Cocaine And Amphetamine-related Transcript Research Articles

Demystifying functional role of cocaine- and amphetamine-related transcript (CART) peptide in control of energy homeostasis: A twenty-five year expedition

Cocaine- and amphetamine-related transcript (CART) is a neuropeptide first discovered in the striatum of the rat brain. Later, the genetic sequence and function of CART peptide (CARTp) was found to be conserved among multiple mammalian species. Over the 25 years, since its discovery, CART mRNA (Cartpt) expression has been reported widely throughout the central and peripheral nervous systems underscoring its role in diverse physiological functions. Here, we review the localization and function of CARTp as it relates to energy homeostasis. We summarize the expression changes of central and peripheral Cartpt in response to metabolic states and make use of available large data sets to gain additional insights into the anatomy of the Cartpt expressing vagal neurons and their expression patterns in the gut. Furthermore, we provide an overview of the role of CARTp as an anorexigenic signal and its effect on energy expenditure and body weight control with insights from both pharmacological and transgenic animal studies. Subsequently, we discuss the role of CARTp in the pathophysiology of obesity and review important new developments towards identifying a candidate receptor for CARTp signalling. Altogether, the field of CARTp research has made rapid and substantial progress recently, and we review the case for considering CARTp as a potential therapeutic target for stemming the obesity epidemic.

Integration of Nutrient Sensing in Fish Hypothalamus

The knowledge regarding hypothalamic integration of metabolic and endocrine signaling resulting in regulation of food intake is scarce in fish. Available studies pointed to a network in which the activation of the nutrient-sensing (glucose, fatty acid, and amino acid) systems would result in AMP-activated protein kinase (AMPK) inhibition and activation of protein kinase B (Akt) and mechanistic target of rapamycin (mTOR). Changes in these signaling pathways would control phosphorylation of transcription factors cAMP response-element binding protein (CREB), forkhead box01 (FoxO1), and brain homeobox transcription factor (BSX) leading to food intake inhibition through changes in the expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opio melanocortin (POMC), and cocaine and amphetamine-related transcript (CART). The present mini-review summarizes information on the topic and identifies gaps for future research.

The Regulatory Role of Apelin on the Appetite and Growth of Common Carp (Cyprinus Carpio L.).

Simple SummaryFood intake and growth are two interrelated physiological processes. Fish obtain energy from the outside to meet their own growth and development through food intake. Apelin, as a new endocrine factor, plays a wide range of biological roles. Among them, apelin’s role in regulating appetite and growth has attracted the attention of researchers. Based on our investigations, in vitro and in vivo experiments showed that Pyr-apelin-13 could induce significant changes in mRNA levels of appetite-related and growth-related genes, suggesting that Pyr-apelin-13 may regulate the appetite and growth of common carp by regulating the expression of these key genes.Apelin, a kind of active polypeptide, has many biological functions, such as promoting food intake, enhancing immunity, and regulating energy balance. In mammals, studies have indicated that apelin is involved in regulating food intake. However, there are relatively few studies about the regulatory effect of apelin on fish feeding, and the specific mechanism is not clear. Therefore, the purpose of this study was to preliminarily investigate the regulatory effects of apelin on key genes of feeding and growth in common carp (Cyprinus Carpio L.) through in vitro and in vivo experiments. In the present study, after incubation with different concentrations of Pyr-apelin-13 (0, 10, 100, and 1000 nM) in hypothalamic fragments, the expressions of Neuropeptide Y (NPY) and Agouti related peptide (AgRP) mRNA were significantly up-regulated at 12 and 3 h, respectively, and the significant down-regulation of Cocaine and amphetamine-related transcript (CART) mRNA expression was observed at 1 and 3 h. In vivo, after Pyr-apelin-13 oral administration (0, 1, 10, and 100 pmol/g), the orexin mRNA level in the hypothalamus of common carp was significantly increased at 1, 6, and 12 h, while CART/(Proopiomelanocortin) POMC mRNA levels in the hypothalamus of common carp were significantly down-regulated. Following incubation with different concentrations of Pyr-apelin-13 (0, 10, 100, and 1000 nM) in primary hepatocytes, GHR (Growth hormone receptor), IGF2 (Insulin-like growth factor 2), IGFBP2 (Insulin like growth factor binding protein 2), and IGFBP3 (Insulin like growth factor binding protein 3) mRNA levels were significantly increased at 3 h. In vivo, the levels of IGF1 (Insulin-like growth factor 1), IGF2, IGFBP2 (Insulin like growth factor binding protein 2), and IGFBP3 mRNA were significantly increased after the oral administration of Pyr-apelin-13 in the hepatopancreas, in a time and dose-dependent manner. These results support the hypothesis that Pyr-apelin-13 might regulate the feeding and growth of common carp through mediating the expressions of appetite- and growth-related genes. Overall, apelin, which is an orexigenic peptide, improves food intake and is involved in the growth of common carp.

