ABSTRACT
Objective:To explore the effects of the γ-aminobutyric acid(GABA) neurons and melanin-concentrating hormone (MCH) neurons of the nucleus accumbens (NAc)-lateral hypothalamic area (LHA) neural pathway on the rewarding feeding(palatable food sweat condensed milk) in the obesity rats.Methods:Total 142 male Wistar rats of SPF grade were divided into normal diet (ND) group ( n=68) and high-fat diet induced obesity (DIO) group ( n=74) according to the principle of body mass matching. The rats in the two groups were given normal diet and high-fat diet for 8 weeks. Eight weeks later, 6 DIO rats were randomly selected to observe the nerve projection from GABA neurons in NAc to MCH neurons in LHA by fluorogold retrograde tracing combined fluorescence immunohistochemistry. And the expressions of c-Fos and MCH in LHA after ingestion of sweet condensed milk(rewarding feeding) were observed by fluorescence immunohistochemistry (6 rats in each group). GABA receptor agonist Musimol or GABA receptor antagonist Bicuculine was microinjected into the nucleus of LHA to observe the effect of GABA on rewarding food intake in ND and DIO rats ( n=8 in each group), and the changes of rewarding food intake after blocking MCH signal ( n=8 in each group). SPSS 17.0 was used for statistical analysis, two-way ANOVA and post hoc Bonferroni test were used for comparison among multiple groups, and t-test was used for comparison between two groups. Results:After 8 weeks of high-fat diet modeling, the intake of delicious food in DIO rats was significantly higher than that in ND rats((12.52±2.29) mL, (7.45±1.23) mL, t=4.778, P<0.01) after satiety.The results of fluorogold retrograde tracing combined with fluorescence immunohistochemistry showed that GABA neurons in NAc projected nerve fibers to neurons in LHA, and GABA A receptors in some neurons in LHA coexisted with MCH.The results of NAc-LHA pathway on delicious food intake showed that the interaction between rat group and drug intervention was significant( F=9.869, P<0.01). Simple effect analysis showed that the intake of delicious food after microinjection of Musimol into LHA nucleus of ND rats was significantly lower than that of microinjection normal saline ((4.25±1.38) mL, (7.29±1.49) mL, P<0.01), while the intake of delicious food after injection of Bicuculine was significantly higher than that of microinjection normal saline((10.72±2.11) mL, (7.29±1.49) mL, P<0.05). The intake of delicious food after microinjection of Musimol into LHA nucleus in DIO group was significantly lower than that of microinjection normal saline((3.51±1.77)mL, (13.68±2.95) mL, P<0.01), but there was no significant difference between microinjection Bicuculine and microinjection normal saline ((14.83±3.44) mL, (13.68±2.95) mL, P>0.05). The results of blocking MCH signal on delicious food intake showed that the interaction effect between SNAP-94847 and Bicuculine intervention was not significant ( F=1.468, P>0.05). The main effect of SNAP-94847 intervention was significant ( F=15.880, P<0.01)and the main effect of Bicuculine intervention was significant ( F=6.930, P<0.05). After intracerebroventricular injection of MCH receptor blocker SNAP-94847, the delicious food intake of ND rats was significantly less than that of injection normal saline((4.78±1.72) mL, (7.63±2.77) mL, P<0.05), and it was not affected by pre injection of Bicuculine in LHA ((6.24±2.18) mL, (4.78±1.72) mL, P>0.05). In the DIO rats, the interaction effect between SNAP-94847 and Bicuculine intervention was not significant( F=0.006, P>0.05). The main effect of SNAP-94847 intervention was significant ( F=18.46, P<0.01) and the main effect of Bicuculine intervention was not significant ( F=2.059, P>0.05). After intracerebroventricular injection of MCH receptor blocker SNAP-94847, the delicious food intake of DIO rats was significantly lower than that of injection normal saline((6.89±2.11) mL, (12.19±4.36) mL, P<0.05), and it was not affected by pre injection of Bicuculine in LHA ((8.72±2.26) mL, (6.89±2.11) mL, P>0.05). Conclusion:GABAergic signal in NAc can regulate the expression of MCH in neurons of LHA. In the DIO rats, the sensitivity of MCH neurons in LHA to satiety signal decreases and the hedonic feeding increases.
