Immunohistochemical determination of the excitatory and inhibitory axonal endings contacting NUCB2/nesfatin-1 neurons

Nesfatin-1 is an anorexigenic peptide suppressing food intake and is synthesized and secreted by neurons located in the hypothalamus. Our study was aimed to demonstrate the effect of excitatory and inhibitory neurotransmitters on NUCB2/nesfatin-1 neurons. In this context, dual peroxidase immunohistochemistry staining was performed using NUCB2/nesfatin-1 primary antibody with each of the primary antibodies of vesicular transporter proteins applied as markers for neurons using glutamate, acetylcholine, and GABA as neurotransmitters. In double labeling applied on floating sections, the NUCB2/nesfatin-1 reaction was determined in brown color with diaminobenzidine, while vesicular carrier proteins were marked in black. Slides were analyzed to determine the ratio of nesfatin-1 neurons in the three hypothalamic nucleus in contact with a relevant vesicular carrier protein. The ratios of NUCB2/nesfatin-1 neurons with the innervation were compared among neurotransmitters. In addition, possible gender differences between males and females were examined. The difference in the number of VGLUT2-contacting NUCB2/nesfatin-1 neurons was significantly higher in males when compared to females. When both genders were compared in different nuclei, it was seen that there was no statistical significance in terms of the percentage of NUCB2/nesfatin-1 neuron apposition with VGLUT3. The statistical evaluation showed that number of NUCB2/nesfatin-1 neurons receiving GABAergic innervation is higher in males when compared to females (*p ≤ 0.05; p = 0.045). When the axonal contact of vesicular neurotransmitter transporter proteins was compared between the neurotransmitters, it was determined that the most prominent innervation is GABAergic. In the supraoptic region, no contacts of VAChT-containing axons were found on NUCB2/nesfatin-1 neurons in both female and male subjects. In conclusion, it is understood that both excitatory and inhibitory neurons can innervate the NUCB2/nesfatin-1 neurons and the glutamatergic system is effective in the excitatory innervation while the GABAergic system plays a role in the inhibitory mechanism.