Anna Menini

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.

THE RELATION BETWEEN STIMULUS AND RESPONSE IN OLFACTORY RECEPTOR CELLS OF THE TIGER SALAMANDER

1. Olfactory receptor cells were isolated from the adult tiger salamander Ambystoma tigrinum and the current in response to odorant stimuli was measured with the whole‐cell voltage‐clamp technique while odorants at known concentrations were rapidly applied for controlled exposure times. 2. Three odorants, cineole, isoamyl acetate and acetophenone, were first applied at 5 x 10(‐4) M. Out of forty‐nine cells tested, 53% responded to one odorant only, 22% to two odorants and 25% to all three odorants. 3. The amplitude of the current in response to a given odorant concentration was found to be dependent on the duration of the odorant stimulus and reached a saturating peak value at 1.2 s of stimulus duration. 4. The current measured at the peak of the response for odorant steps of 1.2 s as a function of odorant concentration was well described by the Hill equation for the three odorants with Hill coefficients higher than 1 and K1/2 (odorant concentration needed to activate half the maximal current) ranging from 3 x 10(‐6) to 9 x 10(‐5) M. 5. It is concluded that olfactory receptor cells are broadly tuned and have a low apparent affinity for odorants, integrate stimulus information over time, and have a narrow dynamic range.

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.

The neurobiology of olfaction

From Odors to Behaviors in Caenorhabditis elegans Anne C. Hart and Michael Y. Chao Odor Coding in Insects C. Giovanni Galizia and Silke Sachse Olfactory Information Processing in Moths S. Shuichi Haupt, Takeshi Sakurai, Shigehiro Namiki, Tomoki Kazawa, and Ryohei Kanzaki Olfactory Coding in Larvae of the African Clawed Frog Xenopus laevis Ivan Manzini and Detlev Schild Development of the Olfactory System Helen B. Treloar, Alexandra M. Miller, Arundhati Ray, and Charles A. Greer Pheromones and Mammalian Behavior Peter A. Brennan Odorant Receptors Bettina Malnic, Daniela C. Gonzalez-Kristeller, and Luciana M. Gutiyama Signal Transduction in Vertebrate Olfactory Cilia Simone Pifferi, Anna Menini, and Takashi Kurahashi Multiple Olfactory Subsystems Convey Various Sensory Signals Minghong Ma Feedback Regulation of Neurogenesis in the Mammalian Olfactory Epithelium: New Insights from Genetics and Systems Biology Kimberly K. Gokoffski, Shimako Kawauchi, Hsiao-Huei Wu, Rosaysela Santos, Piper L.W. Hollenbeck, Arthur D. Lander, and Anne L. Calof Neurogenesis in the Adult Olfactory Bulb Angela Pignatelli and Ottorino Belluzzi Active Sensing in Olfaction Matt Wachowiak Temporal Coding in Olfaction Brice Bathellier, Olivier Gschwend, and Alan Carleton Cortical Activity Evoked by Odors Donald A. Wilson and Robert L. Rennaker Memory and Plasticity in the Olfactory System: From Infancy to Adulthood Anne-Marie Mouly and Regina Sullivan New Perspectives on Olfactory Processing and Human Smell Gordon M. Shepherd

Calcium concentration jumps reveal dynamic ion selectivity of calcium-activated chloride currents in mouse olfactory sensory neurons and TMEM16b-transfected HEK 293T cells.

Ca2+‐activated Cl− channels play relevant roles in several physiological processes, including olfactory transduction, but their molecular identity is still unclear. Recent evidence suggests that members of the transmembrane 16 (TMEM16, also named anoctamin) family form Ca2+‐activated Cl− channels in several cell types. In vertebrate olfactory transduction, TMEM16b/anoctamin2 has been proposed as the major molecular component of Ca2+‐activated Cl− channels. However, a comparison of the functional properties in the whole‐cell configuration between the native and the candidate channel has not yet been performed. In this study, we have used the whole‐cell voltage‐clamp technique to measure functional properties of the native channel in mouse isolated olfactory sensory neurons and compare them with those of mouse TMEM16b/anoctamin2 expressed in HEK 293T cells. We directly activated channels by rapid and reproducible intracellular Ca2+ concentration jumps obtained from photorelease of caged Ca2+ and determined extracellular blocking properties and anion selectivity of the channels. We found that the Cl− channel blockers niflumic acid, 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB) and DIDS applied at the extracellular side of the membrane caused a similar inhibition of the two currents. Anion selectivity measured exchanging external ions and revealed that, in both types of currents, the reversal potential for some anions was time dependent. Furthermore, we confirmed by immunohistochemistry that TMEM16b/anoctamin2 largely co‐localized with adenylyl cyclase III at the surface of the olfactory epithelium. Therefore, we conclude that the measured electrophysiological properties in the whole‐cell configuration are largely similar, and further indicate that TMEM16b/anoctamin2 is likely to be a major subunit of the native olfactory Ca2+‐activated Cl− current.

Fast Adaptation in Mouse Olfactory Sensory Neurons Does Not Require the Activity of Phosphodiesterase

Vertebrate olfactory sensory neurons rapidly adapt to repetitive odorant stimuli. Previous studies have shown that the principal molecular mechanisms for odorant adaptation take place after the odorant-induced production of cAMP, and that one important mechanism is the negative feedback modulation by Ca2+-calmodulin (Ca2+-CaM) of the cyclic nucleotide-gated (CNG) channel. However, the physiological role of the Ca2+-dependent activity of phosphodiesterase (PDE) in adaptation has not been investigated yet. We used the whole-cell voltage-clamp technique to record currents in mouse olfactory sensory neurons elicited by photorelease of 8-Br-cAMP, an analogue of cAMP commonly used as a hydrolysis-resistant compound and known to be a potent agonist of the olfactory CNG channel. We measured currents in response to repetitive photoreleases of cAMP or of 8-Br-cAMP and we observed similar adaptation in response to the second stimulus. Control experiments were conducted in the presence of the PDE inhibitor IBMX, confirming that an increase in PDE activity was not involved in the response decrease. Since the total current activated by 8-Br-cAMP, as well as that physiologically induced by odorants, is composed not only of current carried by Na+ and Ca2+ through CNG channels, but also by a Ca2+-activated Cl− current, we performed control experiments in which the reversal potential of Cl− was set, by ion substitution, at the same value of the holding potential, −50 mV. Adaptation was measured also in these conditions of diminished Ca2+-activated Cl− current. Furthermore, by producing repetitive increases of ciliarys Ca2+ with flash photolysis of caged Ca2+, we showed that Ca2+-activated Cl− channels do not adapt and that there is no Cl− depletion in the cilia. All together, these results indicate that the activity of ciliary PDE is not required for fast adaptation to repetitive stimuli in mouse olfactory sensory neurons.

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.

Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons

The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of −261 pA was measured at −50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction.

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.