Dieter Wicher

Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels

From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. Here we show that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca2+ concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling.

A Conserved Dedicated Olfactory Circuit for Detecting Harmful Microbes in Drosophila

Flies, like all animals, need to find suitable and safe food. Because the principal food source for Drosophila melanogaster is yeast growing on fermenting fruit, flies need to distinguish fruit with safe yeast from yeast covered with toxic microbes. We identify a functionally segregated olfactory circuit in flies that is activated exclusively by geosmin. This microbial odorant constitutes an ecologically relevant stimulus that alerts flies to the presence of harmful microbes. Geosmin activates only a single class of sensory neurons expressing the olfactory receptor Or56a. These neurons target the DA2 glomerulus and connect to projection neurons that respond exclusively to geosmin. Activation of DA2 is sufficient and necessary for aversion, overrides input from other olfactory pathways, and inhibits positive chemotaxis, oviposition, and feeding. The geosmin detection system is a conserved feature in the genus Drosophila that provides flies with a sensitive, specific means of identifying unsuitable feeding and breeding sites.

Antennal transcriptome of Manduca sexta

In recent years, considerable progress has been made in understanding the molecular mechanisms underlying olfaction in insects. Because of the diverse nature of the gene families involved, this process has largely relied on genomic data. As a consequence, studies have focused on a small subset of species with extensive genomic information. For Lepidoptera, a large order historically crucial to olfactory research, this circumstance has mostly limited advances to the domesticated species Bombyx mori, with some progress in the noctuid Heliothis virescens based on a nonpublic partial genome database. Because of the limited behavioral repertoire and nonexistent ecological importance of Bombyx, molecular data on the tobacco hornworm Manduca sexta are of utmost importance, especially with regards to its position as a classical olfactory model and its complex natural behavior. Here we present the use of transcriptomic and microarray data to identify members of the main olfactory gene families of Manduca. To assess the quality of our data, we correlate information on expressed receptor genes with detailed morphological data on the antennal lobe. Finally, we compare the expression of the near-complete transcript sets in male and female antennae.

Non-synaptic ion channels in insects--basic properties of currents and their modulation in neurons and skeletal muscles.

Insects are favoured objects for studying information processing in restricted neuronal networks, e.g. motor pattern generation or sensory perception. The analysis of the underlying processes requires knowledge of the electrical properties of the cells involved. These properties are determined by the expression pattern of ionic channels and by the regulation of their function, e.g. by neuromodulators. We here review the presently available knowledge on insect non-synaptic ion channels and ionic currents in neurons and skeletal muscles. The first part of this article covers genetic and structural informations, the localization of channels, their electrophysiological and pharmacological properties, and known effects of second messengers and modulators such as neuropeptides or biogenic amines. In a second part we describe in detail modulation of ionic currents in three particularly well investigated preparations, i.e. Drosophila photoreceptor, cockroach DUM (dorsal unpaired median) neuron and locust jumping muscle. Ion channel structures are almost exclusively known for the fruitfly Drosophila, and most of the information on their function has also been obtained in this animal, mainly based on mutational analysis and investigation of heterologously expressed channels. Now the entire genome of Drosophila has been sequenced, it seems almost completely known which types of channel genes--and how many of them--exist in this animal. There is much knowledge of the various types of channels formed by 6-transmembrane--spanning segments (6TM channels) including those where four 6TM domains are joined within one large protein (e.g. classical Na+ channel). In comparison, two TM channels and 4TM (or tandem) channels so far have hardly been explored. There are, however, various well characterized ionic conductances, e.g. for Ca2+, Cl- or K+, in other insect preparations for which the channels are not yet known. In some of the larger insects, i.e. bee, cockroach, locust and moth, rather detailed information has been established on the role of ionic currents in certain physiological or behavioural contexts. On the whole, however, knowledge of non-synaptic ion channels in such insects is still fragmentary. Modulation of ion currents usually involves activation of more or less elaborate signal transduction cascades. The three detailed examples for modulation presented in the second part indicate, amongst other things, that one type of modulator usually leads to concerted changes of several ion currents and that the effects of different modulators in one type of cell may overlap. Modulators participate in the adaptive changes of the various cells responsible for different physiological or behavioural states. Further study of their effects on the single cell level should help to understand how small sets of cells cooperate in order to produce the appropriate output.

