Simone Pifferi

From Pheromones to Behavior

In recent years, considerable progress has been achieved in the comprehension of the profound effects of pheromones on reproductive physiology and behavior. Pheromones have been classified as molecules released by individuals and responsible for the elicitation of specific behavioral expressions in members of the same species. These signaling molecules, often chemically unrelated, are contained in body fluids like urine, sweat, specialized exocrine glands, and mucous secretions of genitals. The standard view of pheromone sensing was based on the assumption that most mammals have two separated olfactory systems with different functional roles: the main olfactory system for recognizing conventional odorant molecules and the vomeronasal system specifically dedicated to the detection of pheromones. However, recent studies have reexamined this traditional interpretation showing that both the main olfactory and the vomeronasal systems are actively involved in pheromonal communication. The current knowledge on the behavioral, physiological, and molecular aspects of pheromone detection in mammals is discussed in this review.

Bestrophin-2 is a candidate calcium-activated chloride channel involved in olfactory transduction

Ca-activated Cl channels are an important component of olfactory transduction. Odor binding to olfactory receptors in the cilia of olfactory sensory neurons (OSNs) leads to an increase of intraciliary Ca concentration by Ca entry through cyclic nucleotide-gated (CNG) channels. Ca activates a Cl channel that leads to an efflux of Cl from the cilia, contributing to the amplification of the OSN depolarization. The molecular identity of this Cl channel remains elusive. Recent evidence has indicated that bestrophins are able to form Ca-activated Cl channels in heterologous systems. Here we have analyzed the expression of bestrophins in the mouse olfactory epithelium and demonstrated that only mouse bestrophin-2 (mBest2) was expressed. Single-cell RT-PCR showed that mBest2 was expressed in OSNs but not in supporting cells. Immunohistochemistry revealed that mBest2 was expressed on the cilia of OSNs, the site of olfactory transduction, and colocalized with the main CNGA2 channel subunit. Electrophysiological properties of Ca-activated Cl currents from native channels in dendritic knob/cilia of mouse OSNs were compared with those induced by the expression of mBest2 in HEK-293 cells. We found the same anion permeability sequence, small estimated single-channel conductances, a Ca sensitivity difference of one order of magnitude, and the same side-specific blockage of the two Cl channel blockers commonly used to inhibit the odorant-induced Ca-activated Cl current in OSNs, niflumic acid, and 4-acetamido-4′-isothiocyanato-stilben-2,2′-disulfonate (SITS). Therefore, our data suggest that mBest2 is a good candidate for being a molecular component of the olfactory Ca-activated Cl channel.

Cyclic nucleotide-gated ion channels in sensory transduction

Cyclic nucleotide‐gated (CNG) channels, directly activated by the binding of cyclic nucleotides, were first discovered in retinal rods, cones and olfactory sensory neurons. In the visual and olfactory systems, CNG channels mediate sensory transduction by conducting cationic currents carried primarily by sodium and calcium ions. In olfactory transduction, calcium in combination with calmodulin exerts a negative feedback on CNG channels that is the main molecular mechanism responsible for fast adaptation in olfactory sensory neurons. Six mammalian CNG channel genes are known and some human visual disorders are caused by mutations in retinal rod or cone CNG genes.

Calcium‐activated chloride currents in olfactory sensory neurons from mice lacking bestrophin‐2

Olfactory sensory neurons use a chloride‐based signal amplification mechanism to detect odorants. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide‐gated channels and the entry of Ca2+ into the cilia. Ca2+ activates a Cl− current that produces an efflux of Cl− ions and amplifies the depolarization. The molecular identity of Ca2+‐activated Cl− channels is still elusive, although some bestrophins have been shown to function as Ca2+‐activated Cl− channels when expressed in heterologous systems. In the olfactory epithelium, bestrophin‐2 (Best2) has been indicated as a candidate for being a molecular component of the olfactory Ca2+‐activated Cl− channel. In this study, we have analysed mice lacking Best2. We compared the electrophysiological responses of the olfactory epithelium to odorant stimulation, as well as the properties of Ca2+‐activated Cl− currents in wild‐type (WT) and knockout (KO) mice for Best2. Our results confirm that Best2 is expressed in the cilia of olfactory sensory neurons, while odorant responses and Ca2+‐activated Cl− currents were not significantly different between WT and KO mice. Thus, Best2 does not appear to be the main molecular component of the olfactory channel. Further studies are required to determine the function of Best2 in the cilia of olfactory sensory neurons.

