Yuriy V. Bobkov

Ionotropic Crustacean Olfactory Receptors

The nature of the olfactory receptor in crustaceans, a major group of arthropods, has remained elusive. We report that spiny lobsters, Panulirus argus, express ionotropic receptors (IRs), the insect chemosensory variants of ionotropic glutamate receptors. Unlike insects IRs, which are expressed in a specific subset of olfactory cells, two lobster IR subunits are expressed in most, if not all, lobster olfactory receptor neurons (ORNs), as confirmed by antibody labeling and in situ hybridization. Ligand-specific ORN responses visualized by calcium imaging are consistent with a restricted expression pattern found for other potential subunits, suggesting that cell-specific expression of uncommon IR subunits determines the ligand sensitivity of individual cells. IRs are the only type of olfactory receptor that we have detected in spiny lobster olfactory tissue, suggesting that they likely mediate olfactory signaling. Given long-standing evidence for G protein-mediated signaling in activation of lobster ORNs, this finding raises the interesting specter that IRs act in concert with second messenger-mediated signaling.

Blockade of Insect Odorant Receptor Currents by Amiloride Derivatives

Insect odorant receptors (ORs) function as heteromeric odorant-gated ion channels consisting of a conserved coreceptor, Orco, and an odorant-sensitive tuning subunit. Although some OR modulators have been identified, an extended library of pharmacological tools is currently lacking and would aid in furthering our understanding of insect OR complexes. We now demonstrate that amiloride and several derivatives, which have been extensively used as blockers for various ion channels and transporters, also block odorant-gated currents from 2 OR complexes from the malaria vector mosquito Anopheles gambiae. In addition, both heteromeric and homomeric ORs were susceptible to amiloride blockade when activated by VUAA1, an agonist that targets the Orco channel subunit. Amiloride derivatives therefore represent a valuable class of channel blockers that can be used to investigate the pharmacological and biophysical properties of insect OR function.

Molecular and functional characterization of an Ih‐channel from lobster olfactory receptor neurons

We isolated a cDNA named PAIH encoding a member of the Ih‐channel family expressed in olfactory receptor neurons (ORNs) of the spiny lobster Panulirus argus. Functional expression of recombinant PAIH in HEK293 cells generated a slowly activating, noninactivating inward current under whole‐cell voltage‐clamp to hyperpolarizing voltage steps, the amplitude and activation rate of which increase with increasing hyperpolarization. The channel is weakly selective for K+. Intracellular cAMP or cGMP shifts activation of the current to less negative potentials in a concentration‐dependent manner. Finally, the channel is blocked by the Ih‐channel blocker ZD7288. An Ih‐channel sharing the properties of the recombinant channel occurs in cultured lobster ORNs. PAIH immunoreactivity localizes the protein to the transduction compartment of the ORNs in situ, and selectively applying the blocker to the transduction compartment reduces spontaneous activity in the ORN. Collectively, these results implicate for the first time a functional role for an Ih‐channel in olfactory signal transduction.

The pore properties of human nociceptor channel TRPA1 evaluated in single channel recordings

TRPA channels detect stimuli of different sensory modalities, including a broad spectrum of chemosensory stimuli, noxious stimuli associated with tissue damage and inflammation, mechanical stimuli, and thermal stimuli. Despite a growing understanding of potential modulators, agonists, and antagonists for these channels, the exact mechanisms of channel regulation and activation remain mostly unknown or controversial and widely debated. Relatively little is also known about the basic biophysical parameters of both native and heterologously expressed TRPA channels. Here we use conventional single channel inside-out and outside-out patch recording from the human TRPA1 channel transiently expressed in human embryonic kidney 293T cells to characterize the selectivity of the channel for inorganic mono-/divalent and organic monovalent cations in the presence of allylisothiocyanate (AITC). We show the relative permeability of the hTRPA1 channel to inorganic cations to be:and to organic cations:Na(+)(1.0)≥ dimethylamine (0.99)>trimethylamine (0.7)>tetramethylammonium (0.4)>N-methyl-d-glucamine (0.1). Activation of the hTRPA1 channels by AITC appears to recruit the channels to a conformational state with an increased permeability to large organic cations. The pore of the channels in this state can be characterized as dilated by approximately 1-2.5 Å. These findings provide important insight into the basic fundamental properties and function of TRPA1 channels in general and human TRPA1 channel in particular.

The Na+/Ca2+ Exchanger Inhibitor, KB-R7943, Blocks a Nonselective Cation Channel Implicated in Chemosensory Transduction

The mechanism(s) of olfactory transduction in invertebrates remains to be fully understood. In lobster olfactory receptor neurons (ORNs), a nonselective sodium-gated cation (SGC) channel, a presumptive transient receptor potential (TRP)C channel homolog, plays a crucial role in olfactory transduction, at least in part by amplifying the primary transduction current. To better determine the functional role of the channel, it is important to selectively block the channel independently of other elements of the transduction cascade, causing us to search for specific pharmacological blockers of the SGC channel. Given evidence that the Na(+)/Ca(2+) exchange inhibitor, KB-R7943, blocks mammalian TRPC channels, we studied this probe as a potential blocker of the lobster SGC channel. KB-R7943 reversibly blocked the SGC current in both inside- and outside-out patch recordings in a dose- and voltage-dependent manner. KB-R7943 decreased the channel open probability without changing single channel amplitude. KB-R7943 also reversibly and in a dose-dependent manner inhibited both the odorant-evoked discharge of lobster ORNs and the odorant-evoked whole cell current. Our findings strongly imply that KB-R7943 potently blocks the lobster SGC channel and likely does so directly and not through its ability to block the Na(+)/Ca(2+) exchanger.