Physiological and Morphological Characterization of Local Interneurons in the Drosophila Antennal Lobe

The Drosophila antennal lobe (AL) has become an excellent model for studying early olfactory processing mechanisms. Local interneurons (LNs) connect a large number of glomeruli and are ideally positioned to increase computational capabilities of odor information processing in the AL. Although the neural circuit of the Drosophila AL has been intensively studied at both the input and the output level, the internal circuit is not yet well understood. An unambiguous characterization of LNs is essential to remedy this lack of knowledge. We used whole cell patch-clamp recordings and characterized four classes of LNs in detail using electrophysiological and morphological properties at the single neuron level. Each class of LN displayed unique characteristics in intrinsic electrophysiological properties, showing differences in firing patterns, degree of spike adaptation, and amplitude of spike afterhyperpolarization. Notably, one class of LNs had characteristic burst firing properties, whereas the others were tonically active. Morphologically, neurons from three classes innervated almost all glomeruli, while LNs from one class innervated a specific subpopulation of glomeruli. Three-dimensional reconstruction analyses revealed general characteristics of LN morphology and further differences in dendritic density and distribution within specific glomeruli between the different classes of LNs. Additionally, we found that LNs labeled by a specific enhancer trap line (GAL4-Krasavietz), which had previously been reported as cholinergic LNs, were mostly GABAergic. The current study provides a systematic characterization of olfactory LNs in Drosophila and demonstrates that a variety of inhibitory LNs, characterized by class-specific electrophysiological and morphological properties, construct the neural circuit of the AL.

Phosphorylation via PKC Regulates the Function of the Drosophila Odorant Co-Receptor

Insect odorant receptors (ORs) have a unique design of heterodimers formed by an olfactory receptor protein and the ion channel Orco. Heterologously expressed insect ORs are activated via an ionotropic and a metabotropic pathway that leads to cAMP production and activates the Orco channel. The contribution of metabotropic signaling to the insect odor response remains to be elucidated. Disruption of the Gq protein signaling cascade reduces the odor response (Kain et al., 2008). We investigated this phenomenon in HEK293 cells expressing Drosophila Orco and found that phospholipase C (PLC) inhibition reduced the sensitivity of Orco to cAMP. A similar effect was seen upon inhibition of protein kinase C (PKC), whereas PKC stimulation activated Orco even in the absence of cAMP. Mutation of the five PKC phosphorylation sites in Orco almost completely eliminated sensitivity to cAMP. To test the impact of PKC activity in vivo we combined single sensillum electrophysiological recordings with microinjection of agents affecting PLC and PKC function and observed an altered response of olfactory sensory neurons (OSNs) to odorant stimulation. Injection of the PLC inhibitor U73122 or the PKC inhibitor Gö6976 into sensilla reduced the OSN response to odor pulses. Conversely, injection of the PKC activators OAG, a diacylglycerol analog, or phorbol myristate acetate (PMA) enhanced the odor response. We conclude that metabotropic pathways affecting the phosphorylation state of Orco regulate OR function and thereby shape the OSN odor response.

