Lobster Olfactory Receptor Neurons Research Articles

An inhibitor of Na+/Ca2+ exchange blocks activation of insect olfactory receptors

Earlier we showed that the Na+/Ca2+ exchanger inhibitor, KB-R7943, potently blocks the odor-evoked activity of lobster olfactory receptor neurons. Here we extend that finding to recombinant mosquito olfactory receptors stably expressed in HEK cells. Using whole-cell and outside-out patch clamping and calcium imaging, we demonstrate that KB-R7943 blocks both the odorant-gated current and the odorant-evoked calcium signal from two different OR complexes from the malaria vector mosquito, Anopheles gambiae, AgOr48+AgOrco and AgOr65+AgOrco. Both heteromeric and homomeric (Orco alone) OR complexes were susceptible to KB-R7943 blockade when activated by VUAA1, an agonist that targets the Orco channel subunit, suggesting the Orco subunit may be the target of the drug’s action. KB-R7943 represents a valuable tool to further investigate the functional properties of arthropod olfactory receptors and raises the interesting specter that activation of these ionotropic receptors is directly or indirectly linked to a Na+/Ca2+ exchanger, thereby providing a template for drug design potentially allowing improved control of insect pests and disease vectors.

Mechanisms underlying odorant-induced and spontaneous calcium signals in olfactory receptor neurons of spiny lobsters, Panulirus argus

We determined if a newly developed antennule slice preparation allows studying chemosensory properties of spiny lobster olfactory receptor neurons under in situ conditions with Ca(2+) imaging. We show that chemical stimuli reach the dendrites of olfactory receptor neurons but not their somata, and that odorant-induced Ca(2+) signals in the somata are sufficiently stable over time to allow stimulation with a substantial number of odorants. Pharmacological manipulations served to elucidate the source of odorant-induced Ca(2+) transients and spontaneous Ca(2+) oscillations in the somata of olfactory receptor neurons. Both Ca(2+) signals are primarily mediated by an influx of extracellular Ca(2+) through voltage-activated Ca(2+) channels that can be blocked by CoCl2 and the L-type Ca(2+) channel blocker verapamil. Intracellular Ca(2+) stores contribute little to odorant-induced Ca(2+) transients and spontaneous Ca(2+) oscillations. The odorant-induced Ca(2+) transients as well as the spontaneous Ca(2+) oscillations depend on action potentials mediated by Na(+) channels that are largely TTX-insensitive but blocked by the local anesthetics tetracaine and lidocaine. Collectively, these results corroborate the conclusion that odorant-induced Ca(2+) transients and spontaneous Ca(2+) oscillations in the somata of olfactory receptor neurons closely reflect action potential activity associated with odorant-induced phasic-tonic responses and spontaneous bursting, respectively. Therefore, both types of Ca(2+) signals represent experimentally accessible proxies of spiking.

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.

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 1–2 s 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.

The small molecule phenamil is a modulator of adipocyte differentiation and PPARγ expression

We previously described the use of a cell-based screening approach to identify small molecules that regulate adipocyte differentiation. Here we identify the amiloride derivative phenamil as an adipogenic compound. Phenamil acutely induces expression of the key transcription factor of adipogenesis, peroxisome proliferator-activated receptor γ (PPARγ) and, consequently, promotes the differentiation of multiple preadipocyte cell lines, including 3T3-L1 and F442A. Interestingly, the adipogenic action of phenamil is distinct from and additive with both PPARγ ligands and the previously identified adipogenic small molecule harmine. To identify signaling pathways mediating phenamil's effects, we performed transcriptional profiling of 3T3-F442A preadipocytes. ETS variant 4 (ETV4) was identified as a gene rapidly induced by phenamil but not by other adipogenic small molecules or PPARγ agonists. Transient expression of ETV4 in preadipocytes enhances the expression of PPARγ. Stable overexpression of ETV4 promotes expression of PPARγ and its downstream target genes and enhances morphological differentiation. Finally, knockdown of PPARγ expression by shRNA blocks the effects of phenamil on adipocyte differentiation and gene expression, but it does not block phenamil induction of ETV4, which suggests that ETV4 acts upstream of PPARγ in differentiation processes. These results identify a phenamil as new small molecule tool for the probing of adipocyte differentiation that acts, at least in part, through induction of ETV4 expression.

