The Ca2+-activated Cl− channel TMEM16B regulates action potential firing and axonal targeting in olfactory sensory neurons

The olfactory system detects small volatile molecules, odorants, which enter the nasal cavity via the inhaled air during normal breathing or sniffing. Odorants bind to odorant receptors (ORs) located on the cilia of olfactory sensory neurons (OSNs). Each OSN expresses only one type of OR from ∼1,000 in the mouse genome. Cilia are embedded in the mucus covering the epithelium and are the site of olfactory transduction. Odorant molecules, once bound to ORs, activate a G protein– coupled transduction cascade by activating the olfactory G protein Golf, which in turn activates adenylate cyclase III, leading to the production of cAMP and culminating in the opening of two types of ion channels, CNG and Ca-activated Cl− channels. CNG channels in the ciliary membrane of OSNs have been first described by Nakamura and Gold (1987), they are directly activated by cAMP, and they induce a depolarizing influx of Na and Ca ions (reviewed by Schild and Restrepo, 1998; Pifferi et al., 2006, 2010; Kleene, 2008). The presence of a Ca-activated Cl− conductance was first demonstrated in the cilia of frog OSNs by Kleene and Gesteland (1991), which showed that a rise in intraciliary Ca concentration directly activates an anion-selective current in the ciliary membrane. Subsequent studies showed that Ca-activated Cl− channels are present also in other species, including rodents, and that they are activated by Ca entry through CNG channels producing a large secondary Cl− current (Kleene and Gesteland, 1991; Kleene, 1993, 1997; Kurahashi and Yau, 1993; Lowe and Gold, 1993; Firestein and Shepherd, 1995; Zhainazarov and Ache, 1995; Reisert et al., 2005; Boccaccio and Menini, 2007). In electrophysiological recordings from OSNs isolated from rats or mice, Ca-activated Cl− currents (CaCCs) account for up to 90% of the transduction current (Lowe and Gold, 1993; Boccaccio and Menini, 2007). CaCCs are depolarizing currents as a result of the active Cl− ions accumulation inside OSNs. This The Ca-activated Cl− channel TMEM16B is highly expressed in the cilia of olfactory sensory neurons (OSNs). Although a large portion of the odor-evoked transduction current is carried by Ca-activated Cl− channels, their role in olfaction is still controversial. A previous report (Billig et al. 2011. Nat. Neurosci. http ://dx .doi .org /10 .1038 /nn .2821) showed that disruption of the TMEM16b/Ano2 gene in mice abolished Ca-activated Cl− currents in OSNs but did not produce any major change in olfactory behavior. Here we readdress the role of TMEM16B in olfaction and show that TMEM16B knockout (KO) mice have behavioral deficits in odor-guided food-finding ability. Moreover, as the role of TMEM16B in action potential (AP) firing has not yet been studied, we use electrophysiological recording methods to measure the firing activity of OSNs. Suction electrode recordings from isolated olfactory neurons and on-cell loose-patch recordings from dendritic knobs of neurons in the olfactory epithelium show that randomly selected neurons from TMEM16B KO mice respond to stimulation with increased firing activity than those from wild-type (WT) mice. Because OSNs express different odorant receptors (ORs), we restrict variability by using a mouse line that expresses a GFP-tagged I7 OR, which is known to be activated by heptanal. In response to heptanal, we measure dramatic changes in the firing pattern of I7-expressing neurons from TMEM16B KO mice compared with WT: responses are prolonged and display a higher number of APs. Moreover, lack of TMEM16B causes a markedly reduced basal spiking activity in I7-expressing neurons, together with an alteration of axonal targeting to the olfactory bulb, leading to the appearance of supernumerary I7 glomeruli. Thus, TMEM16B controls AP firing and ensures correct glomerular targeting of OSNs expressing I7. Altogether, these results show that TMEM16B does have a relevant role in normal olfaction. The Ca-activated Cl− channel TMEM16B regulates action potential firing and axonal targeting in olfactory sensory neurons