Characterization Of Neuronal Signaling Underlying Feeding Inhibitory Effects Of Dorsomedial Hypothalamic Cholecystokinin

Cholecystokinin (CCK) is a brain-gut peptide that plays an important role in the control of food intake. Peripheral CCK acts as a satiety signal to limit meal size. CCK is released from the duodenum and jejunum in response to the intra-luminal presence of nutrient-digestive products. Peripheral CCK administration reduces food intake in a dose-related manner across a range of experimental situations and in a variety of species, and the actions of CCK in food intake are specific to a reduction in meal size. The feeding inhibitory effects of exogenously administered CCK appear to mimic a physiological role for endogenous CCK. Administration of CCK receptor-specific antagonists results in an increase in food intake, and this increase is manifested as an increase in meal size. The feeding inhibitory actions of both endogenously released and exogenously administered CCK are mediated through their interaction with CCK1 receptors. In contrast to the well characterized satiety actions of peripheral CCK, the role for brain CCK in the control of food intake has not yet been known. Blevins et al. demonstrated that infusing smaller doses of CCK-8 into specific brain sites resulted in site-specific feeding inhibitory actions in the rat, and this anorexic dose of CCK-8 did not increase plasma CCK-8 levelssufficiently to suppress feeding via a peripheral mechanism. Data from Otsuka Long-Evans Tokushima fatty (OLETF) rats, which have congenital CCK1 receptor deficiency and become hyperphagic and obese, have suggested that both peripheral and brain CCK take roles in the controls of food intake. Analysis of hypothalamic gene expression in OLETF rats have suggested that the dysregulation of dorsomedial hypothalamus neuropeptide Y (DMH NPY) gene expression resulting from CCK1 receptor deficiency may play an etiological role in the hyperphagia and obesity of OLETF rats. Subsequently, Immunohistochemical studies have revealed that CCK1 receptors and NPY were co-localized in DMH neurons. Although we have proposed a role for DMH CCK-NPY signaling in the control of food intake, we have yet to identify the pathways underlying this action. In the present study, we aimed to characterize the feeding inhibition and patterns of brain neuronal activation produced by injection of CCK into the DMH. Firstly,we examined the time course of feeding inhibitory effects of DMH CCK administration. Secondly we also examined whether DMH CCK administration resulted in alterations in hypothalamic corticotrophin-releasing factor (CRF), Pro-opiomelanocortin (POMC) and NPY gene expression. Finally, we assessed brain patterns of c-Fos activation induced by DMH CCK administration to identify candidate brain sites that might mediate the actions of the DMH CCK-NPY signaling system.Methods: Male Sprague-Dawley rats weighing 250-300 g purchased from Charles River Laboratories, Inc. (Wilmington, MA) served as subjects. Rats were individually housed in hanging wire mesh cages and maintained on a 12:12-h light-dark cycle in a temperature-controlled environment (22℃) with ad libitum access to water and feeding schedules as described in each experiment.DMH CCK-8 injection and food intake. Thirteen male Sprague-Dawley rats were implanted with unilateral indwelling DMH cannulae and were randomly divided into two groups. Just before lights off, one group of 6 animals was injectedwith 0.3 μl of artificial cerebral-spinal fluid (aCSF: 147 mM Na⁺, 2.7 mM K⁺, 1.2 mM Ca²⁺, 0.85 mM Mg²⁺ and 153.8 mM Cl^-) and the other group of 7 animals was injected with 0.5 nmol of CCK in 0.3 μl aCSF. Pelleted chow was returned to the cages immediately after the injection. Food intakes were measured at 30 min, 1 h, 2 h, 4 h, and 22 h later. After 7-day recovery, all rats were given a second DMH injection with aCSF or CCK-8 (0.5 nmol), i.e., the rats that had previously received CCK-8 administration were given aCSF injection at this time and vice versa. Food intakes were measured as following the first injections.Analyses of hypothalamic NPY, CRF and POMC gene expression. After feeding tests, 13 DMH cannulated rats were weight matched and randomly divided into two groups: aCSF control (n = 6) and CCK-8 treatment (n = 7), for assessing whether DMH CCK-8 injection affected hypothalamic NPY, CRF and POMC mRNA expression. Animals were maintained on the same feeding schedule as above in which regular chow was removed from the cages 2 hours before lights off and returned to the cages just before dark onset and with access to water ad libitum. Again, rats received either aCSF or CCK-8 injections as described above but food was not returned to the cages. Three hours following injections, rats were sacrificed with an overdose of sodium pentobarbital, and brains were removed rapidly and frozen at -80℃ for subsequent analyses of hypothalamic NPY, CRF and POMC gene expression. 14-μm coronal brain sections ranging from 1.8-3.4 mm caudal to bregma were cut with a cryostat, mounted on superfrost/plus slides and fixed with 4% paraformaldehyde. ³⁵S-labeled NPY, POMC and CRF antisense riboprobes were transcribed by using in vitro transcription systems and purified. Sections for Arc POMC mRNA Arc NPY mRNA, determination, DMH NPY mRNA and for CRF mRNA in the paraventricular nucleus (PVN) were taken. Sections were treated with acetic anhydride and incubated in hybridization buffer containing 500 (J.g/ml yeast tRNA and 10⁸ cpm/ml of ³⁵S-UTP at 55℃ overnight. After hybridization, sectionswere washed, dehydrated, air-dried and exposed with BMR-2 film for 1-3 days. Quantitative analysis of the in situ hybridization data was done with NIH Scion image software (National Institutes of Health). Autoradiographic images were first scanned by EPSON Professional Scanner (EPSON) and saved via a computer for subsequent analyses with Scion image software using autoradiographic ¹⁴C micro scales (Amersham) as a standard. Arc POMC or PVN CRF