Leptin Chronic Effect on Differentiation, Ion Currents and Protein Expression in N1E-115 Neuroblastoma Cells.

<b>Background and Objective:</b> Arcuate nucleus (ARC), a component of appetite-regulatory factors, contains populations of both orexigenic and anorexigenic neurons and one of the fundamental components of its system is leptin. Studies have evidenced the critical neurotrophic role in the development of ARC. To determine such effects on neuron development, N1E-115 neuroblastoma cells were used as an ARC model. <b>Materials and Methods:</b> N1E-115 neuroblastoma cells were treated with leptin [10 nM] for 24, 48 and 72 hrs. Dimethyl sulfoxide (DMSO) 1.5% was used as a known drug that promotes neurite expression. Cells percentage (%) that developed neurites was evaluated by bright field microscopy. Patch-clamp electrophysiology was used to analyze membrane ion currents, RT-PCR for quantifying changes in mRNA expression of anorexic peptides, proopiomelanocortin (POMC) and cocaine and amphetamine-related transcript (CART), in addition to principal Na<sub>v</sub>, Ca<sub>v</sub> ion channel subunits. <b>Results:</b> N1E-115 cells treated with leptin show neurite expression after 24 hrs of treatment, similar effects were obtained with DMSO. Leptin (time-dependent) increases the inward current in comparison with the control value at 72 hrs. Outward currents were not affected by leptin. Leptin and DMSO increased Na<sup>+</sup> and Ca<sup>2+</sup> current without changes in the kinetic properties. Lastly, leptin promotes an increase in mRNA level expression of transcripts to POMC, CART, Na<sub>v</sub>1.2 and Ca<sub>v</sub>1.3. <b>Conclusion:</b> Leptin chronic treatment promotes neurite expression, Up-regulation of Na<sup>+</sup> and Ca<sup>2+</sup> ion channels determining neuronal excitability, besides increasing the mRNA level expression of anorexic peptides POMC and CART in neuroblastoma N1E-115.

Convergent neuronal projections from paraventricular nucleus, parabrachial nucleus, and brainstem onto gastrocnemius muscle, white and brown adipose tissue in male rats.

When energy balance is altered by aerobic exercise, starvation, and cold exposure, for example, there appears to be coordination of the responses of skeletal muscle, white adipose (WAT), and brown adipose (BAT) tissues. We hypothesized that WAT, BAT, and skeletal muscle may share an integrated regulation by the central nervous system (CNS); specifically, that neurons in brain regions associated with energy balance would possess neuroanatomical connections to permit coordination of multiple, complementary responses in these downstream tissues. To study this, we used trans-neuronal viral retrograde tract tracing, using isogenic strains of pseudorabies virus (PRV) with distinct fluorescent reporters (either eGFP or mRFP), injected pairwise into male rat gastrocnemius, subcutaneous WAT and interscapular BAT, coupled with neurochemical characterization of specific cell populations for cocaine- and amphetamine-related transcript (CART), oxytocin (OX), corticotrophin releasing hormone (CRH) and calcitonin gene-related peptide (CGRP). Cells in the paraventricular (PVN) and parabrachial (PBN) nuclei and brainstem showed dual projections to muscle + WAT, muscle + BAT, and WAT + BAT. Dual PRV-labeled cells were found in parvocellular, magnocellular and descending/pre-autonomic regions of the PVN, and multiple structural divisions of the PBN and brainstem. In most PBN subdivisions, more than 50% of CGRP cells dually projected to muscle + WAT and muscle + BAT. Similarly, 31-68% of CGRP cells projected both to WAT + BAT. However, dual PRV-labeled cells in PVN only occasionally expressed OX or CRH but not CART. These studies reveal for the first time both separate and shared outflow circuitries among skeletal muscle and subcutaneous WAT and BAT.

