Accumbal TRH is downstream of the effects of isolation stress on hedonic food intake in rats.

Emotional stress, through elevating corticosterone (CORT) levels may reduce feeding in rodents however when offered palatable food, stressed animals ingest more food compared to non-stressed controls. Nucleus accumbens (NAc) is part of the mesocorticolimbic system and participates in processing rewarding characteristics of food modulating palatable food intake, mainly when glucocorticoids are elevated. A possible mediator of CORT effects is accumbal thyrotropin-releasing hormone (TRH), which reduces chow intake in rats when administered into the NAc. We aimed to study the TRH role in hedonic feeding in stressed rats. For 14 days, animals with ad libitum access to chow or chow plus chocolate milk were either group-housed or singly-housed to induce stress. Rats with access to chocolate milk showed hyperphagia and decreased accumbal TRH mRNA levels, which were potentiated by stress. Results suggest that TRH downregulation was permissive of the increased palatable food intake. TRH injections into NAc of singly-housed animals with palatable food access reduced their food intake and increased serum CORT levels. The accumbal injections of a glucocorticoid receptor antagonist (mifepristone) in stressed rats with palatable food access, reduced only palatable food intake and increased accumbal TRH expression and serum CORT levels. This modulation of TRH mRNA when CORT signaling is modified suggests that accumbal TRH is downstream of glucocorticoids activity, which specifically increase palatable food intake. Our results strengthen the TRH involvement in regulating emotional aspects of hedonic feeding in stressed animals. Finding new therapies directed towards increasing TRHergic activity in NAc may be protective against overeating.

The nociceptin/orphanin FQ-like opioid peptide in nervous periesophageal ganglia of land snail Helix aspersa.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) and its receptor are members of the endogenous opioid peptide family. In mammals N/OFQ modulates a variety of biological functions such as nociception, food intake, endocrine, control of neurotransmitter release, among others. In the molluscs Cepea nemoralis and Helix aspersa the administration of N/OFQ produces a thermopronociceptive effect. However, little is known about its existence and anatomic distribution in invertebrates. The aim of this study was to provide a detailed anatomical distribution of N/OFQ like peptide immunoreactivity (N/OFQ-IL), to quantify the tissue content of this peptide, as well as to demostrate molecular evidence of N/OFQ mRNA in the nervous tissue of periesophageal ganglia of the land snail H. aspersa. Immunohistochemical, immunocytochemical, radioimmunoanalysis (RIA) and reverse transcription-polymerase chain reaction (RT-PCR) techniques were used. With regard to RT-PCR, the primers to detect expression of mRNA transcripts from H. aspersa were derived from the rat N/OFQ opioid peptide. We show a wide distribution of N/OFQ-IL in neurons and fibers in all perioesophageal ganglia, fibers of the neuropile, nerves, periganglionar connective tissue, aortic wall and neurohemal sinuses. The total amount of N/OFQ-IL in the perioesophageal ganglia (7.75 ± 1.75 pmol/g of tissue) quantified by RIA was similar to that found in mouse hypothalamus (10.1 ± 1.6 pmol/g of tissue). In this study, we present molecular evidence of N/OFQ mRNA expression. Some N/OFQ-IL neurons have been identified as neuroendocrine or involved in olfaction, hydro-electrolyte regulation, feeding, and thermonociception. Therefore, we suggest that N/OFQ may participate in these snail functions.

Acute ethanol administration differentially alters enkephalinase and aminopeptidase N activity and mRNA levels in regions of the nigrostriatal pathway.

Opioid peptides play a key role in ethanol reinforcement and may also represent important determinants in brain sensitivity to ethanol through modulation of nigrostriatal dopaminergic activity. Regulation of opioid levels by peptidase-degrading enzymes could be relevant in ethanol's actions. The aim of this work was to study the acute ethanol (2.5 g/kg) effects on the activity and mRNA expression of enkephalinase (NEP) and aminopeptidase N (APN) in the rat substantia nigra (SN) and the anterior-medial (amCP) and medial-posterior (mpCP) regions of the caudate-putamen (CP). Enzymatic activities were measured by fluorometric assays and mRNA expression by reverse transcriptase polymerase chain reaction. Acute ethanol administration differentially altered peptidase activities and mRNA expression with different kinetics. Ethanol increased and decreased NEP mRNA levels in the SN and amCP, respectively, but produced biphasic effects in the mpCP. APN mRNA levels were increased by ethanol in all brain regions. Ethanol induced a transient and long-lasting increase in NEP (mpCP) and APN (amCP) activities, respectively. Peptidase activities were not changed by ethanol in the SN. Our results indicate that striatal NEP and APN are important ethanol targets. Ethanol-induced changes in these neuropeptidases in the CP could contribute to the mechanisms involved in brain sensitivity to ethanol.

