**Abstract**

This chapter elucidates the role of depolarization-induced oxytocin (OT) vs. arginine vasopressin (AVP) secretion in the absence of external calcium, and calcium release from ryanodine-sensitive internal stores as a significant physiological contributor to neuropeptide secretion from hypothalamic neurohypophysial system (HNS) terminals. This has important therapeutic implications, given that exogenous administration of OT to children with autism spectrum disorders (ASD) has shown some success in improving social behavior and lowering anxiety. However, this nonspecific treatment has side effects, including seizures, increased heart rate variability, and psychotic symptoms. Alternatively, facilitating the physiological neuronal release of OT but not AVP from the HNS via modulation of ryanodine vs. inositol triphosphate receptor (IP3R) calcium stores would specifically facilitate central vs. peripheral OT release in ASD patients.

**Keywords:** oxytocin, vasopressin, neurohypophysis, ryanodine receptor, inositol trisphosphate receptor, autism spectrum disorders, ADP-ribosyl cyclase/CD38

### **1. Introduction**

### **1.1 Hypothalamic neurohypophysial system (HNS)**

The main mechanism for neuropeptide release from neurohypophysial terminals (NHT) acts via depolarization-secretion coupling. This refers to the relationship between neuronal depolarization and the subsequent release of hormones, specifically oxytocin (OT) and arginine vasopressin (AVP), from the posterior pituitary gland (**Figure 1**). This hypothalamic-neurohypophysial system (HNS) plays a critical role in regulating various physiological processes, including social behavior, reproduction, and water balance. Within the HNS, specialized neurons located in the hypothalamus synthesize and package OT and AVP into vesicles. These magnocellular neurons (MCNs) extend their axons through the pituitary stalk and terminate in the posterior pituitary (also known as (aka) neurohypophysis) gland, where the hormones are stored and released into the capillary bed for systemic delivery.

The classic understanding of neuropeptide release involves depolarization of the hypothalamic neurons which receive excitatory input which activates action potentials to their terminals. This depolarization leads to the opening of voltage-gated

#### **Figure 1.**

*Mechanisms of [Ca2+]i affecting oxytocin (OT) release. 1) depolarization of L-type voltage-gated calcium channel (VGCC) mechanically opens the ryanodine receptor (RyR), which leads to the release of calcium from ryanodinesensitive stores. 2) activation of the OT receptor (OTR) initiates cyclic adenosine diphosphate ribose (cADPr) signaling, presumably via Gq activation of ADP-ribosyl cyclase/CD38 (cluster of differentiation 38) complex catalyzing the conversion of NAD<sup>+</sup> into cADPr. Cyclic ADPr (cADPr) subsequently leads to the activation of ryanodine receptors (RyRs) on neurosecretory granules (NSGs) in the terminals. 3) inhibition of R-type VGCC. The release of diffusible second messenger Ca2+ from ryanodine-sensitive stores subsequently results in 4) OT release via SNARE complex activation. Notably, CD38, found only in OT terminals, facilitates hormonal secretion by releasing intraterminal Ca2+ and Ca2+-induced Ca2+ release (CICR), other RyR types, and/or other stores [1]. This would traffic more vesicles to release sites, thus, facilitating release.*

calcium channels on the terminal membrane, allowing calcium ions (Ca2+) to enter (**Figure 1**). The influx of calcium into the neurons initiates a cascade of events that ultimately lead to the exocytosis of OT- and AVP-containing vesicles into the bloodstream. The increase in intracellular calcium concentration serves as a key signal for the fusion of vesicles with the neuronal membrane, resulting in the secretion of hormones into the extracellular space. The coupling between depolarization and hormone secretion involves several important steps. When calcium enters the neurons, it binds to proteins such as synaptotagmin, which triggers the fusion of hormone-containing vesicles with the plasma membrane. This fusion process allows the release of OT and AVP. Additionally, calcium influx also activates calcium-dependent enzymes, such as protein kinases, which can modulate the secretion process. These enzymes can regulate the activity of proteins involved in vesicle fusion and neurotransmitter release, thereby fine-tuning the coupling of depolarization and hormone secretion.

In this chapter, we highlight the coupling of depolarization and secretion in the HNS via the release of calcium (Ca2+) from intraterminal Ca2+, specifically voltage-and ryanodine-sensitive, stores as opposed to Ca2+ influx via voltage-gated calcium channels (VGCCs). In the absence of external Ca2+, depolarization-induced secretion of oxytocin

(OT) and arginine vasopressin (AVP) can still occur, albeit with certain limitations. Recent evidence supports the hypothesis that Ca2+ from internal stores plays a crucial role in triggering the exocytosis of hormone-containing vesicles from nerve terminals. These alternative mechanisms can contribute to the release of OT and AVP in the absence of external calcium, thereby helping to shape both the amount and frequency of neuropeptide release differentially from OT vs. AVP terminals.

#### **1.2 Calcium stores**

These internal calcium stores within the HNS have originally referred to the endoplasmic reticulum (ER), which is a specialized organelle within cells involved in calcium storage and release. When an action potential reaches the neurohypophysial terminal, voltage-gated calcium channels on the plasma membrane open, allowing calcium ions (Ca2+) to enter the terminal from the extracellular space. This calcium influx triggers the release of calcium from the internal stores, specifically the ER and the granules containing OT and AVP themselves, through a process known as calcium-induced calcium release (CICR). The released calcium from intraterminal stores amplifies the calcium signal and contributes to the overall calcium concentration in the cytoplasm. This increased calcium concentration is crucial for the fusion of neurotransmitter-containing vesicles with the plasma membrane, facilitating the release of oxytocin and arginine vasopressin into the bloodstream.

Within neurohypophysial terminals (NHT), a distinct intraterminal calcium store is characterized by local, voltage-dependent Ca2+ transients, known as syntillas [2, 3]. Syntillas are unaffected by the removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) within terminals, and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. The physiological role of these syntillas is under continued investigation, with recent important findings adding to the unique mosaic of regulation for OT and AVP release.

#### **1.3 Social behavior and OT**

Autism spectrum disorders (ASDs) are characterized by defects in reciprocal social interaction and communication and occur either sporadically or in a familial pattern [4–6]. The etiology of ASDs remains largely unknown and pharmacological treatments are needed. Oxytocins role in social memory and behavior, communication, and emotional recognition has now been well established [7–10]. Although making inferences about central OT functioning from peripheral measurement is difficult, the data suggest that OT abnormalities may exist in autism and that a more direct investigation of central nervous system's (CNS's) OT function is warranted [11].