Evidence for the presence in rainbow trout brain of amino acid-sensing systems involved in the control of food intake.

To assess the hypothesis of central amino acid-sensing systems involved in the control of food intake in fish, we carried out two experiments in rainbow trout. In the first one, we injected intracerebroventricularly two different branched-chain amino acids (BCAAs), leucine and valine, and assessed food intake up to 48 h later. Leucine decreased and valine increased food intake. In a second experiment, 6 h after similar intracerebroventricular treatment we determined changes in parameters related to putative amino acid-sensing systems. Different areas of rainbow trout brain present amino acid-sensing systems responding to leucine (hypothalamus and telencephalon) and valine (telencephalon), while other areas (midbrain and hindbrain) do not respond to these treatments. The decreased food intake observed in fish treated intracerebroventricularly with leucine could relate to changes in mRNA abundance of hypothalamic neuropeptides [proopiomelanocortin (POMC), cocaine- and amphetamine-related transcript (CART), neuropeptide Y (NPY), and agouti-related peptide (AgRP)]. These in turn could relate to amino acid-sensing systems present in the same area, related to BCAA and glutamine metabolism, as well as mechanistic target of rapamycin (mTOR), taste receptors, and general control nonderepressible 2 (GCN2) kinase signaling. The treatment with valine did not affect amino acid-sensing parameters in the hypothalamus. These responses are comparable to those characterized in mammals. However, clear differences arise when comparing rainbow trout and mammals, in particular with respect to the clear orexigenic effect of valine, which could relate to the finding that valine partially stimulated two amino acid-sensing systems in the telencephalon. Another novel result is the clear effect of leucine on telencephalon, in which amino acid-sensing systems, but not neuropeptides, were activated as in the hypothalamus.

Mild hypothermia ameliorates muscle wasting in septic rats associated with hypothalamic AMPK-induced autophagy and neuropeptides

Sepsis, always developing muscle wasting, contributes to serious complications and mortality. Mild hypothermia has been reported to have protective effects on the prognosis of septic patients. However, the underlying mechanisms remain unclear. We therefore hypothesized that mild hypothermia could ameliorate muscle wasting during sepsis and whether it was associated with hypothalamus AMPK-induced autophagy and neuropeptides. Adult male Sprague-Dawley rats were intraperitoneally injected with lipopolysaccharide (LPS) (5 mg/kg) or saline. Mild hypothermia was instantly induced at 33 °C for 3h after LPS injected. Meanwhile, the control and sepsis groups were simultaneously placed on the thermal mattress to maintain the a normal temperature in control group whatever the changes induced by anesthesia. Twenty-four hours after injection, skeletal muscle and hypothalamus tissues were obtained. Muscle wasting was measured by the mRNA expression of two muscle atrophic genes, muscle ring finger 1 (MuRF-1) and muscle atrophy F-box (MAFbx), as well as 3-methylhistidine (3-MH) and tyrosine release. Hypothalamic AMPK-induced autophagy markers and neuropeptides expression were also detected. Results showed that LPS administration significantly decreased hypothalamic AMPK-induced autophagy together with muscle wasting. Also, increased hypothalamic neuropeptides, proopiomelanocortin (POMC), cocaine and amphetamine-related transcript (CART) and neuro-peptides Y (NPY) and decreased agouti-related protein (AgRP) were observed. Mild hypothermia significantly increased hypothalamic AMPK-induced autophagy and ameliorated LPS-induced muscle wasting, and attenuated the alteration of neuropeptides, POMC, CART and NPY. In conclusion, mild hypothermia could alleviate muscle wasting by LPS injection, which was associated with reversing the level of hypothalamic AMPK-induced autophagy and the alteration of neuropeptides. These results suggested that mild hypothermia could be a potential treatment concept and a novel mechanism in management of muscle wasting in critically ill patients.

Dexmedetomidine ameliorates muscle wasting and attenuates the alteration of hypothalamic neuropeptides and inflammation in endotoxemic rats.

