummaryAmbiguity regarding the role of glucose-dependent insulinotropic polypeptide (GIP) in obesity arises from conflicting reports asserting that both GIP receptor (GIPR) agonism and antagonism are effective strategies for inhibiting weight gain. To enable identification and manipulation of Gipr-expressing (Gipr) cells, we created Gipr-Cre knockin mice. As GIPR-agonists have recently been reported to suppress food intake, we aimed to identify central mediators of this effect. Gipr cells were identified in the arcuate, dorsomedial, and paraventricular nuclei of the hypothalamus, as confirmed by RNAscope in mouse and human. Single-cell RNA-seq identified clusters of hypothalamic Gipr cells exhibiting transcriptomic signatures for vascular, glial, and neuronal cells, the latter expressing somatostatin but little pro-opiomelanocortin or agouti-related peptide. Activation of Gq-DREADDs in hypothalamic Gipr cells suppressed food intake in vivo, which was not obviously additive with concomitant GLP1R activation. These data identify hypothalamic GIPR as a target for the regulation of energy balance.

Crh receptor priming in the bed nucleus of the stria terminalis (BNST) induces tph2 gene expression in the dorsomedial dorsal raphe nucleus and chronic anxiety

The bed nucleus of the stria terminalis (BNST) is a nodal structure in neural circuits controlling anxiety-related defensive behavioral responses. It contains neurons expressing the stress- and anxiety-related neuropeptide corticotropin-releasing hormone (Crh) as well as Crh receptors. Repeated daily subthreshold activation of Crh receptors in the BNST is known to induce a chronic anxiety-like state, but how this affects neurotransmitter-relevant gene expression in target regions of the BNST is still unclear. Since the BNST projects heavily to the dorsal raphe nucleus (DR), the main source of brain serotonin, we here tested the hypothesis that such repeated, anxiety-inducing activation of Crh receptors in the BNST alters the expression of serotonergic genes in the DR, including tph2, the gene encoding the rate-limiting enzyme for brain serotonin synthesis, and slc6a4, the gene encoding the serotonin transporter (SERT). For 5 days, adult male Wistar rats received daily, bilateral, intra-BNST microinjections of vehicle (1% bovine serum albumin in 0.9% saline, n = 11) or behaviorally subthreshold doses of urocortin 1 (Ucn1, n = 11), a potent Crh receptor agonist. Priming with Ucn1 increased tph2 mRNA expression selectively within the anxiety-related dorsal part of the DR (DRD) and decreased social interaction (SI) time, a measure of anxiety-related defensive behavioral responses in rodents. Decreased social interaction was strongly correlated with increased tph2 mRNA expression in the DRD. Together with previous studies, our data are consistent with the hypothesis that Crh-mediated control of the BNST/DRD-serotonergic system plays a key role in the development of chronic anxiety states, possibly also contributing to stress-induced relapses in drug abuse and addiction behavior.

Selective brain neuronal and glial losses without changes in GFAP immunoreactivity: Young versus mature adult Wistar rats

Normal ageing results in brain selective neuronal and glial losses. In the present study we analyze neuronal and glial changes in Wistar rats at two different ages, 45 days (young) and 420 days (mature adult), using Nissl staining and glial fibrillary acidic protein (GFAP) immunohistochemistry associated to the Sholl analysis. Comparing mature adults with young rats we noted the former present a decrease in neuronal density in the cerebral cortex, corpus callosum, pyriform cortex, L.D.D.M., L.D.V.L., central medial thalamic nucleus and zona incerta. A decrease in glial density was found in the dorsomedial and ventromedial hypothalamic nuclei. Additionally, the neuron/glia ratio was reduced in the central medial thalamic nucleus and increased in the habenula. No changes were found in the neuronal and glial densities or neuron/glia ratio in the other studied regions. The number of astrocytic primary processes and the number of intersections counted in the Sholl analysis presented no significant difference in any of the studied regions. Overall, neither GFAP positive astrocytic density nor GFAP immunoreactivity showed alteration.