La hormona liberadora de tirotropina (TRH) del núcleo paraventricular hipotalámico y sistema límbico como reguladora de la homeostasis energética y de la conducta alimentaria en animales con ayuno, restricción alimentaria y anorexia / Thyrotropin-releasing hormone from the hypothalamic paraventricular nucleus and limbic system regulates energy homeostasis and feeding behavior in fasted, food retricted and anorexic animals

TRH expression and release from hypothalamic paraventricular nucleus (PVN) change with environmental stimuli. Fasted and food-restricted animals present decreased TRH synthesis and release, decelerating metabolic rate and utilization of energy stores, which is an advantageous adaptation of animals with nutrient deficit. Comparing thyroid axis function between prepuberal vs. adult male fasted animals, we found a greater body weight reduction than in adults (30% vs.11%) and TRH release was not decreased; TRH degradation by pituitary PPII enzyme decreased, which maintained energy waste. TRH content of fasted-prepuberal animals changed in hippocampus and nucleus accumbens, and in amygdala of adults vs. ad libitum fed animals. PVN TRH role in food-avoiding behavior was studied by comparing its expression levels and of adolescent, adult females and male animals with anorexic conduct when drinking 2.5% of NaCl solution (AN) vs. a group forced to ingest the amount of food consumed by AN (FFR); also vs. a control group fed ad libitum (C). PVN TRH mRNA and TSH serum levels increased in AN vs. C; both decreased in FFR, supporting the putative anorexigenic role for the peptide. TRH content differentially changed in hippocampus and in frontal cortex of AN and FFR, suggesting its participation in taste perception and memory association. Orexinergic and NPYergic pathways are inactive in anorexic animals. Blocking corticotrophin-releasing hormone signal by an antagonist of CRH-R2 in the PVN reverses TRH high expression and TSH serum levels in AN.

La expresión y liberación de la TRH del núcleo paraventicular hipotalámico (NPV) cambia con estímulos ambientales; en ayuno y restricción de alimentos la liberación del péptido disminuye, reduciéndose la tasa del metabolismo y la degradación de reservas energéticas. Esto es una adaptación ventajosa para los animales con balance negativo de energía. Al comparar el contenido de TRH en la eminencia media entre animales prepúberes y adultos en ayuno de 48 horas, observamos que los jóvenes no tienen una adaptación al déficit de nutrimentos. Su peso baja más que en adultos (30% vs. 11%) y la liberación de TRH no disminuye; la degradación de TRH por PPII en la adenohipófisis (PPII) disminuye, manteniéndose el gasto energético. El contenido de TRH de animales prepúberes en ayuno cambió en el hipocampo y en el núcleo accumbens, así como en la amígdala de los adultos comparado contra los animales con alimentación ad libitum. La TRH se ha propuesto como agente anorexigénico. Evaluamos su contenido y expresión en el NPV de animales que evitan el alimento al beber una solución de NaCl (2.5%)(AN), en otros con restricción de alimento forzada (RAF) que ingieren la misma cantidad que AN y en aquéllos (C) con alimentación ad libitum. La síntesis de TRH en el NPV y el contenido sérico de TSH disminuyen en RAF pero aumentan en AN. La vía orexinérgica y la de NPY de AN están inactivas. La inyección de un antagonista a CRH revierte las alteraciones de TRH y TSH y atenúa la anorexia de AN.

Activity and expression of enkephalinase and aminopeptidase N in regions of the mesocorticolimbic system are selectively modified by acute ethanol administration.

Opioid peptides play a key role in ethanol reinforcement and alcohol drinking behavior. However, regulation of opioid levels by peptidase-degrading activities in ethanol's actions in brain is still unclear. The aim of this work was to study the acute effects of ethanol (2.5 g/kg) on enkephalinase (NEP) and aminopeptidase N (APN) activities and expression in regions of the mesocorticolimbic system, as well as on corticosterone levels in serum for up to 24 h after administration. Enzymatic activities were measured by fluorometric assays, mRNA's expression by reverse transcriptase polymerase chain reaction (RT-PCR) and corticosterone levels by radioimmunoassay. Acute ethanol administration modified peptidase activity and expression with different kinetics. Ethanol induced a transitory increase and decrease in NEP and APN activities in the frontal cortex (FC) and ventral tegmental area (VTA), whereas only increases in these activities were observed in the nucleus accumbens (NAcc). Ethanol induced an increase in NEP mRNA in the FC and decreases in APN mRNA in the FC and NAcc. In contrast, ethanol produced biphasic effects on both enzymes expression in the VTA. Corticosterone levels were not changed by ethanol. Our results suggest that NEP and APN could play a main role in ethanol reinforcement through regulation of opioid levels in mesolimbic areas.