Phosphatidylinositol 4,5-bisphosphate-dependent regulation of the output in lobster olfactory receptor neurons

Transient receptor potential (TRP) channels often play a role in sensory transduction, including chemosensory transduction. TRP channels, a common downstream target of phosphoinositide (PI) signaling, can be modulated by exogenous phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and/or diacylglycerol (DAG). Lobster olfactory receptor neurons (ORNs) express a TRP-related, non-selective, calcium/magnesium-permeable, sodium/calcium-gated cation (SGC) channel. Here we report that PIs regulate the function of the calcium-activated form of the lobster channel. Sequestering of endogenous PI(4,5)P2, either with an anti-PI(4,5)P2 antibody or by electrostatic screening with polyvalent cations, blocks the channel. Exogenous PI(3,4,5)P3 activates the channel independently of intracellular sodium and/or calcium. Exogenous non-hydrolysable DAG analogs fail to change the gating parameters of the channel, suggesting the channel is insensitive to DAG. Electrophysiological recording from lobster ORNs in situ using a panel of pharmacological tools targeting the key components of both PI and DAG metabolism (phospholipase C, phosphoinositide 4-kinase and DAG kinase) extend these findings to the intact ORN. PI(4,5)P2 depletion suppresses both the odorant-evoked discharge and whole-cell current of the cells, and does so possibly independently of DAG production. Collectively, our results argue that PIs can regulate output in lobster ORNs, at least in part through their action on the lobster SGC channel.

Phosphoinositide 3‐kinase mediated signaling in lobster olfactory receptor neurons

In vertebrates and some invertebrates, odorant molecules bind to G protein-coupled receptors on olfactory receptor neurons (ORNs) to initiate signal transduction. Phosphoinositide 3-kinase (PI3K) activity has been implicated physiologically in olfactory signal transduction, suggesting a potential role for a G protein-coupled receptor-activated class I PI3K. Using isoform-specific antibodies, we identified a protein in the olfactory signal transduction compartment of lobster ORNs that is antigenically similar to mammalian PI3Kgamma and cloned a gene for a PI3K with amino acid homology with PI3Kbeta. The lobster olfactory PI3K co-immunoprecipitates with the G protein alpha and beta subunits, and an odorant-evoked increase in phosphatidylinositol (3,4,5)-trisphosphate can be detected in the signal transduction compartment of the ORNs. PI3Kgamma and beta isoform-specific inhibitors reduce the odorant-evoked output of lobster ORNs in vivo. Collectively, these findings provide evidence that PI3K is indeed activated by odorant receptors in lobster ORNs and further support the potential involvement of G protein activated PI3K signaling in olfactory transduction.

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.

Block by Amiloride Derivatives of Odor-Evoked Discharge in Lobster Olfactory Receptor Neurons through Action on a Presumptive TRP Channel

Amiloride and its derivatives inhibit a number of sensory transduction processes, including some types of chemosensory transduction. Here, we report that pyrazine derivatives of amiloride reversibly inhibit odorant-evoked activity in lobster olfactory receptor neurons. The potency sequence is as follows-(IC50, mM): 5-(N,N-hexamethylene)amiloride (0.015) approximately 5-(N-methyl-N-isobutyl)amiloride (0.02) approximately 5-(N-ethyl-N-isopropyl)amiloride (0.03) > 5-(N,N-dimethyl)amiloride (0.48); 3',4'-dichlorobenzamil (0.4), phenamil (0.5), and amiloride itself (2) are ineffective. The same derivatives with the similar potency sequence also block a presumptive transient receptor potential (TRP) channel that is the likely downstream target of phosphoinositide signaling in these cells. Our results suggest that pyrazine derivatives of amiloride are useful probes to study more detailed mechanisms of chemosensory transduction in this system and possibly in other chemosensory systems in which TRP channels are the known or suspected downstream effector.

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

We isolated a cDNA named PAIH encoding a member of the I(h)-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 I(h)-channel blocker ZD7288. An I(h)-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 I(h)-channel in olfactory signal transduction.