Vulnerability to stress consequences induced by repeated social defeat in rats: Contribution of the angiotensin II type 1 receptor in cardiovascular alterations associated to low brain derived neurotrophic factor

After social stress, rats become vulnerable to depression, and this state is characterized by persistent low blood levels of brain-derived neurotrophic factor (BDNF). The aim of this study was to determine whether low BDNF levels are associated with long term autonomic changes. Defeated animals were subjected to four daily episodes of social defeats. Twenty five days later, defeated rats with low BDNF levels (Dlow) still displayed elevated sympathetic tone (as indicated by an elevated low frequency to high frequency ratio (LF/HF) in heart rate) and elevated blood pressure, as well as reduced baroreflex sensitivity (BRS). In contrast, those with higher BDNF levels (Dhigh) similar to controls, did not. Dlow animals persistent cardiovascular changes were abolished by acute inhibition of the dorsomedial nucleus of the hypothalamus (DMH). These cardiovascular changes were also prevented by chronic sub-cutaneous osmotic infusion of losartan, an angiotensin II type 1 receptor (AT1) receptor antagonist, started immediately after social defeat. In conclusion, the results show that greater vulnerability to stress consequences following a traumatic event is associated with an elevated LF/HF ratio, a persistent high blood pressure and a low BRS, all due to an AT1 receptor activation.

The dromedary camel displays annual variation in hypothalamic kisspeptin and Arg-Phe-amide-related peptide-3 according to sex, season, and breeding activity.

The dromedary camel (Camelus dromedarius) is a desert mammal whose cycles in reproductive activity ensure that the offspring's birth and weaning coincide with periods of abundant food resources and favorable climate conditions. In this study, we assessed whether kisspeptin (Kp) and arginine-phenylalanine (RF)-amide related peptide-3 (RFRP-3), two hypothalamic peptides known to regulate the mammalian hypothalamo-pituitary gonadal axis, may be involved in the seasonal control of camel's reproduction. Using specific antibodies and riboprobes, we found that Kp neurons are present in the preoptic area (POA), suprachiasmatic (SCN), and arcuate (ARC) nuclei, and that RFRP-3 neurons are present in the paraventricular (PVN), dorsomedial (DMH), and ventromedial (VMH) hypothalamic nuclei. Kp fibers are found in various hypothalamic areas, notably the POA, SCN, PVN, DMH, VMH, supraoptic nucleus, and the ventral and dorsal premammillary nucleus. RFRP-3 fibers are found in the POA, SCN, PVN, DMH, VMH, and ARC. POA and ARC Kp neurons and DMH RFRP-3 neurons display sexual dimorphism with more neurons in female than in male. Both neuronal populations display opposed seasonal variations with more Kp neurons and less RFRP-3 neurons during the breeding (December-January) than the nonbreeding (July-August) season. This study is the first describing Kp and RFRP-3 in the camel's brain with, during the winter period lower RFRP-3 expression and higher Kp expression possibly responsible for the HPG axis activation. Altogether, our data indicate the involvement of both Kp and RFRP-3 in the seasonal control of the dromedary camel's breeding activity.

1816-P: Sex-Specific Metabolic Imbalance and Hypothalamic Inflammation Triggered by Low-Grade Benzene Exposure

Current studies have shown that insulin resistance, T2D have been associated with long- term exposure to air and traffic pollution. Benzene is a highly volatile liquid, which is a constituent of crude petroleum. Given its ubiquitous utilization in industry as well as consumer products, it is classified as a common airborne pollutant. We hypothesized that benzene, at levels below carcinogenic contributes to insulin resistance and inflammatory responses linking persistent organic pollutants exposure to T2D mellitus. For this purpose, C57BL/6 mice in inhalation chambers were exposed to benzene concentration of 50 ppm for 6h/day for 4 weeks. We found that under these conditions, exposure to benzene did not significantly influence mice body weight, neither had it trigger any toxic responses in these animals. Despite normal fasting glucose and insulin levels, benzene-exposed male mice displayed significantly impaired glucose tolerance. Consequently, we detected an increase in hepatic genes associated with gluconeogenesis, G6Pase and Pck1, and the expression of SREBP-1c and SREBP-2, genes associated with lipid synthesis was significantly elevated in livers from benzene exposed male mice as compared to control mice. Female mice were completely resistant to the negative metabolic consequences of chronic benzene exposure. Further sex differences were apparent in neuroinflammatory responses, with greater male vulnerability observed in the hypothalamus of benzene- exposed animals. Benzene exposure promoted hypothalamic gliosis and robust neuronal activation in the arcuate, paraventricular, ventromedial and dorsomedial hypothalamic nucleus. These findings demonstrate male vulnerability to air pollution, and provide evidence that exposure to benzene induces metabolic imbalance and severe neuroinflammatory responses in a sex-specific manner. Persistent ambient benzene exposure may be a heretofore unrecognized contributor to metabolic diseases with male bias. Disclosure L.K. Debarba: None. A. Mulka: None. A.A. Awada: None. O. Didyuk: None. U. Klueh: Research Support; Self; The Leona M. and Harry B. Helmsley Charitable Trust. Other Relationship; Self; Cell and Molecular Tissue Engineering, LLC. M. Sadagurski: None. Funding The Leona M. and Harry B. Helmsley Charitable Trust