Dexmedetomidine is generally used for sedaton in critically ill, it could shorten duration of mechanical ventilation, ICU stay and lower basic metabolism. However, the exact mechanism of these positive effects remains unkown. Here we investigated the hypothesis that dexmedetomidine could ameliorate muscle wasting in endotoxemic rats and whether it was related to hypothalamic neuropeptides alteration and inflammation. Fourty-eight adult male Sprague–Dawley rats were intraperitoneally injected with lipopolysaccharide (LPS) (5 mg/kg) or saline, followed by 50 μg/kg dexmedetomidine or saline administration via the femoral vein catheter (infusion at 5 μg·kg-1·hr-1). Twenty-four hours after injection, hypothalamus tissues and skeletal muscle were obtained. Muscle wasting was measured by the mRNA expression of two E3 ubiquitin ligases, muscle atrophy F-box (MAFbx) and muscle ring finger 1 (MuRF-1) as well as 3-methylhistidine (3-MH) and tyrosine release. Hypothalamic inflammatory markers and neuropeptides expression were also detected in all four groups. Results showed that LPS administration led to significant increase in hypothalamic inflammation together with muscle wasting. Increased hypothalamic neuropeptides, proopiomelanocortin (POMC), cocaine and amphetamine-related transcript (CART) and neuropeptides Y (NPY) and decreased agouti-related protein (AgRP) were also observed. Meanwhile dexmedetomidine administration ameliorated muscle wasting, hypothalamic inflammation and modulated the alteration of neuropeptides, POMC, CART and AgRP, in endotoxemic rats. In conclusion, dexmedetomidine could alleviate muscle wasting in endotoxemic rats, and it could also attenuate the alteration of hypothalamic neuropeptides and reduce hypothalamic inflammation.

Impact of Orexin-A Treatment on Food Intake, Energy Metabolism and Body Weight in Mice.

Orexin-A and -B are hypothalamic neuropeptides of 33 and 28-amino acids, which regulate many homeostatic systems including sleep/wakefulness states, energy balance, energy homeostasis, reward seeking and drug addiction. Orexin-A treatment was also shown to reduce tumor development in xenografted nude mice and is thus a potential treatment for carcinogenesis. The aim of this work was to explore in healthy mice the consequences on energy expenditure components of an orexin-A treatment at a dose previously shown to be efficient to reduce tumor development. Physiological approaches were used to evaluate the effect of orexin-A on food intake pattern, energy metabolism body weight and body adiposity. Modulation of the expression of brain neuropeptides and receptors including NPY, POMC, AgRP, cocaine- and amphetamine related transcript (CART), corticotropin-releasing hormone (CRH) and prepro-orexin (HCRT), and Y2 and Y5 neuropeptide Y, MC4 (melanocortin), OX1 and OX2 orexin receptors (Y2R, Y5R, MC4R, OX1R and OX2R, respectively) was also explored. Our results show that orexin-A treatment does not significantly affect the components of energy expenditure, and glucose metabolism but reduces intraperitoneal fat deposit, adiposity and the expression of several brain neuropeptide receptors suggesting that peripheral orexin-A was able to reach the central nervous system. These findings establish that orexin-A treatment which is known for its activity as an inducer of tumor cell death, do have minor parallel consequence on energy homeostasis control.

Behind melanocortin antagonist overexpression in the zebrafish brain: A behavioral and transcriptomic approach

Melanocortin signaling is regulated by the binding of naturally occurring antagonists, agouti-signaling protein (ASIP) and agouti-related protein (AGRP) that compete with melanocortin peptides by binding to melanocortin receptors to regulate energy balance and growth. Using a transgenic model overexpressing ASIP, we studied the involvement of melanocortin system in the feeding behaviour, growth and stress response of zebrafish. Our data demonstrate that ASIP overexpression results in enhanced growth but not obesity. The differential growth is explained by increased food intake and feeding efficiency mediated by a differential sensitivity of the satiety system that seems to involve the cocaine- and amphetamine- related transcript (CART). Stress response was similar in both genotypes. Brain transcriptome of transgenic (ASIP) vs wild type (WT) fish was compared using microarrays. WT females and males exhibited 255 genes differentially expressed (DEG) but this difference was reduced to 31 after ASIP overexpression. Statistical analysis revealed 1122 DEG when considering only fish genotype but 1066 and 981 DEG when comparing ASIP males or females with their WT counterparts, respectively. Interaction between genotype and sex significantly affected the expression of 97 genes. Several neuronal systems involved in the control of food intake were identified which displayed a differential expression according to the genotype of the fish that unravelling the flow of melanocortinergic information through the central pathways that controls the energy balance. The information provided herein will help to elucidate new central systems involved in control of obesity and should be of invaluable use for sustaining fish production systems.

