The vomeronasal organ (VNO) is a peripheral sensory organ present in many mammals that is involved in the detection of pheromones, substances released by animals affecting behavior or physiology of other individuals of the same species. The VNO contains specialized neurons, called vomeronasal sensory neurons (VSNs), expressing individual types of vomeronasal receptors from large families and capable to detect chemical stimuli by transducing their binding into electrical signals that are transferred to the central nervous system to be processed. Ligand binding to vomeronasal receptors on microvilli of the dendritic knobs of VSNs activates a PLC-dependent second messenger transduction cascade that produces an increase in intracellular calcium concentration. The increase in cytosolic calcium concentration plays several roles in signal transduction, involving the activation of other ion channels and enzymes. Previous studies showed that calcium-activated chloride currents are activated by cytosolic calcium increase in mouse VSNs, and that both TMEM16A and TMEM16B, two proteins forming calcium-activated chloride channels, are expressed in microvilli of VSNs.
Here, we used whole-cell and inside-out patch-clamp recordings to provide a functional characterization of currents activated by calcium in isolated mouse VSNs. We found that intracellular calcium activated anionic currents in whole-cell and inside-out patches from the dendritic knob/microvilli. These currents were activated at sub-micromolar calcium concentration, were voltage-dependent and were blocked by commonly used chloride channel blockers. We compared the electrophysiological properties of the native currents with those mediated by heterologously expressed TMEM16A or TMEM16B calcium-activated chloride channels, which are co-expressed in microvilli of mouse VSNs, and found a closer resemblance to those of TMEM16A. We used the Cre\u2013loxP system to selectively knock out TMEM16A in mature VSNs. We showed that calcium-activated currents were abolished in VSNs of TMEM16A conditional knockout mice (TMEM16A cKO), demonstrating that TMEM16A is an essential component of calcium-activated chloride currents in mouse VSNs.
As TMEM16A cKO VSNs do not present calcium-activated chloride currents, this mouse line is a good model to study the role of calcium-activated chloride currents in vomeronasal physiology. We performed electrophysiological recordings to compare the properties of the membrane and the spontaneous and evoked activity in WT and TMEM16A cKO VSNs. In whole-cell experiments in the voltage-clamp configuration, we found that deleting TMEM16A channel in VSNs did not affect the membrane input resistance, resting membrane potential or current-voltage relations of voltage-activated inward and outward currents. Extracellular recordings in loose-patch configuration showed that firing pattern of spontaneous activity was affected in VSNs from TMEM16A cKO mice, showing less activity coded in burst with respect to WT neurons, while the mean frequency was not affected. We recorded the capability of VSNs to respond to urine at 1:50 dilution presented for a 10 s period. We found urine responses both in WT and TMEM16A cKO VSNs, indicating that neurons lacking calcium-activated chloride currents were still able to activate signal transduction after stimulus presentation and to increase firing activity. When we compared the firing activity of evoked activity, we found that mean frequency was not altered, while the firing pattern was strongly affected. Inter-spike interval distribution of evoked activity showed that VSNs from TMEM16A cKO fired with shorter intervals than WT neurons. We conclude that the calcium-activated chloride current in VSNs depends on TMEM16A expression and that it regulates the spike firing pattern both during spontaneous and evoked activity