Involvement of neuropeptide CART in the central effects of insulin on feeding and body weight

While insulin secreted from pancreas plays a pivotal role in the control of glucose homeostasis, it also interacts with hypothalamic sites and negatively influences the energy balance. The present study was undertaken to reveal the functional interaction between cocaine- and amphetamine-regulated transcript (CART), a well-known anorexic peptide, and insulin within the framework of hypothalamus in the regulation of feeding behavior and body weight. Insulin was administered daily by intracerebroventricular (icv) route, alone or in combination with CART (icv) for a period of seven days. Immediately thereafter, preweighed food was offered to the animals at the commencement of the dark phase. The food intake and body weight were measured daily just prior to next injection. Furthermore, brains of insulin-treated rats were processed for the immunohistochemical analysis of CART-containing elements in the hypothalamus. Treatment with insulin (6 mU, icv) for a period of 7 days caused a significant decrease in food intake and body weight as compared to control. Concomitant administration of CART (0.5 μg, icv) potentiated insulin-induced anorexia and weight loss. Insulin administration resulted in a significant increase in CART immunoreactivity in the hypothalamic arcuate, paraventricular, dorsomedial and ventromedial nuclei. We suggest that increased CART contents in the hypothalamus may be causally linked with anorexia and weight loss induced by insulin.

A spontaneous leptin receptor point mutation causes obesity and differentially affects leptin signaling in hypothalamic nuclei resulting in metabolic dysfunctions distinct from db/db mice

ObjectiveLeptin (Lep) plays a crucial role in controlling food intake and energy expenditure. Defective Lep/LepRb-signaling leads to fat accumulation, massive obesity, and the development of diabetes. We serendipitously noticed spontaneous development of obesity similar to LepR-deficient (db/db) mice in offspring from a C57BL/6J breeding and transmittance of the phenotype in a Mendelian manner. Candidate gene sequencing revealed a spontaneous point mutation in the LepRb gene. We investigated leptin responsiveness, leptin receptor signaling and metabolic phenotype of this novel LepRb mutant mouse variant. MethodsOverexpression and functional tests of the mutant LepRb in 3T3 cells. Measurement of leptin responsiveness in hypothalamic nuclei, glucose tolerance, food uptake and energy expenditure in the mutant mice. ResultsThe mutation results in the exchange of a glycine for serine (G506S) and introduces an alternative splice acceptor which, when used, encodes for a protein with a 40aa deletion that is retained in the cytoplasm. LepRb signaling was abrogated in the hypothalamic ventromedial nucleus (VMN) and dorsomedial nucleus (DMN), but only partially reduced in the hypothalamic arcuate nucleus (ARC) of LepRbG506S/G506S mice, most likely due to differential splicing in neurons located in the respective regions of the hypothalamus. Extensive metabolic characterization of these mice revealed interesting differences in the control of food intake, glucose tolerance, energy expenditure, and fat accumulation in LepRbG506S/G506S compared with LepRb-deficient db/db mice. ConclusionsThis study provides further insight into differences of the leptin responsiveness in VMN, DMN, and ARC and its metabolic consequences.

SAT-179 Low Grade Benzene Exposure Induces Metabolic Dysbalance and Hypothalamic Inflammation in Mice