Orectic And Anorectic Peptides And Their Implication In Obesity And The Metabolic Syndrome

Abstract Background and aims: Cardiovascular diseases, diabetes mellitus, the metabolic syndrome and obesity are now globally widespread clinical conditions, addressing different ages, lately extending to young and children. The causes are multiple, involving an interaction between individual genetic risk factors and environmental factors. Many studies showed the importance of the hypothalamic neuropeptides and other neuropeptides in the regulation of the balance between food intake and energy consumption. We reviewed 25 recent research studies describing the physiological and physiopathological mechanisms of the orectic and anorectic peptides and their interaction to adjust the balance between food intake and energy expenditure. Conclusions: The hypothalamus, through its nuclei (arcuate and paraventricular) controls the balance between food intake and energy expenditure. The proopiomelanocortin (POMC) / Cocaine and amphetamine-related transcript (CART) neurons represent the anorectic centre. The neurons that release neuropeptide Y (NPY) and agouti-related protein (AgRP) by stimulation form the orectic centre. The neuropeptide Y (NPY) is the main hypothalamic orectic neuropeptide. Its action, besides stimulating the orectic effect, is to modulate the release of other hypothalamic orectic and anorectic neuropeptides. In addition, the energy balance is regulated by adipokines released by the adipose cells, hormones and neurotransmitters, blood glucose level and other metabolites.

Structural and functional consequences of buserelin-induced enteric neuropathy in rat.

BackgroundWomen treated with gonadotropin-releasing hormone (GnRH) analogs may develop enteric neuropathy and dysmotility. Administration of a GnRH analog to rats leads to similar degenerative neuropathy and ganglioneuritis. The aim of this study on rat was to evaluate the early GnRH-induced enteric neuropathy in terms of distribution of neuronal subpopulations and gastrointestinal (GI) function.MethodsForty rats were given the GnRH analog buserelin (20 μg, 1 mg/ml) or saline subcutaneously, once daily for 5 days, followed by 3 weeks of recovery, representing one treatment session. Two weeks after the fourth treatment session, the animals were tested for GI transit time and galactose absorption, and fecal weight and fat content was analyzed. After sacrifice, enteric neuronal subpopulations were analyzed. Blood samples were analyzed for zonulin and antibodies against GnRH and luteinizing hormone, and their receptors.ResultsBuserelin treatment transiently increased the body weight after 5 and 9 weeks (p < 0.001). Increased estradiol in plasma and thickened uterine muscle layers indicate high estrogen activity. The numbers of both submucous and myenteric neurons were reduced by 27%–61% in ileum and colon. The relative numbers of neurons containing calcitonin gene-related peptide (CGRP), cocaine- and amphetamine-related transcript (CART), galanin, gastrin-releasing peptide (GRP), neuropeptide Y (NPY), nitric oxide synthase (NOS), serotonin, substance P (SP), vasoactive intestinal peptide (VIP) or vesicular acetylcholine transporter (VAchT), and their nerve fiber density, were unchanged after buserelin treatment, but the relative number of submucous neurons containing somatostatin tended to be increased (p = 0.062). The feces weight decreased in buserelin-treated rats (p < 0.01), whereas feces fat content increased (p < 0.05), compared to control rats. Total GI transit time, galactose absorption, zonulin levels in plasma, and antibody titers in serum were unaffected by buserelin treatment.ConclusionsA marked enteric neuronal loss with modest effects on GI function is found after buserelin treatment. Increased feces fat content is suggested an early sign of dysfunction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12876-014-0209-7) contains supplementary material, which is available to authorized users.

Insulin ameliorating endotoxaemia-induced muscle wasting is associated with the alteration of hypothalamic neuropeptides and inflammation in rats.