Cyclic Nucleotide Signaling Mediates an Odorant-suppressible Chloride Conductance in Lobster Olfactory Receptor Neurons

We have previously shown that lobster olfactory receptor neurons (ORNs) express an odorant-suppressible Cl(-) conductance that modulates the output of the cells. Here, we develop a more complete pharmacological profile of this conductance, showing it is blockable by the Cl(-) channel blockers DIDS, 9-AC and flufenamic acid, but not by niflumic acid. We then show that a conductance with this pharmacological profile is mediated by cyclic nucleotide signaling. These findings further our understanding of the cellular mechanisms through which odorants can modulate the output of lobster ORNs.

Specificity of Odorant-evoked Inhibition in Lobster Olfactory Receptor Neurons

Lobster olfactory receptor neurons, like those of many animals, use two modes of olfactory signaling, excitation and inhibition to code olfactory information. Inhibition appears to act through two distinct ionic mechanisms. Here we show that neither ionic mechanism is odor-specific, providing further support for the emerging understanding that there are no inhibitory odorants per se, but rather that the action of a particular odorant is inherent in the olfactory receptor cell on which an odorant acts.

Pharmacological Properties and Functional Role of a TRP-Related Ion Channel in Lobster Olfactory Receptor Neurons

Odors activate lobster olfactory receptor neurons (ORNs) through phosphoinositide signaling that appears to target a Na(+)-gated nonselective cation channel. The Na(+)-gated channel is a potential member of the growing family of transient receptor potential (TRP) channels. Here, we test the effect of potential antagonists on the channel in cell-free patches from cultured lobster ORNs. We show that the channel is antagonized by H+ and the TRP channel blockers 2-aminoethoxydiphenyl borate, SKF96365, ruthenium red, Al3+, Gd3+, and La3+. We then use this enhanced antagonist profile together with the agonists Na+ and Ca2+ to implicate the channel in signal amplification in the cells.

Calcium sensitivity of a sodium-activated nonselective cation channel in lobster olfactory receptor neurons.

We report that a Na+-activated nonselective cation channel described previously in lobster olfactory neurons, in which phosphoinositide signaling mediates olfactory transduction, can also be activated by Ca2+. Ca2+ activates the channel in the presence of Na+, increasing the open probability of the channel with a K1/2 of 490 nM and a Hill coefficient of 1.3. Ca2+ also increases the sensitivity of the channel to Na+. In some cells, the same channel is Ca2+ insensitive in a cell-specific manner. The nonspecific activator of protein phosphatases, protamine, applied to the intracellular face of patches containing the channel irreversibly eliminates the sensitivity to Ca2+. This effect can be blocked by okadaic acid, a nonspecific blocker of protein phosphatases, and restored by the catalytic subunit of protein kinase A in the presence of MgATP. The Ca2+-sensitive form of the channel is predominantly expressed in the transduction zone of the cells in situ. These findings imply that the Ca2+ sensitivity of the channel, and possibly its regulation by phosphorylation, play a role in olfactory transduction and help tie activation of the channel to the canonical phosphoinositide turnover pathway.

Calcium regulation of cyclic nucleotide signaling in lobster olfactory receptor neurons.

An elevated free Ca2+ concentration reduces odor-stimulated production of cyclic AMP (cAMP) in the outer dendritic membranes of lobster olfactory receptor neurons in vitro. This effect can occur within 50 ms of odor stimulation. The effect is concentration-dependent at submicromolar concentrations of free Ca2+. An elevated free Ca2+ concentration also reduces basal and forskolin-stimulated cAMP levels in a concentration-dependent manner, suggesting that Ca2+ is not targeting the activation of the odor receptor/G protein complex. The degradation of synthetic cAMP by phosphodiesterases is not enhanced by an increased free Ca2+ concentration, suggesting that Ca2+ acts by down-regulating the olfactory adenylyl cyclase. Western blot analysis of the lobster olfactory sensilla that contain the outer dendrites reveals a protein in the transduction zone with a molecular mass of approximately 138 kDa that is immunoreactive to an antiserum against adenylyl cyclase type III. Given earlier evidence that Ca2+ potentially enters the receptor cell through odor-activated inositol 1,4,5-trisphosphate-gated channels, our results suggest a possible route for cross talk between the cyclic nucleotide and the inositol phospholipid signaling pathways in lobster olfactory receptor neurons.

An odorant-suppressed Cl- conductance in lobster olfactory receptor cells.