Type 2-diabetes mellitus (T2D) has become a global pandemic resulting in a reduced quality of life due to comorbidity and premature mortality. The World Health Organization estimates the current global prevalence at 8.4%, which represents a fourfold increase over the last four decades. In this same interval, the US prevalence has increased by 160%, such that it affects 1 in 11 Americans with a disproportionate impact on minorities. The choice of lifestyle, diet, lack of physical activity and the growing aging population is often used to explain the rise of the diabetes pandemic. However, it is now increasingly accepted that insulin resistance, T2D prevalence and mortality have been associated with long- term exposure to air and traffic pollution. Benzene is a highly volatile liquid, which is a constituent of crude petroleum. Given its ubiquitous utilization in industry as well as consumer products, it is classified as a common airborne pollutant. As such, any exposure to petroleum or its products can result in significant occupational benzene exposure. While it is well accepted that excessive benzene exposure is carcinogenic in animals and humans, little is known regarding the effect of non-carcinogenic benzene level exposure and its metabolic effects. Notably, benzene exposure from motor vehicle exhaust is higher in inner city populations, which suggests a causative role in the pathogenesis of T2D. We hypothesized that benzene, at levels below carcinogenic contributes to insulin resistance and inflammatory responses linking persistent organic pollutants (POP) exposure to type 2 diabetes mellitus. For this purpose, C57BL/6 mice in inhalation chambers were exposed to benzene concentration of 50 ppm for 6h/day for 4 weeks. We found that under these conditions, exposure to benzene did not significantly change male mice body weight, neither had it trigger any toxic responses in these animals. However, despite normal fasting glucose and insulin levels, glucose tolerance test (GTT) of benzene exposed male mice displayed significantly impaired IP glucose tolerance. Consequently, we detected an increase in hepatic genes associated with gluconeogenesis, G6Pase and Pck1, and the expression of SREBP-1c and SREBP-2, genes associated with lipid synthesis was significantly elevated in livers from benzene exposed mice as compared to control mice. Additionally, we detected significant neuroinflammatory response in the hypothalamus of benzene exposed mice. Benzene exposure promoted a significant increase on microglia and astrocytes numbers in the arcuate, ventromedial and dorsomedial hypothalamic nucleus. We conclude that exposure to benzene induces hyperglycemia and that hypothalamic inflammatory responses may be a sensitive indicator of inhaled benzene toxicity.

Accumbal Adenosine A2A Receptors Enhance Cognitive Flexibility by Facilitating Strategy Shifting.

The deficits of cognitive flexibility (including attentional set-shifting and reversal learning) concomitant with dysfunction of the striatum are observed in several neuropsychiatric disorders. Rodent and human studies have identified the striatum [particularly the dorsomedial striatum (DMS) and nucleus accumbens (NAc)] as the critical locus for control of cognitive flexibility, but the effective neuromodulator and pharmacological control of cognitive flexibility remains to be determined. The adenosine A2A receptors (A2ARs) are highly enriched in the striatopallidal neurons where they integrate dopamine and glutamate signals to modulate several cognitive behaviors, but their contribution to cognitive flexibility control is unclear. In this study, by coupling an automated operant cognitive flexibility task with striatal subregional knockdown (KD) of the A2AR via the Cre-loxP strategy, we demonstrated that NAc A2AR KD improved cognitive flexibility with enhanced attentional set-shifting and reversal learning by decreasing regressive and perseverative errors, respectively. This facilitation was not attributed to mnemonic process or motor activity as NAc A2AR KD did not affect the visual discrimination, lever-pressing acquisition, and locomotor activity, but was associated with increased attention and motivation as evident by the progressive ratio test (PRT). In contrast to NAc A2ARs, DMS A2ARs KD neither affected visual discrimination nor improved set-shifting nor reversal learning, but promoted the effort-related motivation. Thus, NAc and DMS A2ARs exert dissociable controls of cognitive flexibility with NAc A2ARs KD selectively enhancing cognitive flexibility by facilitating strategy shifting with increased motivation/attention.

Nicotine-induced neuroplasticity in striatum is subregion-specific and reversed by motor training on the rotarod.

Nicotine is recognized as one of the most addictive drugs, which in part could be attributed to progressive neuroadaptations and rewiring of dorsal striatal circuits. Since motor‐skill learning produces neuroplasticity in the same circuits, we postulate that rotarod training could be sufficient to block nicotine‐induced rewiring and thereby prevent long‐lasting impairments of neuronal functioning. To test this hypothesis, Wistar rats were subjected to 15 days of treatment with either nicotine (0.36 mg/kg) or vehicle. After treatment, a subset of animals was trained on the rotarod. Ex vivo electrophysiology was performed 1 week after the nicotine treatment period and after up to 3 months of withdrawal to define neurophysiological transformations in circuits of the striatum and amygdala. Our data demonstrate that nicotine alters striatal neurotransmission in a distinct temporal and spatial sequence, where acute transformations are initiated in dorsomedial striatum (DMS) and nucleus accumbens (nAc) core. Following 3 months of withdrawal, synaptic plasticity in the form of endocannabinoid‐mediated long‐term depression (eCB‐LTD) is impaired in the dorsolateral striatum (DLS), and neurotransmission is altered in DLS, nAc shell, and the central nucleus of the amygdala (CeA). Training on the rotarod, performed after nicotine treatment, blocks neurophysiological transformations in striatal subregions, and prevents nicotine‐induced impairment of eCB‐LTD. These datasets suggest that nicotine‐induced rewiring of striatal circuits can be extinguished by other behaviors that induce neuroplasticity. It remains to be determined if motor‐skill training could be used to prevent escalating patterns of drug use in experienced users or facilitate the recovery from addiction.

