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It is hoped that other groups may test and, where suitable, adopt this method to reduce the use of experimental animals in basic cardiology research. None declared. This work was funded Moroxydine by the British Heart Foundation. We would like to thank Professor Ed White for useful discussion of this technique. ""1.?The aim of the present study was to investigate the orexigenic effect of cocaine- and amphetamine-regulated transcript (CART) peptide on feeding regulation following its injection into discrete nuclei of the hypothalamus. 2.?Male Sprague-Dawley diabetic rats were injected with 0.06 or 0.2?nmol CART (55�C102) or an equal volume of saline into various hypothalamic areas and food intake was then measured 1, 2, 4, 8 and 24?h after injection. Changes in hypothalamic CART mRNA expression in response check details to dietary intervention (2?weeks feeding of a high-fat diet) were assessed using quantitative real-time reverse transcription�Cpolymerase chain reaction. Possible interactions between neuropeptide Y (NPY), agouti-related protein (AGRP), ��-melanocyte-stimulating hormone (MSH) and corticotropin-releasing hormone (CRH) were evaluated in an in vitro hypothalamic explant system. Neuropeptide immunoreactivities (IR) were detertmined using radioimmunassays (RIAs). 3.?At 0.2?nmol, CART (55�C102) significantly increased feeding in fasted diabetic rats after injection into the dorsomedial hypothalamic nucleus and arcuate nucleus (ARC). Injection of 0.2?nmol CART (55�C102) into the ARC of satiated diabetic rats also increased food intake that was similar in both magnitude and time-course to the response seen in Temozolomide chemical structure fasted diabetic rats. Food intake in diabetic rats on a high-fat diet was clearly increased after injection of 0.2?nmol CART (55�C102) into the ARC, as was CART mRNA expression. Incubation of hypothalamic explants with 0.4, 4 and 40?nmol/L CART (55�C102) for 45?min significantly increased NPY IR, whereas exposure of explants to 4?nmol/L CART (55�C102) increaesd AGRP IR and CRH IR. None of the concentrations of CART (55�C102) tested had any effect on ��-MSH IR. 4.?Together, these data provide further evidence that hypothalamic CART has an orexigenic effect, which, in the ARC, may stimulate the release of hypothalamic orexigenic neuropeptides. The hypothalamus is essential in the integration of energy homeostasis and endocrine function and hypothalamic neuropeptides play an important role in the control of both appetite and the hypothalamo�Cpituitary axis.1 Cocaine- and amphetamine-regulated transcript (CART) peptides are one of the most abundant hypothalamic-specific transcripts and have emerged as major neurotransmitters and hormones.1 The CART peptides are widely distributed throughout the central nervous system (CNS) and regulate many processes, including food intake, maintenance of bodyweight and reward and endocrine functions.