The aim of this review is to explore the relationship between melatonin, free radicals, and non-excitatory amino acids, and their role in stroke and aging. Melatonin has garnered significant attention in recent years due to its diverse physiological functions and potential therapeutic benefits by reducing oxidative stress, inflammation, and apoptosis. Melatonin has been found to mitigate ischemic brain damage caused by stroke. By scavenging free radicals and reducing oxidative damage, melatonin may help slow down the aging process and protect against age-related cognitive decline. Additionally, non-excitatory amino acids have been shown to possess neuroprotective properties, including antioxidant and anti-inflammatory in stroke and aging-related conditions. They can attenuate oxidative stress, modulate calcium homeostasis, and inhibit apoptosis, thereby safeguarding neurons against damage induced by stroke and aging processes. The intracellular accumulation of certain non-excitatory amino acids could promote harmful effects during hypoxia-ischemia episodes and thus, the blockade of the amino acid transporters involved in the process could be an alternative therapeutic strategy to reduce ischemic damage. On the other hand, the accumulation of free radicals, specifically mitochondrial reactive oxygen and nitrogen species, accelerates cellular senescence and contributes to age-related decline. Recent research suggests a complex interplay between melatonin, free radicals, and non-excitatory amino acids in stroke and aging. The neuroprotective actions of melatonin and non-excitatory amino acids converge on multiple pathways, including the regulation of calcium homeostasis, modulation of apoptosis, and reduction of inflammation. These mechanisms collectively contribute to the preservation of neuronal integrity and functions, making them promising targets for therapeutic interventions in stroke and age-related disorders.

The calyx of Held is a giant axo-somatic synapse classically confined to the auditory brainstem. We recently identified morphologically similar calyx-like terminals in the extended amygdala (EA) that arise from the ventrolateral parabrachial complex and co-express PACAP, CGRP, VAChT, VGluT1, and VGluT2, targeting PKCδ⁺/GluD1⁺ EA neurons. Here, we asked whether this parabrachial–EA pathway participates in compensation during acute hypotension. In rats given hydralazine (10 mg/kg, i.p.), we quantified Fos protein during an early phase (60 min) and a late phase (120 min). Early after hypotension, Fos surged in a discrete subpopulation of the parabrachial Kölliker–Fuse (KF) region and in the EA, whereas magnocellular neurons of the supraoptic and paraventricular nuclei (SON/PVN) remained largely silent. By 120 min, magnocellular SON/PVN neurons were robustly Fos-positive. Confocal immunohistochemistry showed that most Fos+ PKCδ⁺/GluD1⁺ EA neurons were encircled by PACAP+ perisomatic terminals (80.8%), of which the majority co-expressed VGluT1 (88.1%). RNAscope in situ hybridization further identified a selective KF population co-expressing Adcyap1 (PACAP) and Slc17a7 (VGluT1) that became fos-positive during the early phase. Together, these data suggest that a KF PACAP⁺/VGluT1⁺ projection forms calyceal terminals around PKCδ⁺/GluD1⁺ EA neurons, providing a high-fidelity route for rapid autonomic rebound to falling blood pressure, while slower endocrine support is subsequently recruited via neurohormone-magnocellular activation. This work links multimodal parabrachial output to temporally layered autonomic–neuroendocrine control.

Adrenal chromaffin cells are key effectors of the sympathoadrenal response and play a central role in the organism adaptation to environmental and physiological challenges. While cholinergic and pituitary adenylate cyclase-activating polypeptide (PACAP)-dependent mechanisms have long been recognized as major regulators of catecholamine secretion, increasing evidence indicates that connexin-mediated gap junctional communication provides an additional and highly dynamic level of control. Whether early-life experience modifies the adult capacity of chromaffin-cell networks to undergo stress-induced connexin remodeling remains unclear. Here, we examined adrenal medullary connexin expression in adult rats exposed to neonatal maternal separation (MS; 3 h daily, postnatal days 2-15) and later challenged with an 8-day unpredictable mild stress (UMS) protocol. Under basal adult conditions, MS did not produce an overt change in adrenal medullary Cx36 or Cx43 immunoreactivity relative to animal-facility-reared controls. In contrast, UMS increased connexin immunoreactivity in the adrenal medulla, and this response was amplified in animals with a history of MS. MS+UMS animals also displayed enhanced corticosterone responses to acute restraint stress. These findings suggest that neonatal MS does not impose a constitutively altered adult chromaffin-cell phenotype, but instead primes the future stress responsiveness of adrenal medullary connexin remodeling. We propose that chromaffin-cell gap junctions represent a substrate sensitive to stress history, through which developmental experience may influence sympathoadrenal and endocrine adaptation in adulthood.

The bed nucleus of the anterior commissure (BAC) is a small pituitary adenylate cyclase-activating polypeptide (PACAP)-rich glutamatergic cell population located at the intersection of the anterior commissure, stria terminalis, stria medullaris, and fornix. Here, we provide functional neuroanatomical characterization of the BAC using PACAP-Cre mice and Cre-dependent viral tracing. BAC axons traverse these major forebrain conduits to engage distributed limbic and diencephalic networks, with dense innervation of the posterior basolateral amygdala (pBLA) and anterior dorsal thalamic nucleus (AD), both implicated in emotional processing and spatial orientation. To determine the functional significance of the BAC, we developed a novel looming-threat memory test (LTMT) paradigm in which mice learned the location of a shelter within a dual-maze labyrinth before exposure to an overhead looming stimulus. Chemogenetic inhibition of BAC PACAP neurons impaired shelter-directed escape, increased freezing behavior, prolonged escape trajectories, and disrupted efficient safety-seeking responses. Selective deletion of PACAP from BAC neurons also blocked key aspects of the looming-induced behavioral phenotype. Fos mapping revealed robust neuronal activation by the looming stimulus, within the AD, pBLA, and dorsal periaqueductal gray (dPAG). BAC silencing blocked Fos induction. PACAPBAC depletion blocked fos induction in AD, but not in pBLA or dPAG. Preliminary ex-vivo recordings further indicated that PACAP modulates the intrinsic excitability of AD (and pBLA) neurons. Together, these findings identify the BAC as a previously unrecognized PACAPergic forebrain hub that links emotional and spatial-orientation networks to coordinate looming-threat learning, memory, and adaptive defensive behavior.

Background: Adrenal chromaffin cells integrate cholinergic and peptidergic signaling to regulate catecholamine secretion during stress. Although pituitary adenylate cyclase-activating polypeptide (PACAP] sustains adrenal secretion during intense sympathetic activation, the systems-level mechanisms coordinating chromaffin-cell activity remain incompletely understood. Perspective: We suggest that chromaffin cells function as dynamically recruited chromaffin-cell ensembles rather than independent endocrine units. Within this framework, connexin-mediated gap junctions act as activity-dependent amplifiers that enable PACAP- and acetylcholine-dependent recruitment of progressively larger ensembles according to physiological demand. We further suggest that ensemble recruitability is continuously modified by physiological state and previous experience. Conclusions: Building upon recent evidence and our experimental observations, we propose that early-life adversity remodels hypothalamic autonomic circuits, altering subsequent PACAP-dependent recruitment of chromaffin-cell ensembles during adult stress. This unified framework integrates peptidergic signaling, gap junction plasticity, and developmental programming, generating experimentally testable hypotheses and providing a new systems-level view of sympathoadrenal regulation.