Septic patients always develop muscle wasting, which delays the rehabilitation and contributes to the increased complications and mortality. Previous studies have implied the crucial role of central inflammation and neuropeptides in the energy balance and muscle metabolism. Insulin has been confirmed to attenuate muscle degradation and inhibit inflammation. We tested the hypothesis whether insulin ameliorating muscle wasting was associated with modulating hypothalamic inflammation and neuropeptides. Thirty-two adult male Sprague-Dawley rats were in intraperitoneally injected with lipopolysaccharide (LPS) (5 mg/kg) or saline, followed by subcutaneous injection of insulin (5 IU/kg) or saline. Twenty-four hours after injection, skeletal muscle and hypothalamus tissues were harvested. Muscle wasting was measured by the mRNA expression of two E3 ubiquitin ligases, muscle ring finger 1 (MuRF-1) and muscle atrophy F-box (MAFbx), as well as 3-methylhistidine (3-MH) and tyrosine release. Hypothalamic inflammatory markers and neuropeptides expression were also measured in four groups. LPS injection led to significant increase in hypothalamic inflammation as well as muscle wasting. Also, increased hypothalamic neuropeptides, proopiomelanocortin (POMC), cocaine and amphetamine-related transcript (CART) and neuropeptides Y (NPY) and decreased agouti-related protein (AgRP) were observed. Insulin treatment ameliorated endotoxaemia-induced muscle wasting and hypothalamic inflammation, and attenuated the alteration of neuropeptides, POMC, CART and AgRP. Hypothalamic inflammation and neuropeptides are involved in the endotoxaemia-induced muscle wasting. Insulin treatment can reduce muscle wasting, which is associated with reduced hypothalamic inflammation and alteration of hypothalamic neuropeptides.

Kinin B1 receptor gene ablation affects hypothalamic CART productionb

A role for the kinin B1 receptor in energy-homeostatic processes was implicated in previous studies; notably, the studies where kinin B1 receptor knockout mice (B1-/-) were shown to have impaired adiposity, impaired leptin and insulin production, lower feed efficiency, protection from liver steatosis and diet-induced obesity when fed a high fat diet (HFD). In particular, in a model where the B1 receptor is expressed exclusively in the adipose tissue, it rescues the plasma insulin concentration and the weight gain seen in wild type mice. Taking into consideration that leptin participates in the formation of hypothalamic nuclei, which modulate energy expenditure, and feeding behavior, we hypothesized that these brain regions could also be altered in B1-/- mice. We observed for the first time a difference in the gene expression pattern of cocaine and amphetamine related transcript (CART) in the (lateral hypothalamic area (LHA) resulting from the deletion of the kinin B1 receptor gene. The correlation between CART expression in the LHA and the thwarting of diet-induced obesity corroborates independent correlations between CART and obesity. Furthermore, it seems to indicate that the mechanism underlying the 'lean' phenotype of B1-/- mice does not stem solely from changes in peripheral tissues but may also receive contributions from changes in the hypothalamic machinery involved in energy homeostasis processes.

Fasting Induces CART Down-Regulation in the Zebrafish Nervous System in a Cannabinoid Receptor 1-Dependent Manner

Central and peripheral mechanisms modulate food intake and energy balance in mammals and the precise role of the type 1 cannabinoid receptor (CB1) in these processes is still being explored. Using the zebrafish, Danio rerio, we show that rimonabant, a CB1-specific antagonist with an EC(50) of 5.15 × 10(-8) m, decreases embryonic yolk sac reserve use. We reveal a developmental overlap between CART genes and CB1 expression in the hypothalamus and medulla oblongata, two brain structures that play crucial roles in appetite regulation in mammals. We show that morpholino knockdown of CB1 or fasting decreases cocaine- and amphetamine-related transcript (CART)-3 expression. Strikingly, this down-regulation occurs only in regions coexpressing CB1 and CART3, reinforcing the link between CB1, CART, and appetite regulation. We show that rimonabant treatment impairs the fasting-induced down-regulation of CART expression in specific brain regions, whereas vehicle alone-treated embryos do not display this rescue of CART expression. Our data reveal that CB1 lies upstream of CART and signals the appetite through the down-regulation of CART expression. Thus, our results establish the zebrafish as a promising system to study appetite regulation.

