Peripherally injected CCK-8S activates CART positive neurons of the paraventricular nucleus in rats

Abstract

Cholecystokinin (CCK) plays a role in the short-term inhibition of food intake. Cocaine- and amphetamine-regulated transcript (CART) peptide has been observed in neurons of the paraventricular nucleus (PVN). It has been reported that intracerebroventricular injection of CART peptide inhibits food intake in rodents. The aim of the study was to determine whether intraperitoneally (ip) injected CCK-8S affects neuronal activity of PVN-CART neurons. Ad libitum fed male Sprague-Dawley rats received 6 or 10 microg/kg CCK-8S or 0.15M NaCl ip (n=4/group). The number of c-Fos-immunoreactive neurons was determined in the PVN, arcuate nucleus (ARC), and the nucleus of the solitary tract (NTS). CCK-8S dose-dependently increased the number of c-Fos-immunoreactive neurons in the PVN (mean+/-SEM: 102+/-6 vs. 150+/-5 neurons/section, p<0.05) and compared to vehicle treated rats (18+/-7, p<0.05 vs. 6 and 10 microg/kg CCK-8S). CCK-8S at both doses induced an increase in the number of c-Fos-immunoreactive neurons in the NTS (65+/-13, p<0.05, and 182+/-16, p<0.05). No effect on the number of c-Fos neurons was observed in the ARC. Immunostaining for CART and c-Fos revealed a dose-dependent increase of activated CART neurons (19+/-3 vs. 29+/-7; p<0.05), only few activated CART neuron were observed in the vehicle group (1+/-0). The present observation shows that CCK-8S injected ip induces an increase in neuronal activity in PVN-CART neurons and suggests that CART neurons in the PVN may play a role in the mediation of peripheral CCK-8S's anorexigenic effects.

Fig. 1

CCK-8S injected intraperitoneally increases the number of c-Fos-positive neurons in the paraventricular nucleus of the hypothalamus (PVN) and in the nucleus of the solitary tract (NTS), whereas no effects were observed in the arcuate nucleus of the hypothalamus (ARC). Data are expressed as mean ± SEM of the number of rats indicated in parentheses. *p < 0.05 vs. 6 μg/kg CCK-8S and vs. vehicle; ^#p < 0.05 vs. 10 μg/kg CCK-S and vs. vehicle.

Fig. 2

In the arcuate nucleus of the hypothalamus (ARC) no effects were observed on the number of activated neurons (green staining) at both doses of CCK-8S. The white outer line delineates the area of the ARC. 3V, third brain ventricle.

Fig. 3

Total number of CART-immunoreactive neurons in all treatment groups in the paraventricular nucleus of the hypothalamus (A) as well as number of double labeled neurons (c-Fos/CART) in the PVN (B). Double staining revealed that CCK-8S significantly and dose-dependently increased the number of c-Fos-positive CART cells in the PVN. Data are expressed as mean ± SEM of the number of rats indicated in parentheses.

Fig. 4

Double labeled neurons at low magnification with antibodies against c-Fos (green staining) and CART (red staining) in the paraventricular nucleus 90 min after intraperitoneal injection of vehicle, 6 or 10 μg/kg CCK-8S. 3V, third brain ventricle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.).

Fig. 5

Magnification of c-Fos-positive neurons co-localized with CART immunoreactivity in the PVN after injection of 6 μg/kg CCK-8S (the region of the magnification is indicated in Fig. 4).

Fig. 6

CCK-8S injected intraperitoneally at both doses (6 and 10 μg/kg) significantly increases the number of c-Fos-positive neurons in the nucleus of the solitary tract (NTS) in a dose-dependent manner. AP, area postrema.