Expression of the dopaminergic D1 and D2 receptors in the anterior cingulate cortex in a model of neuropathic pain.

BACKGROUND: The anterior cingulate cortex (ACC) has been related to the affective component of pain. Dopaminergic mesocortical circuits, including the ACC, are able to inhibit neuropathic nociception measured as autotomy behaviour. We determined the changes in dopamine D1 and D2 (D1R and D2R) receptor expression in the ACC (cg1 and cg2) in an animal model of neuropathic pain. The neuropathic group had noxious heat applied in the right hind paw followed 30 min. later by right sciatic denervation. Autotomy score (AS) was recorded for eight days and subsequently classified in low, medium and high AS groups. The control consisted of naïve animals.A semiquantitative RT-PCR procedure was done to determine mRNA levels for D1R and D2R in cg1 and cg2, and protein levels were measured by Western Blot. RESULTS: The results of D1R mRNA in cg1 showed a decrease in all groups. D2R mRNA levels in cg1 decreased in low AS and increased in medium and high AS. Regarding D1R in cg2, there was an increase in all groups. D2R expression levels in cg2 decreased in all groups. In cg1, the D2R mRNA correlated positively with autotomy behaviour. Protein levels of D2R in cg1 increased in all groups but to a higher degree in low AS. In cg2 D2R protein only decreased discretely. D1R protein was not found in either ACC region. CONCLUSIONS: This is the first evidence of an increase of inhibitory dopaminergic receptor (D2R) mRNA and protein in cg1 in correlation with nociceptive behaviour in a neuropathic model of pain in the rat.

Bone morphogenetic protein 15 and growth differentiation factor 9 expression in the ovary of European sea bass (Dicentrarchus labrax): cellular localization, developmental profiles, and response to unilateral ovariectomy.

Vertebrate oocytes actively contribute to follicle development by secreting a variety of growth factors, among which bone morphogenetic protein 15 (BMP15/Bmp15) and growth differentiation factor 9 (GDF9/Gdf9) have been paid particular attention. In the present study, we describe the cellular localization, the developmental profiles, and the response to unilateral ovariectomy (a procedure implying the surgical removal of one of the ovaries) of protein and mRNA steady-state levels of Bmp15 and Gdf9 in the ovary of European sea bass, an important fish species for marine aquaculture industry. In situ hybridization and immunohistochemistry demonstrated that the oocyte is the main production site of Bmp15 and Gdf9 in European sea bass ovary. During oocyte development, Bmp15 protein expression started to be detected only from the lipid vesicle stage onwards but not in primary pre-vitellogenic (i.e. perinucleolar) oocytes as the bmp15 mRNA already did. Gdf9 protein and gdf9 mRNA expression were both detected in primary perinucleolar oocytes and followed similar decreasing patterns thereafter. Unilateral ovariectomy induced a full compensatory growth of the remaining ovary in the 2-month period following surgery (Á. García-López, M.I. Sánchez-Amaya, C.R. Tyler, F. Prat 2011). The compensatory growth elicited different changes in the expression levels of mRNA and protein of both factors, although the involvement of Bmp15 and Gdf9 in the regulatory network orchestrating such process remains unclear at present. Altogether, our results establish a solid base for further studies focused on elucidating the specific functions of Bmp15 and Gdf9 during primary and secondary oocyte growth in European sea bass.

Targeted gene expression profiling in European sea bass (Dicentrarchus labrax, L.) follicles from primary growth to late vitellogenesis.

A real-time PCR-based gene expression survey was performed on isolated European sea bass follicles from primary growth to late vitellogenesis. Expression levels of 18 transcripts with demonstrated relevance during oogenesis, encoding gonadotropin, thyrotropin, estrogen, androgen, and vitellogenin receptors, steroidogenesis-related as well as growth and transcription factors were measured. Primary oocytes showed high mRNA levels of insulin-like growth factors 1 and 2, bone morphogenetic protein 4, estrogen receptor 2b, androgen receptor b, and SRY-box containing gene 17 together with low transcript amounts of gonadotropin receptors. Follicles at the lipid vesicles stage (i.e., the beginning of the secondary growth phase) showed elevated mRNA amounts of follicle stimulating hormone receptor (fshr) and anti-Mullerian hormone. Early-to-mid vitellogenic follicles showed high mRNA levels of fshr and cytochrome P450, family 19, subfamily A, polypeptide 1a while mid-to-late vitellogenic follicles expressed increasing transcript amounts of luteinizing hormone/choriogonadotropin receptor, steroidogenic acute regulatory protein, and estrogen receptors 1 and 2a. The molecular data presented here may serve as a solid base for future studies focused on unraveling the specific mechanisms orchestrating follicular development in teleost fish.