Molecular ontogenesis of digestive capability and associated endocrine control in Atlantic cod (Gadus morhua) larvae

We have profiled the expression of twelve genes, in order to provide an overview on the molecular ontogeny of digestive capability with the associated endocrine control during Atlantic cod (Gadus morhua) larval development. Enzyme activity levels for the key digestive enzyme, trypsin, was also measured. Specifically, transcripts for trypsin, amylase, lipolytic enzymes: bile salt activated lipase (BAL), phospholipase A2 (PLA2) and Acyl CoA dehydrogenase (ACADM), regulatory peptides: neuropeptide Y (NPY), orexin (OX) cholecystokinin (CCK) and cocaine and amphetamine-related transcript (CART), the somatotropic factors: growth hormone (GH), preprosomatostatin 1 (PPSS1) and thyroid hormone receptors (TRα and TRβ) were analyzed using quatitative (real-time) polymerase chain reaction (qPCR). Trypsin and BAL mRNA levels peaked at approximately day 17 and 25 post-hatch, respectively, and thereafter displayed a decreasing pattern until metamorphosis. GH mRNA levels decreased moderately from 3 to 33dph, and thereafter, an increase was observed until 46dph. TRα mRNA levels showed a fluctuating pattern peaking at day 39 post-hatch. TRβ mRNA levels were too low to obtain quantitative measurements. Amylase mRNA slightly increased from day 3 to 17 post-hatch, and thereafter showed a steady decrease until day 60. Interestingly, PLA2 mRNA expression showed a consistent increase throughout the study period, indicating an increasingly important role during larval development. Overall, data from this study indicate that cod larvae show differential developmental mode of expression patterns for key genes and endocrine factors that regulate digestive capability, growth and development. These data are discussed in relation to larval trypsin enzyme activity and previous reports for other teleost species.

Accumbens Shell–Hypothalamus Interactions Mediate Extinction of Alcohol Seeking

The nucleus accumbens shell (AcbSh) is required to inhibit drug seeking after extinction training. Conversely, the lateral hypothalamus (LH), which receives projections from AcbSh, mediates reinstatement of previously extinguished drug seeking. We hypothesized that reversible inactivation of AcbSh using GABA agonists (baclofen/muscimol) would reinstate extinguished alcohol seeking and increase neuronal activation in LH. Rats underwent self-administration training for 4% (v/v) alcoholic beer followed by extinction. AcbSh inactivation reinstated extinguished alcohol seeking when infusions were made after, but not before, extinction training. We then used immunohistochemical detection of c-Fos as a marker of neuronal activity, combined with immunohistochemical detection of the orexin and cocaine- and amphetamine-related transcript (CART) peptides, to study the profile and phenotype of neural activation during reinstatement produced by AcbSh inactivation. AcbSh inactivation increased c-Fos expression in hypothalamus, as well as in paraventricular thalamus and amygdala. Within hypothalamus, there was an increase in the number of orexin and CART cells expressing c-Fos. Finally, we hypothesized that concurrent inactivation of LH would prevent reinstatement produced by inactivation of AcbSh alone. Our results confirmed this. Together, these findings suggest that AcbSh mediates extinction of reward seeking by inhibiting hypothalamic neuropeptide neurons. Reversible inactivation of the AcbSh removes this influence, thereby releasing hypothalamus from AcbSh inhibition and enabling reinstatement of reward seeking. These ventral striatal-hypothalamic circuits for extinction overlap with those that mediate satiety, and we suggest that extinction training inhibits drug seeking because it co-opts neural circuits originally selected to produce satiety.

Cocaine and amphetamine related transcript (CART) inhibits context induced reinstatement of reward seeking.

We studied the role of cocaine and amphetamine related transcript (CART) in regulating context induced reinstatement (renewal) of reward seeking. Rats were trained to respond for alcoholic beer in context A before extinction in context B. Rats were tested for responding in context A (ABA) and context B (ABB). Intracerebroventricular (ICV) infusions of the active fragment CART55-102 but not the inactive fragment CART1-27 before test prevented ABA renewal of extinguished responding. ICV CART55-102 had no effect on responding in the extinction context (ABB). ICV CART55-102 also altered the profile of behavioral responses observed on test in the training (ABA) but not extinction (ABB) context. These results identify a novel role for CART in preventing reinstatement of reward seeking.

