Immunohistochemical determination of excitatory and inhibitory axonal endings on nesfatin-1 neurons

Nesfatin-1 is an anorexigenic peptide suppressing food intake and is synthesized and secreted by neurons located in the hypothalamus. In our study, it was aimed to demonstrate the effect of excitatory and inhibitory neurotransmitters on nesfatin-1 neurons. In this context, dual peroxidase immunohistochemistry staining was performed using 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 nesfatin-1 reaction was determined in brown with diaminobenzidine, while vesicular carrier proteins were marked in black. Slides were analyzed to determine the ratio of nesfatin-1 neurons in the supraoptic nucleus contacting with relevant vesicular carrier protein. The ratios of nesfatin-1 neurons with the innervation was compared among neurotransmitters. In addition, possible gender differences between male and female were examined. While 69% of nesfatin-1 neurons in the supraoptic nucleus contact VGLUT2 in female, this rate was 88% in male. VGLUT3 contacts in nesfatin-1 neurons was 53% in female and 66% in male. The difference in the number of VGLUT2-contacting nesfatin-1 neurons was significantly higher in males when compared to female. The difference between male and female groups was insignificant in the number of nesfatin-1 neurons contacting VGLUT3-containing endings. The contacting ratio of nesfatin-1 neurons with VGAT-positive axons was 98% in female and 97% in male with no significance between genders. In the same region no contacts of VAChT-containing axons were found on nesfatin-1 neurons in both female and male subjects. 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 conclusion it is understood that the nesfatin-1 neurons can be innervated by both excitatory and inhibitory neurons and glumatergic system is effective in the excitatory innervation while GABAergic system plays a role in the inhibitory mechanism. Key words: Nesfatin-1, vesicular neurotransmitter transporter protein, supraoptic nucleus, GABA, glutamate