mRNA levels were determined by a mean of the product of hybridization area x density (background density was subtracted) for each rat. Data from each group were normalized to vehicle aCSF treated controls as 100%, and all data were presented as mean ± SEM.DMH CCK injection and c-Fos immunohistochemistry. Twenty-eight male Sprague-Dawley rats were implanted with unilateral indwelling DMH cannulae as described above. After postoperative recovery and habituation to the injection procedure, rats were randomly divided into two groups (n=14): one group was injected with 0.3 μl of aCSF and the other with 0.5 nmol of CCK-8 in 0.3 μl aCSF. All DMH injections were performed as described above, but rats were not allowed to access to chow food after DMH injection. Ninety minutes following injections, rats were anesthesized with Euthasol (pentobarbital sodium and phenytoin, Delmarva Laboratories, Midlothian, VA) and perfused transcardially with phosphate buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in PBS. Brains were removed and stored in 25% sucrose containing 4% paraformaldehyde at 4℃ for subsequent c-Fos immunoreactivity determinations. In the initial step, 3 animals per group were examined for determination of the regions where c-Fos activation was potentially induced by DMH CCK injection. Since the initial c-Fos immunoreactivity determination revealed that DMH CCK-induced c-Fos positive cells were exclusively localized to hypothalamic areas, subsequent quantitative c-Fos immunoreactivity was only determined in brain regions over the hypothalamus in the remaining animals (11 rats per group). Forty μm coronal sections extending from0.48 mm anterior to bregma to 4.36 mm posterior to bregma were cut, and every other section was collected in PBS for c-Fos immunoreactivity determination. The number of c-Fos positive cells was counted in the following areas: the suprachiasmatic neucleus (SCh), the retrochiasmatic area (RCh), the supraoptic nucleus (SON), the PVN, the DMH, the Arc, the medial eminence (ME), the ventromedial hypothalamus (VMH), and the lateral hypothalamus (LH), as well as the central neucleus of amygdala (CeA). Images of sections were captured by digital camera attached to Zeiss Axio Imager. The area of interest was outlined based on cellular morphology and c-Fos positive cells were automatically counted by the imaging program (IPLab, Scanalytics, Fairfax, VA) by setting minimum and maximum optical density levels. Cell counts of c-Fos immunoreactivity were made separately in the ipsilateral (iDMH) and contralateral (cDMH) to the site of DMH CCK injection. Data for c-Fos activation in all other areas were bilaterally assessed, and were presented as the total number of c-Fos positive cells per section.Results: i. Effects of DMH CCK injection on food intake. Parenchymal injection of CCK into the DMH decreased food intake during the entire 22 hour observation period. The magnitude of feeding inhibitory effect was time dependent. DMH CCK administration resulted in a 51% reduction in the first 30 min as compared to vehicle treated rats. In contrast to the time course of the effect of peripherally administered CCK, the feeding inhibition produced by DMH CCK injection was long lasting with a 38.4% suppression maintained at 4 hours. Compensation for this reduction did not occurred within the next 18 hours. Food intake of DMH CCK administered rats remained significantly reduced at 22 hours as compared to the vehicle controls. ii. Effects of DMH CCK injection on Arc NPY,DMH NPY,PVN CRF and Arc POMC gene expression. Relative to vehicle-treated rats, DMH CCK administration elevated PVN CRF gene expression with a 38% increase in mRNA levels, down-regulated DMH NPY gene expressionwith a 27% decrease in mRNA levels and Arc NPY gene expression a with 24% decrease in m RNA levels. DMH CCK administration did not affect Arc POMC mRNA levels as compared to vehicle treatment iii. Characterization of brain c-Fos activation induced by DMH CCK administration. Examination of c-Fos immunoreactivity throughout the entire brain revealed that DMH CCK-induced c-Fos activation was exclusively localized to the hypothalamus. The positive sites included the SCh, RCh, PVN, cDMH, and Arc, but not the SON, VMH, LH and ME. DMH CCK injection resulted in a 5-fold increase in the number of c-Fos positive cells in the cDMH as compared to that of vehicle treated rats. This c-Fos immunoreactivity was detected in all three subregions, i.e., the dorsal, ventral and compact part of the cDMH. Within the PVN, DMH CCK-induced c-Fos activation was primarily located in the medial parvicellular part of the PVN,and with a 4-fold increase as compared to the vehicle treatment, whereas very few c-Fos positive neurons were detected in the lateral magnocellular part of the PVN. DMH CCK injection also significantly increased c-Fos immunoreactivity in the Arc, with a majority of c-Fos positive cells in the medial part DMH CCK administration increased the number of c-Fos positive cells by 3.8 folds in the SCh and 5.3 folds in the RCh relative to vehicle treatment. DMH CCK injection did not induce c-Fos activation in the CeA, NTS, and AP.Conclusion: Injection of CCK into the DMH results in a rapid decrease in food intake, and this feeding inhibition maintained at least 22 hours. In response to DMH CCK injection, PVN CRF gene expression is significantly elevated, DMH NPY and Arc NPY gene expression is significantly reduced, while Arc POMC gene expression is not affected. In response to DMH CCK administration, c-Fos is activated in various hypothalamic areas including the cDMH, PVN, Arc, SCh and RCh, but not in the SON, VMH, LH and ME or in the CeA and the brain stem NTS and AP. In all, these data suggest that multiple hypothalamic signaling pathwaysmay underlie the actions of DMH CCK. DMH CCK-NPY signaling system plays an important role in the control of food intake and energy balance. Its actions seem to be mediated through multiple hypothalamic pathways, which depend upon PVN CRF and Arc NPY.