Effects of orchidectomy on adaptation of arcuate neuropeptide Y, proopiomelanocortin, and cocaine- and amphetamine-related transcript gene profiles to recurring insulin-induced hypoglycemia in the male rat

Our studies show that recurring insulin-induced hypoglycemia (RIIH) diminishes neuronal activation in several key components of the central metabolic regulatory circuitry, including the hypothalamic arcuate nucleus (ARH), and that orchidectomy (ORDX) modifies this impact of RIIH on Fos protein expression in this and other select neural structures. The testicular hormone, testosterone, regulates the expression of ARH neuropeptide genes of characterized metabolic relevance, including neuropeptide Y (NPY) and proopiomelanocortin (POMC). We investigated the hypothesis that acute hypoglycemia-associated patterns of ARH NPY, POMC, and cocaine and amphetamine-related transcript (CART) gene transcription, and potential RIIH-associated adaptive modifications in these expression profiles are regulated by testes-dependent mechanisms. ARH tissue was micropunched from serial frozen brains sections obtained from sham-operated (SHAM) or bilaterally ORDX male rats after sc injection of one or four doses of neutral protamine Hagedorn insulin, over as many days, or vehicle alone, and analyzed by quantitative real-time RT-PCR. In SHAM rats, acute hypoglycemia increased ARH NPY mRNA; precedent hypoglycemia elevated baseline gene expression in this group, but suppressed transcription during RIIH. In ORDX rats, ARH NPY mRNA was decreased during acute hypoglycemia and after multiple exposures; however, gene expression was not further suppressed by RIIH. ARH POMC gene transcription was not modified by acute or recurring hypoglycemia in the SHAM group. ORDX caused a reduction in both basal and acute hypoglycemic patterns of POMC transcription, relative to the SHAM controls, but enhanced baseline and RIIH-associated patterns of gene expression. ARH CART transcripts were not altered by acute or recurring hypoglycemia in SHAM rats, whereas ORDX animals exhibited elevated CART gene expression during RIIH. These data show that acute and recurring hypoglycemia exert opposite effects on ARH NPY gene expression in testes-intact male rats, and that these transcriptional responses are abolished by ORDX. Hypoglycemia had no impact on POMC nor CART mRNA profiles in the SHAM group, but both genes were upregulated during RIIH in ORDX rats. Collectively, these results demonstrate that in the male rat, testes-dependent mechanisms underlie patterns of acclimated or unvarying reactivity to RIIH of the specific ARH metabolic neuropeptide genes evaluated here.

Hyperphagia and obesity produced by arcuate injection of NPY–saporin do not require upregulation of lateral hypothalamic orexigenic peptide genes

Neuropeptide Y (NPY) conjugated with a ribosomal inactivating toxin, saporin (SAP), is a toxin that targets NPY receptor-expressing cells. Injection of NPY–SAP into the rat arcuate nucleus (Arc) and basomedial hypothalamus (BMH) destroys two populations of NPY-receptor-expressing neurons important for the control of food intake and body weight, NPY and pro-opiomelanocortin (POMC) and cocaine and amphetamine related transcript (CART) neurons, and produces profound hyperphagia and obesity. Here, we investigated the contribution of lateral hypothalamus (LHA) orexigenic peptides, orexins and melanocortin concentrating hormone (MCH), to these lesion effects. We microinjected NPY–SAP into two sites on each side of the Arc, causing a loss of NPY and POMC/CART neurons that was limited to the Arc. Lesioned rats rapidly became hyperphagic and obese. However, MCH and prepro-orexin mRNA expression were not increased in the LHA in the lesioned rats, but were decreased at some levels of the LHA or were unchanged. NPY–SAP-induced obesity therefore differs from dietary obesity and from obesity associated with leptin or leptin receptor deficiency in which MCH gene expression is increased. The Arc NPY–SAP lesion produces obesity and hyperphagia that does not require overexpression of hypothalamic neuropeptides currently considered to provide major stimulatory drive for food intake: NPY, agouti gene-related protein, MCH or orexins. The source of the seemingly unregulated stimulatory drive for feeding in these animals has not been identified, but may be associated with hindbrain or endocrine mechanisms.