Imaging ensemble activity in arthropod olfactory receptor neurons in situ

We show that lobster olfactory receptor neurons (ORNs), much like their vertebrate counterparts, generate a transient elevation of intracellular calcium (Ca(i)) in response to odorant activation that can be used to monitor ensemble ORN activity. This is done in antennal slice preparation in situ maintaining the polarity of the cells and the normal micro-environment of the olfactory cilia. The Ca(i) signal is ligand-specific and increases in a dose-dependent manner in response to odorant stimulation. Saturating stimulation elicits a robust increase of up to 1 μM free Ca(i) within 1-2s of stimulation. The odor-induced Ca(i) response closely follows the discharge pattern of extracellular spikes elicited by odorant application, with the maximal rise in Ca(i) matching the peak of the spike generation. The Ca(i) signal can be used to track neuronal activity in a functional subpopulation of rhythmically active ORNs and discriminate it from that of neighboring tonically active ORNs. Being able to record from many ORNs simultaneously over an extended period of time not only allows more accurate estimates of neuronal population activity but also dramatically improves the ability to identify potential new functional subpopulations of ORNs, especially those with more subtle differences in responsiveness, ligand specificity, and/or transduction mechanisms.

Intermittency Coding in the Primary Olfactory System: A Neural Substrate for Olfactory Scene Analysis

The spatial and temporal characteristics of the visual and acoustic sensory input are indispensable attributes for animals to perform scene analysis. In contrast, research in olfaction has focused almost exclusively on how the nervous system analyzes the quality and quantity of the sensory signal and largely ignored the spatiotemporal dimension especially in longer time scales. Yet, detailed analyses of the turbulent, intermittent structure of water- and air-borne odor plumes strongly suggest that spatio-temporal information in longer time scales can provide major cues for olfactory scene analysis for animals. We show that a bursting subset of primary olfactory receptor neurons (bORNs) in lobster has the unexpected capacity to encode the temporal properties of intermittent odor signals. Each bORN is tuned to a specific range of stimulus intervals, and collectively bORNs can instantaneously encode a wide spectrum of intermittencies. Our theory argues for the existence of a novel peripheral mechanism for encoding the temporal pattern of odor that potentially serves as a neural substrate for olfactory scene analysis.

Quantifying bursting neuron activity from calcium signals using blind deconvolution

Advances in calcium imaging have enabled studies of the dynamic activity of both individual neurons and neuronal assemblies. However, challenges, such as unknown nonlinearities in the spike-calcium relationship, noise, and the often relatively low temporal resolution of the calcium signal compared to the time-scale of spike generation, restrict the accurate estimation of action potentials from the calcium signal. Complex neuronal discharge, such as the activity demonstrated by bursting and rhythmically active neurons, represents an even greater challenge for reconstructing spike trains based on calcium signals. We propose a method using blind calcium signal deconvolution based on an information-theoretic approach. This model is meant to maximise the output entropy of a nonlinear filter where the nonlinearity is defined by the cumulative distribution function of the spike signal. We tested our maximum entropy (ME) algorithm using bursting olfactory receptor neurons (bORNs) of the lobster olfactory organ. The advantage of the ME algorithm is that the filter can be trained online based only on the statistics of the spike signal, without any assumptions regarding the unknown transfer function characterizing the relation between the spike and calcium signal. We show that the ME method is able to more accurately reconstruct the timing of the first and last spikes of a burst compared to other methods and that it improves the temporal precision fivefold compared to direct timing resolution of calcium signal.

Candidate pheromone receptors of codling moth Cydia pomonella respond to pheromones and kairomones

Olfaction plays a dominant role in the mate-finding and host selection behaviours of the codling moth (Cydia pomonella), an important pest of apple, pear and walnut orchards worldwide. Antennal transcriptome analysis revealed a number of abundantly expressed genes related to the moth olfactory system, including those encoding the olfactory receptors (ORs) CpomOR1, CpomOR3 and CpomOR6a, which belong to the pheromone receptor (PR) lineage, and the co-receptor (CpomOrco). Using heterologous expression, in both Drosophila olfactory sensory neurones and in human embryonic kidney cells, together with electrophysiological recordings and calcium imaging, we characterize the basic physiological and pharmacological properties of these receptors and demonstrate that they form functional ionotropic receptor channels. Both the homomeric CpomOrco and heteromeric CpomOrco + OR complexes can be activated by the common Orco agonists VUAA1 and VUAA3, as well as inhibited by the common Orco antagonists amiloride derivatives. CpomOR3 responds to the plant volatile compound pear ester ethyl-(E,Z)-2,4-decadienoate, while CpomOR6a responds to the strong pheromone antagonist codlemone acetate (E,E)-8,10-dodecadien-1-yl acetate. These findings represent important breakthroughs in the deorphanization of codling moth pheromone receptors, as well as more broadly into insect ecology and evolution and, consequently, for the development of sustainable pest control strategies based on manipulating chemosensory communication.

Cellular basis for response diversity in the olfactory periphery.

An emerging idea in olfaction is that temporal coding of odor specificity can be intrinsic to the primary olfactory receptor neurons (ORNs). As a first step towards understanding whether lobster ORNs are capable of generating odor-specific temporal activity and what mechanisms underlie any such heterogeneity in discharge pattern, we characterized different patterns of activity in lobster ORNs individually and ensemble using patch-clamp recording and calcium imaging. We demonstrate that lobster ORNs show tonic excitation, tonic inhibition, phaso-tonic excitation, and bursting, and that these patterns are faithfully reflected in the calcium signal. We then demonstrate that the various dynamic patterns of response are inherent in the cells, and that this inherent heterogeneity is largely determined by heterogeneity in the underlying intrinsic conductances.