Purinergic receptors are membrane ion channels that are activated by extracellular ATP. In vertebrates, seven genes encode subunits of P2X receptors. The subunits, designated P2X1-7, are 40 - 50% identical in amino acid sequences. P2X receptors are composed of three subunits and are found as homo- and heterotrimers in tissues of vertebrates. P2X receptors have a wide distribution in the organism, functional receptors are found in neurons, glial cells, muscle cells and also in nonexcitable tissues as epithelial, endothelial, and in hemopoietic tissue. Purinergic signalling plays an important role in pain transmission, at CNS injury and immune processes. P2X receptor subunit consists of two transmembrane domains, extracellular domain and intracellular N-and C-termini. Each transmembrane domain contains two amino acids conserved across all P2X subunits. In the first transmembrane domain receptor P2X2 are that Gly30 and Tyr43. In previous experiments performed on P2X2 receptor, electrophysiological measurements demonstrated that substitution of conserved Tyr43 in the first transmembrane domain with alanine prolongs the deactivation time of ion channel after agonist wash out. This work is focused on clarifying the role of conserved tyrosine in the process of opening and closing of ion channel of P2X...

Aging is the main risk factor for cognitive decline. The hippocampus, a brain region critical for learning and memory formation, is especially vulnerable to normal and pathological age-related cognitive decline. Dysregulation of both insulin and intracellular Ca2+ signaling appear to coexist and their compromised actions may synergistically contribute to neuronal dysfunction with aging. This dissertation focused on the interaction between insulin, Ca2+ dysregulation, and cognition in hippocampal neurons by examining the contributions of insulin to Ca2+ signaling events that influence memory formation. I tested the hypothesis that insulin would increase cognition in aged animals by altering Ca2+-dependent physiological mechanisms involved in learning. The possible effects of insulin on learning and memory in young and aged rats were studied. In addition, the effects of insulin on the Ca2+-dependent afterhyperpolarization in CA1 pyramidal hippocampal neurons from young and aged animals were compared. Further, primary hippocampal cultures were used to examine the possible effects of insulin on voltage-gated Ca2+ channel activity and Ca2+-induced Ca2+-release; mechanisms known to influence the AHP. We found that intranasal insulin improved memory in aged F344 rats. Young and aged F344 rats were treated with Humalog®, a short-acting insulin analog, or Levemir®, a long-acting insulin analog. The aged rats performed similar to young rats in the Morris Water Maze, a hippocampal dependent spatial learning and memory task. Electrophysiological recordings from CA1 hippocampal neurons revealed that insulin reduced the age-related increase in the Ca2+-dependent afterhyperpolarization, a prominent biomarker of brain aging that is associated with cognitive decline. Patch clamping recording from hippocampal cultured neurons showed that insulin reduced Ca2+ channel currents. Intracellular Ca2+ levels were also monitored using Fura-2 in response to cellular depolarization. Results indicated that a reduction in Ca2+-induced Ca2+-release from intracellular stores occurred in the presence of insulin. These results suggest that increasing brain insulin levels in aged rats may have improved memory by reducing the AHP and intracellular Ca2+concentrations. This study indicates a possible mechanism responsible for the beneficial effects of intranasal insulin on cognitive function absorbed in selective Alzheimer’s patients. Thus, insulin therapy may reduce or prevent age-related compromises to Ca2+ regulatory pathways typically associated with cognitive decline.

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<p>Aortic bodies (ABs) are putative peripheral arterial chemoreceptors located near the aortic arch. They are hypothesized to contribute to O₂ homeostasis by sensing arterial O₂ content and initiating cardiovascular reflexes during hypoxia; however, information on their cellular physiology is lacking. The primary goal of this thesis was to elucidate chemosensory mechanisms among mammalian (rat) AB cells, located specifically at the bifurcation of the left vagus nerve and recurrent laryngeal nerve (RLN), where they are found in association with a group of local neurons (>30). In vagus nerve-RLN whole-mounts, AB chemoreceptor (type I) cells were immunoreactive against the vesicular acetylcholine (ACh) transporter, and were surrounded by nerve terminals immunopositive for purinergic P2X2 and P2X3 receptor subunits, suggesting that ACh and ATP may act as neurotransmitters as in the related carotid body. In a novel dissociated AB culture model, subsets of type I cells exhibited elevated intracellular Ca²⁺ responses to hypoxia, isohydric hypercapnia, isocapnic acidosis, and acidic hypercapnia, demonstrating their direct chemosensitivity for the first time. Interestingly, surviving local neurons also responded to these chemostimuli, suggesting that they are sensory. Patch clamp electrophysiological and Ca²⁺ imaging studies revealed functional heteromeric P2X2/3 and nicotinic ACh receptors on local neurons, consistent with ACh and/or ATP mediating chemotransmission between receptor cells and local neurons. These neurons were also found to be interconnected by electrical synapses. Finally, the short-term survival of red blood cells (RBCs) in AB cultures, along with the finding that blood-borne factors (e.g. ATP released from RBCs) may have access to AB nerve terminals <em>in situ</em>, implicates RBCs as O₂-sensors in AB function. Altogether, these results suggest an important role for purinergic P2X2/3 receptors on local neurons/nerve terminals and ATP release from type I cells and RBCs, in the unique ability of ABs to sense and process information about blood O₂ content.</p> / Doctor of Philosophy (PhD)