Sensory receptors—design principles revisited

This research topic was aimed toward collecting the present knowledge of structure and function of sensory receptors in animal kingdom as well as the mechanisms of signal transduction and amplification. To translate external signals such as light, sound, smell, etc., into an appropriate intracellular signal, sensory receptors use either a fast, direct or a slow, indirect way. These qualitatively different signal transduction pathways are now usually called ionotopic or metabotropic. Historically, the term metabotropic receptor has been introduced to distinguish a subtype of glutamate receptors that triggers chemical reactions (cell metabolism) in the postsynaptic cell from other glutamate receptors that pass an ion current (ionotropic) (Eccles and McGeer, 1979). Metabotropic glutamate receptors were found to be linked to inositol phospholipid metabolism (Sugiyama et al., 1987), and were subsequently identified as G-protein-coupled receptors (GPCRs) (Masu et al., 1991). The terminology ionotropic/metabotropic has been extended to other neurotransmitter receptors, such as for nicotinic/muscarinergic acetyl choline or GABAA/GABAB receptors. All metabotropic neurotransmitter receptors are GPCRs. There are, however, a large number of non-GPCRs that also fulfill the original definition for a metabotropic receptor, namely “that the transmitter acts indirectly, by triggering a chemical reaction or a series of reactions” (Eccles and McGeer, 1979). Accordingly, it has been used to extent the term metabotropic receptor to receptor kinases, receptor cyclases, etc., as well. Sensory receptors are often part of complex signal transduction cascades. An ion current through an ionotropic receptor may initiate metabotropic signaling, as well as a metabotropic receptor may downstream affect the function of ion channels. An example for protein–protein interaction in chemosensation is given in the original article by Liu et al. (2012). The authors identified so far unknown binding partners of Gγ13, a G-protein subunit expressed in mammalian taste and olfactory receptor cells. These binding partners are PDZ-domain containing proteins assumed to target Gγ13 to specific subcellular locations or represent parts of the chemosensory signal transduction cascade. The evolution of chemoreceptors shows that—from bacteria to mammals—both, ionotropic as well as metabotropic mechanisms were conserved. Functional aspects of chemoreceptors, including the interaction of electrical and chemical signaling, and the amplification of sensory information are discussed in the perspective article (Wicher, 2012). Intriguingly, insect chemoreceptors operate as ionotropic receptors, namely odorant receptors (ORs), ionotropic glutamate-like receptors (IRs), and gustatory receptors (GRs). Getahun et al. (2012) investigate the temporal response dynamics of insect chemoreceptors and demonstrate that olfactory sensory neurons (OSNs) expressing ORs, GRs, or IRs differ in their response kinetics to brief stimuli. OR-expressing neurons respond faster and with higher sensitivity, while IR-expressing neurons do not adapt to long stimulations. Although ORs primarily operate as ionotropic receptors, metabotropic signaling was seen to modulate the ionotropic odor response (Olsson et al., 2011; Sargsyan et al., 2011). Stimulation of cAMP production enhanced the response to a given odor concentration, corresponding to an increased sensitivity. This type of modulation may constitute the mechanistic basis for the higher sensitivity of ORs compared with IRs. Chemical information released from different sources may interfere during processing in the nervous system and affect the response of an organism. Odor mixtures can act in synergistic or in an inhibitory way. On the level of the chemoreceptors the existence of a huge number of different chemical signal molecules leads to the intriguing question of receptor specificity and whether a given chemical signal is perceived independent of the background. The interaction of odorant and pheromone detection in moths is reported by Pregitzer et al. (2012) and commented by Anton and Renou (2012). Certain plant odors are known to inhibit the activation of pheromone receptors. The reported investigations provide evidence that the odorant-pheromone interaction already takes place at the receptor level. Since the first editorial to this topic was written in 2010 recent progress shed new light on structure and function of certain receptors. Channelrhodopsins, for example, are photoreceptors in green algae which conduct a current upon illumination. They are seven transmembrane (7-TM)-spanning proteins as typical for GPCRs but do not couple to a heterotrimeric G-protein. With the given 7-TM topology it was as yet not clear how the channelrhodopsin proteins have to arrange to form an ion channel. Recently, the non-selective cation channel, channelrhodopsin-2 from Chlamydomonas reinhardtii has been successfully crystallized (Muller et al., 2011; Kato et al., 2012). The channelrhodopsin-2 proteins were found to stably dimerize in such an arrangement that the third and the fourth TM helix of each protein align to a tetramer thereby lining the cation-permeable pore. Another example for ion channel-forming 7-TM proteins are the above mentioned insect ORs. In contrast to homodimeric channelrhodopsin channels they are heterodimers, composed of variable, odorant-binding protein OrX, and an ubiquitous co-receptor OrCo. There is growing evidence that both OR proteins contribute to channel pore formation and determine their properties such as the ion permeability and pharmacological properties (Nichols et al., 2011; Pask et al., 2011; Nakagawa et al., 2012). It remains to be established whether OrCo form homomeric channels in the receptor neurons as seen in the heterologous expression system and whether they represent the metabotropic pathway used to tune the sensitivity of the ionotropic receptor (Olsson et al., 2011; Sargsyan et al., 2011). The role of stimulatory G-proteins in olfactory signaling has been demonstrated (Deng et al., 2011), and also downstream signaling such as cAMP production were seen to affect the odor response of receptor neurons (Olsson et al., 2011). These recent findings on insect OR function modify the view to classify them. While in the first editorial they have been considered as combined metabotropic and ionotropic receptors, they might now be more appropriately characterized as metabotropically regulated ionotropic receptors. This change of view illustrates the highly dynamic development in the field.