Endogenous amylin contributes to birth of microglial cells in arcuate nucleus of hypothalamus and area postrema during fetal development.

Amylin acts in the area postrema (AP) and arcuate nucleus (ARC) to control food intake. Amylin also increases axonal fiber outgrowth from the AP→nucleus tractus solitarius and from ARC→hypothalamic paraventricular nucleus. More recently, exogenous amylin infusion for 4 wk was shown to increase neurogenesis in adult rats in the AP. Furthermore, amylin has been shown to enhance leptin signaling in the ARC and ventromedial nucleus of the hypothalamus (VMN). Thus, we hypothesized that endogenous amylin could be a critical factor in regulating cell birth in the ARC and AP and that amylin could also be involved in the birth of leptin-sensitive neurons. Amylin+/- dams were injected with BrdU at embryonic day 12 and at postnatalday 2; BrdU+ cells were quantified in wild-type (WT) and amylin knockout (KO) mice. The number of BrdU+HuC/D+ neurons was similar in ARC and AP, but the number of BrdU+Iba1+ microglia was significantly decreased in both nuclei. Five-week-old WT and KO littermates were injected with leptin to test whether amylin is involved in the birth of leptin-sensitive neurons. Although there was no difference in the number of BrdU+c-Fos+ neurons in the ARC and dorsomedial nucleus, an increase in BrdU+c-Fos+ neurons was seen in VMN and lateral hypothalamus (LH) in amylin KO mice. In conclusion, these data suggest that during fetal development, endogenous amylin favors the birth of microglial cells in the ARC and AP and that it decreases the birth of leptin-sensitive neurons in the VMN and LH.

Lateral parabrachial neurons innervate orexin neurons projecting to brainstem arousal areas in the rat

Orexin (ORX) neurons in the hypothalamus send their axons to arousal-promoting areas. We have previously shown that glutamatergic neurons in the lateral parabrachial nucleus (LPB) innervate ORX neurons. In this study, we examined potential pathways from the LPB to ORX neurons projecting to arousal-promoting areas in the brainstem by a combination of tract-tracing techniques in male Wistar rats. We injected the anterograde tracer biotinylated dextranamine (BDA) into the LPB and the retrograde tracer cholera toxin B subunit (CTb) into the ventral tegmental area, dorsal raphe nucleus, pedunculopontine tegmental nucleus, laterodorsal tegmental area, or locus coeruleus (LC). We then analyzed the BDA-labeled fibers and ORX-immunoreactive neurons in the hypothalamus. We found that double-labeled ORX and CTb neurons were the most abundant after CTb was injected into the LC. We also observed prominently overlapping distribution of BDA-labeled fibers, arising from neurons located in the lateral-most part of the dorsomedial nucleus and adjacent dorsal perifornical area. In these areas, we confirmed by confocal microscopy that BDA-labeled synaptophysin-immunoreactive axon terminals were in contiguity with cell bodies and dendrites of CTb-labeled ORX-immunoreactive neurons. These results suggest that the LPB innervates arousal-promoting areas via ORX neurons and is likely to promote arousal responses to stimuli.