Anoctamin 2/TMEM16B: a calcium‐activated chloride channel in olfactory transduction

In vertebrate olfactory transduction, a Ca2+‐dependent Cl− efflux greatly amplifies the odorant response. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide‐gated channels and the entry of Ca2+ into the cilia. The Ca2+ activates a Cl− current that, in the presence of a maintained elevated intracellular Cl− concentration, produces an efflux of Cl− ions and amplifies the depolarization. In this review, we summarize evidence supporting the hypothesis that anoctamin 2/TMEM16B is the main, or perhaps the only, constituent of the Ca2+‐activated Cl− channels involved in olfactory transduction. Indeed, studies from several laboratories have shown that anoctamin 2/TMEM16B is expressed in the ciliary layer of the olfactory epithelium, that there are remarkable functional similarities between currents in olfactory sensory neurons and in HEK 293 cells transfected with anoctamin 2/TMEM16B, and that knockout mice for anoctamin 2/TMEM16B did not show any detectable Ca2+‐activated Cl− current. Finally, we discuss the involvement of Ca2+‐activated Cl− channels in the transduction process of vomeronasal sensory neurons and the physiological role of these channels in olfaction.

Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel

Extracellular anions more permeant than Cl− modulate TMEM16B gating to promote channel opening, whereas less permeant anions favor channel closure.

The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop

Ca2+-activated Cl− channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure–function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca2+ dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild-type and mutant TMEM16B channels expressed in HEK 293T cells using the whole cell voltage-clamp technique in the presence of various intracellular Ca2+ concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates 386EEEEE390 and 399EYE401. The EYE deletion did not significantly modify the apparent Ca2+ dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca2+ affinity but modified the voltage dependence, shifting the conductance–voltage relations toward more positive voltages. These findings indicate that glutamates E367 and 386EEEEE390 in the first intracellular putative loop play an important role in the voltage dependence of TMEM16B, thus providing an initial structure–function study for this channel.

Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons.

TMEM16A is an essential component of Ca2+-activated Cl− currents in mouse vomeronasal sensory neurons.

TrkB signaling directs the incorporation of newly generated periglomerular cells in the adult olfactory bulb.

In the adult rodent brain, the olfactory bulb (OB) is continuously supplied with new neurons which survival critically depends on their successful integration into pre-existing networks. Yet, the extracellular signals that determine the selection which neurons will be ultimately incorporated into these circuits are largely unknown. Here, we show that immature neurons express the catalytic form of the brain-derived neurotrophic factor receptor TrkB [full-length TrkB (TrkB-FL)] only after their arrival in the OB, at the time when integration commences. To unravel the role of TrkB signaling in newborn neurons, we conditionally ablated TrkB-FL in mice via Cre expression in adult neural stem and progenitor cells. TrkB-deficient neurons displayed a marked impairment in dendritic arborization and spine growth. By selectively manipulating the signaling pathways initiated by TrkB in vivo, we identified the transducers Shc/PI3K to be required for dendritic growth, whereas the activation of phospholipase C-γ was found to be responsible for spine formation. Furthermore, long-term genetic fate mapping revealed that TrkB deletion severely compromised the survival of new dopaminergic neurons, leading to a substantial reduction in the overall number of adult-generated periglomerular cells (PGCs), but not of granule cells (GCs). Surprisingly, this loss of dopaminergic PGCs was mirrored by a corresponding increase in the number of calretinin+ PGCs, suggesting that distinct subsets of adult-born PGCs may respond differentially to common extracellular signals. Thus, our results identify TrkB signaling to be essential for balancing the incorporation of defined classes of adult-born PGCs and not GCs, reflecting their different mode of integration in the OB.

Human Cord Blood CD133+ Stem Cells Transplanted to Nod-Scid Mice Provide Conditions for Regeneration of Olfactory Neuroepithelium After Permanent Damage Induced by Dichlobenil†‡

The herbicide dichlobenil selectively causes necrosis of the dorsomedial part of olfactory neuroepithelium (NE) with permanent damage to the underlying mucosa, whereas the lateral part of the olfactory region and the nasal respiratory mucosa remain undamaged. We investigated here whether human umbilical cord blood CD133+ stem cells (HSC) injected intravenously to nod‐scid mice pretreated with dichlobenil may engraft the olfactory mucosa and contribute to the regeneration of the damaged NE. We tested HLA‐DQα1 DNA and three human microsatellites (Combined DNA Index System) as indicators of engrafted cells, finding polymerase chain reaction evidence of chimaerism in various tissues of the host, including the olfactory mucosa and bulb, at 7 and 31 days following HSC transplantation. Histology, immunohistochemistry, and lectin staining revealed the morphological recovery of the dorsomedial region of the NE in dichlobenil‐treated mice that received HSC, contrasting with the lack of regeneration in similarly injured areas as these remained damaged in control nontransplanted mice. FISH analysis, to detect human genomic sequences from different chromosomes, confirmed persistent engraftment of the regenerating olfactory area with chimeric cells. Electro‐olfactograms in response to odorants, to test the functionality of the olfactory NE, confirmed the functional damage of the dorsomedial area in dichlobenil‐treated mice and the functional recovery of the same area in transplanted mice. These findings support the concept that transplanted HSC migrating to the damaged olfactory area provide conditions facilitating the recovery from olfactory receptor cell loss. STEM CELLS 2009;27:825–835