Insect odorant response sensitivity is tuned by metabotropically autoregulated olfactory receptors

Insects possess one of the most exquisitely sensitive olfactory systems in the animal kingdom, consisting of three different types of chemosensory receptors: ionotropic glutamate-like receptors (IRs), gustatory receptors (GRs) and odorant receptors (ORs). Both insect ORs and IRs are ligand-gated ion channels, but ORs possess a unique configuration composed of an odorant-specific protein OrX and a ubiquitous coreceptor (Orco). In addition, these two ionotropic receptors confer different tuning properties for the neurons in which they are expressed. Unlike IRs, neurons expressing ORs are more sensitive and can also be sensitized by sub-threshold concentrations of stimuli. What is the mechanistic basis for these differences in tuning? We show that intrinsic regulation of Orco enhances neuronal response to odorants and sensitizes the ORs. We also demonstrate that inhibition of metabotropic regulation prevents receptor sensitization. Our results indicate that Orco-mediated regulation of OR sensitivity provides tunable ionotropic receptors capable of detecting odors over a wider range of concentrations, providing broadened sensitivity over IRs themselves.

Sex-Specific Odorant Receptors of the Tobacco Hornworm Manduca Sexta

As odor information plays a vital role in the life of moths, their olfactory sense has evolved into a highly specific and sensitive apparatus relevant to reproduction and survival. The key players in the detection of odorants are olfactory receptor (OR) proteins. Here we identify four OR-encoding genes differentially expressed in the antennae of males and females of the sphingid moth Manduca sexta. Two male-specific receptors (the previously reported MsexOR-1 and the newly identified MsexOR-4) show great resemblance to other male moth pheromone ORs. The putative pheromone receptors are co-expressed with the co-receptor involved in general odorant signal transduction, the DmelOr83b homolog MsexOR-2. One female-specific receptor (MsexOR-5) displays similarities to BmorOR-19, a receptor in Bombyx mori tuned to the detection of the plant odor linalool.

Octopamine modulates ionic currents and spiking in dorsal unpaired median (dum) neurons

MODULATORY effects of octopamine on ionic currents and spiking in isolated cockroach dorsal unpaired median neurons were investigated by means of the chopped voltage-/current-clamp and the patch-clamp technique. Octopamine increased the spiking frequency at concentrations ≤ 10 μM and reduced it at >10 μM. It enhanced a low voltage-activated Ca2+ current at 1 to 100 μM. At concentrations up to 10 μM two components of Ca2+-activated K+ current were potentiated. At >10 μM octopamine in addition reduced a high voltage-activated Ca2+ current and the Ca2+-activated K+ current. A membrane permeant cAMP-analogue imitated the effects obtained at high octopamine concentration. Octopamine and cAMP in addition reduced a depolarizing resting current at both low and high concentration. Possible mechanisms and physiological significance are discussed for the opposite effects of octopamine observed at low versus high concentrations.