Online

Vick, Jonathan

Biology

2014

PhD

Olfaction, the sense of smell, allows animals to perceive the multitude of volatile and nonvolatile molecules present in the environment. In many mammals, such as mice and rats, there are four unique chemosensory organs including the (1) main olfactory epithelium (MOE), (2) septal organ, (3) Grüneberg ganglion, and (4) vomeronasal organ (VNO). While the VNO detects some general volatile odorants, it is further specialized for the detection of behaviorally relevant nonvolatile odorants or pheromones. In rodents, the VNO is encased within a bony capsule and located at the base of the nasal cavity. Odorants are detected by vomeronasal sensory neuron (VSN)s, bipolar neurons with a single axon that projects to the accessory olfactory bulb of the brain and a single dendrite capped with microvilli that project into the lumen of the VNO. In the MOE, purinergic signaling through adenosine 5'-triphosphate (ATP) gated ionotropic P2X and G-protein coupled P2Y receptors contributes a neuroprotective and neuroregenerative pathway. As virtually nothing was known about purinergic signaling in the VNO, I set out to characterize the (1) presence of the purinergic receptors and (2) ATP release pathways. In isolated VSNs, ATP elicited an increase in intracellular calcium ([Ca2+]I) and an inward current with similar potency. Adenosine and the P2Y receptor agonists adenosine 5'-diphosphate (ADP), uridine 5'-triphosphate (UTP), and uridine 5'-diphosphate (UDP) were ineffective. The increase in [Ca2+]I was dependent upon extracellular calcium and the inward current elicited by ATP was partially blocked by the P2X receptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS) and 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP). When coapplied with the natural stimulus dilute urine, ATP increased the inward current above that elicited by either dilute urine or ATP alone. Furthermore, ADP hyperpolarized the voltage dependence of steady state inactivation of voltage activated sodium current (INa) in a subset of VSNs. The hyperpolarization in the voltage dependence of steady state inactivation elicited by ADP was blocked in the presence of suramin, a purinergic receptor antagonist, but similar to that produced by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable protein kinase C (PKC) activator. Neither ATP nor ADP affected the voltage dependence of activation, fast inactivation, or time dependent recovery from inactivation. Interestingly, ADP reversibly increased spike frequency but did not change an action potential's amplitude, latency, halfwidth, or threshold voltage. Accordingly, we detected gene expression of the P2X1 and 3 as well as P2Y1, 2, and 6 receptors in the VNO and localized the P2Y1 and 2 receptors to isolated VSNs. Thus, excitability in VSNs can be enhanced by (1) ATP eliciting an inward current through P2X receptors and (2) ADP decreasing spike adaptation during persistent firing presumably through P2Y receptors. Moreover, one possible source of ATP may be from mechanical stimulation of the VNO that accompanies vasomotor pump activation.