ABSTRACT
Organisms with source-populations restricted to the subterranean biotope (troglobites) are excellent models for comparative evolutionary studies, due to their specialization to permanent absence of light. Eye and dark pigment regression are characteristics of most troglobites. In spite of the advance in knowledge on the mechanisms behind eye regression in cave fishes, very little is known about pigmentation changes. Studies were focused on three species of the genus Pimelodella. Exemplars of the troglobitic P. spelaea and P. kronei were compared with the epigean (surface) P. transitoria, putative sister-species of the latter. Melanophore areas and densities are significantly lower in the troglobitic species. Evaluating the in vitro response of these cells to adrenaline, acetylcholine and MCH, we observed a reduced response in both troglobites to adrenaline. The same trend was observed with MCH, but not statistically significant. No response to acetilcholine was detected in all the three. Contrary to expectations, even though eye-regression in P. spelaea was much lower than in P. kronei, pigmentation regression was more advanced. Multiple mechanisms of loss showing a mosaic of traits in troglobitic fishes are discussed here.(AU)
Organismos com populações-fonte restritas ao biótopo subterrâneo (troglóbios) são excelentes modelos para estudos evolutivos comparativos, devido à especialização resultante do isolamento sob um regime seletivo particular, com ênfase na permanente falta de luz. A regressão do olho e da pigmentação são características dos troglobites. Apesar do avanço do conhecimento sobre os mecanismos subjacentes à regressão ocular em peixes de caverna, pouco se sabe sobre mudanças de pigmentação. Os estudos foram focados em três espécies do gênero Pimelodella. Exemplares das espécies troglóbias P. spelaea e P. kronei foram comparados com a epígea P. transitoria, provável espécie-irmã dessa última. As áreas e densidades dos melanóforos são significativamente menores nas espécies troglóbias. Avaliando a resposta in vitro dessas células à adrenalina, acetilcolina e MCH, observamos uma resposta reduzida em ambos os troglóbios à adrenalina. A mesma tendência foi observado com o MCH, mas não estatisticamente. Nenhuma resposta à acetilcolina foi detectada três. Contrariamente às expectativas, embora a regressão ocular em P. spelaea seja bem menor do que em P. kronei, a regressão na pigmentação foi mais acentuada. Múltiplos mecanismos de regressão, mostrando um mosaico de características em peixes troglóbios, são discutidos aqui.(AU)
Subject(s)
Animals , Catfishes/physiology , Pigmentation , Color , Neurotransmitter Agents , Hormones , FishesABSTRACT
Introducción: la depresión mayor (DM) es una enfermedad psiquiátrica frecuente, con importante morbilidad y una relación estrecha con el suicidio. Objetivo: hacer una puesta a punto de los avances en el estudio de la neurobiología de la DM, enfocándonos en el posible rol de la hormona concentradora de melanina (MCH) en esta patología. Metodología: revisión de la bibliografía con énfasis en nuestros propios trabajos originales. Resultados: la MCH es un neuromodulador peptídico sintetizado por neuronas del hipotálamo. Las neuronas MCHérgicas envían proyecciones hacia diversas regiones del sistema nervioso central, incluyendo las áreas vinculadas con la regulación de la vigilia y del sueño, así como a diversas estructuras del sistema límbico que participan en la regulación del humor. Aunque numerosos estudios han relacionado el sistema MCHérgico con el control de la homeostasis energética, hallazgos recientes han permitido señalar un rol de este sistema en los mecanismos de generación del sueño. A su vez, una convergencia de datos provenientes de diversos estudios sugiere que la MCH estaría involucrada en la fisiopatología de la DM. Nuestros propios estudios preclínicos tienden a indicar que la MCH promueve la generación del sueño REM y un estado tipo depresivo. Ambos efectos estarían siendo mediados a través de la modulación de la actividad de las neuronas serotoninérgicas del núcleo dorsal del rafe. Conclusiones: estudios preclínicos sugieren un rol protagónico del sistema MCHérgico en la fisiopatología de la depresión. Resta confirmar si esta afirmación es cierta en pacientes con DM...