Dopamine D1-like receptors in the dorsomedial prefrontal cortex regulate contextual fear conditioning

RationaleDopamine D1 receptor (D1R) signalling is involved in contextual fear conditioning. The D1R antagonist SCH23390 impairs the acquisition of contextual fear when administered systemically or infused locally into the dorsal hippocampus or basolateral amygdala.ObjectivesWe determined if state dependency may account for the impairment in contextual fear conditioning caused by systemic SCH23390 administration. We also examined if the dorsomedial prefrontal cortex (dmPFC), nucleus accumbens (NAc), and ventral hippocampus (VH) are involved in mediating the effect of systemic SCH23390 treatment on contextual fear conditioning.MethodsIn experiment 1, SCH23390 (0.1 mg/kg) or vehicle was given before contextual fear conditioning and/or retrieval. In experiment 2, SCH23390 (2.5 μg/0.5 uL) or vehicle was infused locally into dmPFC, NAc, or VH before contextual fear conditioning, and retrieval was tested drug-free. Freezing was quantified as a measure of contextual fear.ResultsIn experiment 1, SCH23390 given before conditioning or before both conditioning and retrieval decreased freezing at retrieval, whereas SCH23390 given only before retrieval had no effect. In experiment 2, SCH23390 infused into dmPFC before conditioning decreased freezing at retrieval, while infusion of SCH23390 into NAc or VH had no effect.ConclusionsThe results of experiment 1 confirm those of previous studies indicating that D1Rs are required for the acquisition but not retrieval of contextual fear and rule out state dependency as an explanation for these findings. Moreover, the results of experiment 2 provide evidence that dmPFC is also part of the neural circuitry through which D1R signalling regulates contextual fear conditioning.

Hypothalamic mechanisms associated with corticotropin-releasing factor-induced anorexia in chicks

Central administration of corticotropin-releasing factor (CRF), a 41-amino acid peptide, is associated with potent anorexigenic effects in rodents and chickens. However, the mechanism underlying this effect remains unclear. Hence, the objective of the current study was to elucidate the hypothalamic mechanisms that mediate CRF-induced anorexia in 4 day-old Cobb-500 chicks. After intracerebroventricular (ICV) injection of 0.02 nmol of CRF, CRF-injected chicks ate less than vehicle chicks while no effect on water intake was observed at 30 min post-injection. In subsequent experiments, the hypothalamus samples were processed at 60 min post-injection. The CRF-injected chicks had more c-Fos immunoreactive cells in the arcuate nucleus (ARC), dorsomedial nucleus (DMN), ventromedial hypothalamus (VMH), and paraventricular nucleus (PVN) of the hypothalamus than vehicle-treated chicks. CRF injection was associated with decreased whole hypothalamic mRNA abundance of neuropeptide Y receptor sub-type 1 (NPYR1). In the ARC, CRF-injected chicks expressed more CRF and CRF receptor sub-type 2 (CRFR2) mRNA but less agouti-related peptide (AgRP), NPY, and NPYR1 mRNA than vehicle-injected chicks. CRF-treated chicks expressed greater amounts of CRFR2 and mesotocin mRNA than vehicle chicks in the PVN and VMH, respectively. In the DMN, CRF injection was associated with reduced NPYR1 mRNA. In conclusion, the results provide insights into understanding CRF-induced hypothalamic actions and suggest that the anorexigenic effect of CRF involves increased CRFR2-mediated signaling in the ARC and PVN that overrides the effects of NPY and other orexigenic factors.

2-arachidonylglycerol interacts with nitric oxide in the dorsomedial hypothalamus to increase food intake and body weight in young male rats

The dorsomedial nucleus of the hypothalamus (DMH) is an important appetite regulatory center in the brain. In young rats, neural communication in the DMH is modulated by two interacting signals: endocannabinoids (eCBs) and nitric oxide (NO), both of which are known to modulate appetite. It remains unknown, however, whether eCBs and NO interact in the DMH to regulate food intake and body weight in young rats. We developed stereotaxic coordinates for the DMH in young, male Sprague-Dawley rats and conducted surgeries to implant bilateral guide cannulas for microinjection of vehicle, eCBs [2-arachidonylglycerol (2-AG) or anandamide]; NO (via the precursor l-arginine), or a combination of the two, with and without prior subcutaneous injections of drugs to block cannabinoid receptors or NO synthesis. Food intake and body weight of animals were measured two hours following the injection and brains were subsequently removed and sliced to verify placement of the cannulas relative to the DMH. Here we show that 2-AG, when administered in combination with l-arginine, significantly increased food intake and body weight, an effect that required type I cannabinoid receptors and NO synthesis. 2-AG and l-arginine had no effect on food intake or body weight when administered into the DMH independently. Anandamide also failed to affect these parameters when administered alone or with l-arginine. Together, these data suggest that 2-AG and NO interact in the DMH to increase food intake in young male rats and provide insight into a possible mechanism by which 2-AG increases appetite.