Inflammatory nociception diminishes dopamine release and increases dopamine D2 receptor mRNA in the rat's insular cortex.

BACKGROUND: The insular cortex (IC) receives somatosensory afferent input and has been related to nociceptive input. It has dopaminergic terminals and D1 (D1R) -excitatory- and D2 (D2R) -inhibitory- receptors. D2R activation with a selective agonist, as well as D1R blockade with antagonists in the IC, diminish neuropathic nociception in a nerve transection model. An intraplantar injection of carrageenan and acute thermonociception (plantar test) were performed to measure the response to inflammation (paw withdrawal latency, PWL). Simultaneously, a freely moving microdyalisis technique and HPLC were used to measure the release of dopamine and its metabolites in the IC. Plantar test was applied prior, one and three hours after inflammation. Also, mRNA levels of D1 and D2R's were measured in the IC after three hours of inflammation. RESULTS: The results showed a gradual decrease in the release of dopamine, Dopac and HVA after inflammation. The decrease correlates with a decrease in PWL. D2R's increased their mRNA expression compared to the controls. In regard of D1R's, there was a decrease in their mRNA levels compared to the controls. CONCLUSIONS: Our results showed that the decreased extracellular levels of dopamine induced by inflammation correlated with the level of pain-related behaviour. These results also showed the increase in dopaminergic mediated inhibition by an increase in D2R's and a decrease in D1R's mRNA. There is a possible differential mechanism regarding the regulation of excitatory and inhibitory dopaminergic receptors triggered by inflammation.

Expression of muscarinic M1 and M2 receptors in the anterior cingulate cortex associated with neuropathic pain.

The anterior cingulate cortex (ACC) and muscarinic receptors modulate pain. This study investigates changes in the expression of muscarinic-1 and -2 receptors (M1R, M2R) in rats' ACC (cg1-rostral- and cg2-caudal) using a model of neuropathic pain by denervation, measured as autotomy score (AS) for 8 days. Changes were analysed with painful stimuli and with scopolamine into the ACC prior to this scheme. We used reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence to determine M1R and M2R's mRNA and protein levels, respectively. Animals were divided in low, medium and high AS groups. Cg1 showed decreased mRNA levels for both M1R and M2R in the low AS group, as opposed to an increased expression in the medium and high AS groups. Both receptors correlated positively with AS in these groups. In the scopolamine-treated animals there was an increase in mRNA levels for both receptors in cg1, whereas in cg2, mRNA levels of M1R decreased in all the AS and scopolamine groups. The increased M2R mRNA in cg2 correlated with AS in the low, medium and high AS groups whereas all the scopolamine groups showed an increase. Immunoreactivity of the M2R in cg1 decreased in the medium AS group in comparison to controls but scopolamine treatment produced an increase in the medium scopolamine AS group compared to the medium AS group. The M1R in cg1 and both receptors in cg2 showed no immunoreactivity changes. These results highlight the role of the M2R in cg1 related to the degree of autotomy.

Involvement of CRH-R2 receptor in eating behavior and in the response of the HPT axis in rats subjected to dehydration-induced anorexia.

Wistar rats subjected to dehydration-induced anorexia (DIA), with 2.5% NaCl solution as drinking water for 7 days, decrease by 80% their food intake and present some changes common to pair-fed food restricted rats (FFR) such as: weight loss, decreased serum leptin and expression of orexigenic arcuate peptides, increasing the anorexigenic ones and serum corticosterone levels. In contrast, the response of the HPT axis differs: DIA animals have increased TRH expression in PVN and present primary as opposed to the tertiary hypothyroidism of the FFR. Exclusive to DIA is the activation of CRHergic neurons in the lateral hypothalamus (LH) that project to PVN. Since TRH neurons of the PVN contain CRH receptors, we hypothesized that the differences in the response of the HPT axis to DIA could be due to CRH regulating TRHergic neurons. CRH effect was first evaluated on TRH expression of cultured hypothalamic cells where TRH mRNA levels increased after 1h with 0.1nM of CRH. We then measured the mRNA levels of CRH receptors in the PVN of male and female rats subjected to DIA; only those of CRH-R2 were modulated (down-regulated). The CRH-R2 antagonist antisauvagine-30 was therefore injected into the PVN of male rats, during the 7 days of DIA. Antisauvagine-30 induced a higher food intake than controls, and impeded the changes produced by DIA on the HPT axis: PVN TRH mRNA, and serum TH and TSH levels were decreased to similar values of FFR animals. Results corroborate the anorexigenic effect of CRH and show its role, acting through CRH-R2 receptors, in the activation of TRHergic PVN neurons caused by DIA. These new data further supports clinical trials with CRH-R2 antagonists in anorexia nervosa patients.