Subject(s)
Humans , Depression/physiopathology , Neurobiology , Depressive Disorder, Major/physiopathologyABSTRACT
Objective To express and purify the epitope peptide of human melanin-concentrating hormone receptor 1, and to evaluate its performance in the detection of autoantibodies in vitiligo patients. Methods The target gene encoding the epitope peptide of human melanin-concentrating hormone receptor 1 was synthesized, cloned to prokaryotic expression vector pGEX-4T-2 which was then transferred to E. coli BL21. The protein expression was induced by isopropy-β-D-thiogalactoside (IPTG) and identified with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Blocking ELISA was carried out with membrane proteins extracted from melanocytes as the blocking antigen. The antigenicity of the peptide was detected in sera from 100 patients with progressive vitiligo and 30 healthy human controls. Results The recombinant expression vector was successfully constructed, and the target protein was successfully expressed in E.coli, which was evidenced by SDS-PAGE and Western blot. With the glutathione S-transferase (GST) purification kit, the purity of the recombinant protein reached 100% when the sampling weight was less than 0.625 μg.The binding of the target protein with serum IgG antibodies from vitiligo patients could be blocked by natural membrane antigen of melanocytes. Of the 100 sera from patients with progressive vitiligo, 36 were reactive with the target protein. Conclusions The epitope peptide of human melanin-concentrating hormone receptor 1 has been successfully expressed and purified. The purified protein can bind with serum IgG antibodies from vitiligo patients, and may be applied to the detection of autoantibodies against human melanin-concentrating hormone receptor 1.
ABSTRACT
Objective To construct eukaryotic expressing vector of human melanin-concentrating hormone receptor1(MCHR1) and transfect HEK293 cells so as to establish stable HEK293 cell line.Methods The full-length MCHR1 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and was inserted into pcDNA3.1(+).After the identification of digestion and sequencing on the recombinant pcDNA3.1(+)/MCHR1,we transfected the recombinant into HEK293 cell by lipofectamineTM2000.After screening culture by G418,stable transfected HEK293 cell line was established,and the expression of MCHR1 was identified by RT-PCR and Western blotting.Results The eukaryotic expression vector pcDNA3.1(+)/MCHR1 was constructed,stable transfected HEK293 cell line was established,and MCHR1 protein was expressed successfully.Conclusion The construction of the eukaryotic expression vector pcDNA3.1(+)/MCHR1 and the establishment of stable transfected HEK293 cell line have provided solid experiment foundation for further studies on the function of MCHR1.
ABSTRACT
Objective To construct eukaryotic expressing vector of human melanin-concentrating hormone receptor 1(MCHR1), then to transfect CHO cells with the vector for establishment of stable CHO cell line. Methods The full-length MCHR1 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and then inserted into eukaryotic expression vector pcDNA3.1(+).The recombinant was transfected into CHO cells by lipofectamine TM 2000 after identification of digestion and sequencing on the recombinant eukaryotic expression vector pcDNA3.1(+)/MCHR1. The stable transfected CHO cell line was then established by screening cultures with G418, and the transcription and expression of MCHR1 were identified by RT-PCR, Western blot and immunofluorescence. Results The eukaryotic expression vector pcDNA3.1(+)/MCHR1 was constructed successfully, stable transfected CHO cell line was established, the MCHR1 protein was expressed successfully. Conclusion The construction of eukaryotic expression vector pcDNA3.1(+)/MCHR1 and the establishment of stable transfected CHO cell line provided a solid experimental foundation for further studies on the function of MCHR1.