The basic principle in maintaining energy balance is food intake and use. Regulation of energy homeostasis is implemented by the neuroendocrine system. With the identification of the hormone leptin, which is known to play a role in food intake and appetite control, research in this area has gradually increased and has led to the discovery of many new molecules (Sobrino Crespo et al., 2014). One of these peptides is the nesfatin-1 molecule. Nesfatin-1 has been shown to play a role in energy hemostasis and especially in food intake control via a leptin-independent pathway (Oh-I et al., 2006).

Nesfatin-1 was first discovered in 2006 in a study on rats by Oh-I et al. (Oh-I et al., 2006). It was found in the head-brain hypothalamic region of rats and was characterized as an anorexigenic peptide. The precursor of nesfatin-1, which consists of 82 amino acids, is the nucleobindin 2 (NUCB2) protein. NUCB2/nesfatin-1 has been detected in the cortex, hypothalamic nuclei, zona postrema, the dorsal motor nucleus of the vagus nerve, and the cerebellum (Oh-I et al., 2006; Stengel and Taché, 2013). Specific binding sites for NUCB2/nesfatin-1 could be detected both in the central nervous system (CNS) (e.g., hypothalamus, cortex) and peripheral organs (e.g., gastrointestinal tract, pituitary, pancreas) (Shimizu et al., 2009). Studies have shown that nesfatin-1 conducts intracellular signal transduction not only in neurons located in the CNS but also in peripheral tissues (e.g., pancreatic and cardiac muscle cells). Nesfatin-1 carries out signal transmission in the cell by increasing the entry of intracellular calcium ions, and it can use different Ca+2 channels for this (Brailoiu et al., 2007).

The anorexigenic effect of nesfatin-1 was shown to be independent of the leptin pathway in the Zucker rat model. After the nesfatin-1 peptide was administered to the third ventricle region, a decrease in food intake was observed in leptin-resistant animals (Brunner et al., 1997). At the same time, an increase in food intake was shown after daily injection of an appropriate NUCB2 antisense oligonucleotide for 10 days, thus suggesting that endogenous NUCB2/nesfatin-1 plays a role in the regulation of feeding behaviors (Oh-I et al., 2006; Stengel and Taché, 2013). Studies conducted in our laboratory have determined that NUCB2/nesfatin-1 neurons are activated by glutamatergic agonists known as excitatory neurotransmitters (Gok et al., 2020; Gok Yurtseven et al., 2018).

In addition to its anorexigenic properties, nesfatin-1 has functions related to cardiovascular and reproductive functions, glucose homeostasis, lipid metabolism, sleep and mood regulation (Dore et al., 2017). Studies have shown that nesfatin-1 can affect glucose metabolism by increasing insulin sensitivity (Yang et al., 2012). It has also been shown that peripheral infusion of nesfatin-1 can reduce adiposity and plasma levels of triglycerides and cholesterol and increase heart rate and blood pressure in rats (Yosten and Samson, 2009a). It is known that nesfatin-1 also plays a role in the regulation of the reproductive axis in the rats (Schalla and Stengel, 2018) and NUCB2/nesfatin-1 neurons express estrogen receptors in order to receive peripheral gonadal signals (Oy et al., 2023).

Studies conducted in subsequent years have shown that this peptidergic system may also play a role in the regulation of psychopathological behaviors such as depression and anxiety (Emmerzaal and Kozicz, 2013). It has been reported that intracerebroventricular injection of nesfatin-1 has an anxiety- and fear-increasing effect (Merali et al., 2008), and intraperitoneal administration similarly stimulates anxiety-like behaviors in rats (Ge et al., 2015). Possible relationships between nesfatin-1 and anxiety have also been examined in human studies and an increase in NUCB2/nesfatin-1 levels has been observed in patients with high anxiety levels (Hofmann et al., 2015a; Weibert et al., 2019). NUCB2/nesfatin-1 levels also showed a positive correlation with anxiety only in women suggesting a possible gender difference in regulation (Hofmann et al., 2015b). Plasma NUCB2/nesfatin-1 levels were measured in patients suffering from anorexia nervosa and reported to positively be related to perceived anxiety and may vary depending on the course of the eating disorder (Hofmann et al., 2015c; Pałasz et al., 2020). Additionally NUCB2/nesfatin-1 neurons receive peripheral stress signals via glucocorticoid receptors (Ekizceli et al., 2021) and exhibit activation against stress which can be diminished by glutamate antagonists (Goebel et al., 2009; Minbay et al., 2019).

Glutamate is one of the leading amino acid neurotransmitters that play an important role in the excitatory interaction in the hypothalamus. The findings from our previous studies show that the number of NUCB2/nesfatin-1 neurons expressing c-Fos increases after the injection of glutamate agonists, and this increase is suppressed by the agonist-specific antagonist administration performed before the injection of the glutamate agonist (Gok et al., 2020). In fact, these results indicate the existence of a direct interaction between the glutamatergic system and NUCB2/nesfatin-1 neurons. This interaction may occur with the presence of synaptic formation between glutamate-containing axon terminations and the neurons under study. In our study, we aimed to demonstrate the presence of axon terminations labeled with VGLUT 2, VGLUT 3, VGAT, and VAChT proteins, which we think are expressed in NUCB2/nesfatin-1 neurons.

Except for glutamate, there is no information in the literature about neurotransmitter systems involved in the regulation of NUCB2/nesfatin-1 neurons. In our study, the relationship of acetylcholine, another excitatory neurotransmitter, with NUCB2/nesfatin-1 neurons at the level of axon terminations was investigated. The existence of cholinergic axon terminations in the hypothalamus has been shown in various studies, but the relationship of these terminations with NUCB2/nesfatin-1 neurons is unknown. NUCB2/nesfatin-1 neurons are the cells that lose their activation by inhibition in the fasting state. Based on the hypothesis that GABA neurotransmission may affect the access and inhibition of peripheral hunger signals to these neurons, our study investigated the presence of GABAergic endings at the level of NUCB2/nesfatin-1-expressing neurons.

The original aim of the study was to determine the presence of excitatory and inhibitory neurotransmitter inputs on NUCB2/nesfatin-1 neurons. For this purpose, our hypothesis was determined as ‘NUCB2/nesfatin-1 neurons perform their anorexigenic functions under the control of upper central neurons containing different neurotransmitters’. In order to test this hypothesis, it is aimed to demonstrate the appositions of axon endings of neurons that use glutamate, acetylcholine, or GABA as neurotransmitters on NUCB2/nesfatin-1 neurons by dual immunohistochemical technique. Within the scope of the study, 3 goals were determined: (a) To determine the density of such neurons in the hypothalamus by demonstrating the presence of excitatory axon endings containing vesicular glutamate transporter (VGLUT) proteins on NUCB2/nesfatin-1 neurons, (b) To determine the density of such neurons in the hypothalamus by demonstrating the presence of excitatory axon endings containing vesicular acetylcholine transporter (VAChT) proteins on NUCB2/nesfatin-1 neurons, (c) To determine the density of such neurons in the hypothalamus by demonstrating the presence of inhibitory axon endings containing vesicular GABA transporter (VGAT) proteins on NUCB2/nesfatin-1 neurons.