TDK conference 2012

Melanin-concentrating hormone (MCH) is a cyclic 19-amino acid hypothalamic peptide originally isolated from the hypophysis of fish controlling skin pigmentation. In mammals it is involved among others in the regulation of feed intake. MCH-expressing neurons were identified within the lateral hypothalamus and zona incerta, however their axons are present in many other brain areas. The hypothalamic regulatory centers were shown to be in reciprocal connection with the lateral septal area (LS). Previous studies proved that the LS is rich in several orexigenic and anorexigenic neuropeptides such as galanin, neuropeptide Y, opioid peptides and CART peptide. Feed deprivation experiments revealed that the density of the immunocytochemically detectable peptide-containing axonal meshworks is selectively increasing or decreasing upon partial feed deprivation. As MCH is also a well-known orexigenic neuropeptide, our aim was to reveal, whether these neural elements of the LS also react to feed deprivation, and if so, is it an increase or decrease of their density.

Young adult male Wistar rats were used in our experiments.Controls had access to regular rat chow and water ad libitum, whereas the members of the experimental group received only 60 % of the average daily amount of feed for one week. Then the animals were anaesthesized by intraperitoneal injection of Nembutal and transcardially perfused with 4% paraformaldehyde in 0.2 M phosphate-buffer (pH=7.4). 70 micron-thick vibratome serial sections were cut from the septal area of the brain in the coronal plane. Free-floating sections were processed according the normal protocol of pre-embedding immunocytochemistry (primary antiserum was MCH (SIGMA) diluted in 1:10.000, secondary antiserum was biotinylated anti-rabbit IgG followed by ABC, both in a dilution 1:100, and the chromogen was DAB). Light microscopic serial sections were then digitally photographed, and the photos were evaluated with computer-assisted densitometry (Scion Image). Some sections containing immunpositive elements were processed for electron microscopic analysis.

MCH-immunopositive axons were present in each part of the septum. The varicose axons enmeshed the whole septal complex, with the densest network in the medial septum and the dorsal subnucleus of the LS. MCH-immunopositive cell bodies were not detected. More than probable that these axons originate from the somata identified earlier in the lateral hypothalamus. Varicosities detected in the light microscopic sections appeared in the electron microscopic material as synaptic terminals filled with electron dense precipitate. Most of them established asymmetrical synaptic contacts with local immunonegative dendritic spines. The immunoreactive axons were found to be present in the same density along the rostrocaudal axis of the septal complex as it was measured in serial sections. The feed deprivation caused a significant increase in the density of MCH-immunopositive axons. Our results are in good agreement with the data published earlier about the hypothalamic feed-intake centers. These data confirm previous observations, according to which besides hypothalamus, the LS also plays an important role in the regulation of apetite and feed intake.