Odorants evoke an outward current in cultured lobster olfactory receptor neurons voltage clamped at -60 mV. The reversal potential of the outward current is independent of the reversal potential of potassium, but shifts with imposed changes in the reversal potential of chloride. The slope of the current-voltage relationship is negative, suggesting that the current is mediated by the odorant suppressing a steady-state conductance. Anthracene-9-carboxylic acid, a specific chloride channel blocker, reversibly inhibits the steady-state conductance. Local application of odorants to the outer dendrites evokes a hyperpolarizing receptor potential in lobster olfactory receptor neurons current-clamped at -70 mV in situ. Consistent with the current characterized in the cultured cells, hyperpolarizing receptor potentials in some cells are voltage sensitive, blocked by anthracene-9-carboxylic acid and associated with a decrease in membrane conductance. These results support the hypothesis that odorants suppress a steady-state chloride conductance in lobster olfactory receptor neurons. Evidence that the chloride conductance can coexist with a 4-aminopyridine-blockable potassium conductance reported earlier in these cells suggests that two distinct mechanisms can mediate odorant-evoked inhibition in lobster olfactory receptor neurons.

Odor-stimulated phosphatidylinositol 3-kinase in lobster olfactory receptor cells.

Two antagonists of phosphoinositide 3-OH kinases (PI3Ks), LY294002 and Wortmannin, reduced the magnitude of the receptor potential in lobster olfactory receptor neurons (ORNs) recorded by patch clamping the cells in vivo. An antibody directed against the c-terminus of human PI3K-P110 beta detected a molecule of predicted size in the outer dendrites of the ORNs. Two 3-phosphoinositides, PI(3,4)P(2) (1--4 microM) and PI(3,4,5)P(3) (1--4 microM) applied to the cytoplasmic side of inside-out patches taken from cultured lobster ORNs, reversibly activated a Na(+)-gated channel previously implicated in the transduction cascade in these cells. 3-Phosphoinositides were the most effective phosphoinositide (1 microM) in enhancing the open probability of the channel. Collectively, these results implicate 3-phosphoinositides in lobster olfactory transduction and raise the need to consider the 3-phosphoinositide pathway in olfactory transduction.

Characterization of a Phosphoinositide-mediated Odor Transduction Pathway Reveals Plasma Membrane Localization of an Inositol 1,4,5-Trisphosphate Receptor in Lobster Olfactory Receptor Neurons

The role of phosphoinositide signaling in olfactory transduction is still being resolved. Compelling functional evidence for the transduction of odor signals via phosphoinositide pathways in olfactory transduction comes from invertebrate olfactory systems, in particular lobster olfactory receptor neurons. We now provide molecular evidence for two components of the phosphoinositide signaling pathway in lobster olfactory receptor neurons, a G protein alpha subunit of the G(q) family and an inositol 1,4, 5-trisphosphate-gated channel or an inositol 1,4,5-trisphosphate (IP(3)) receptor. Both proteins localize to the site of olfactory transduction, the outer dendrite of the olfactory receptor neurons. Furthermore, the IP(3) receptor localizes to membranes in the ciliary transduction compartment of these cells at both the light microscopic and electron microscopic levels. Given the absence of intracellular organelles in the sub-micron diameter olfactory cilia, this finding indicates that the IP(3) receptor is associated with the plasma membrane and provides the first definitive evidence for plasma membrane localization of an IP(3)R in neurons. The association of the IP(3) receptor with the plasma membrane may be a novel mechanism for regulating intracellular cations in restricted cellular compartments of neurons.

Inhibitory receptor binding events among the components of complex mixtures contribute to mixture suppression in responses of olfactory receptor neurons of spiny lobsters.

Responses of olfactory receptor neurons of spiny lobsters Panulirus argus to two-component mixtures can be shaped by inhibitory events such as odor-activated hyperpolarizations and inhibition of odor-receptor binding (Daniel et al. 1996). In the current study, we extend this analysis to complex mixtures by examining responses of spiny lobster olfactory receptor neurons to mixtures containing up to seven odorants, consisting of adenosine-5'-monophosphate, ammonium, betaine, L-cysteine, L-glutamate, DL-succinate, and taurine. The response to a mixture was often less than the response to its most excitatory component. The effect of adding an excitatory odorant to a mixture depended on olfactory receptor neuron type, composition of the mixture, and which compound was added. In some cases the added excitatory compound had no effect or even decreased the mixture's response intensity, thus demonstrating nonlinear contributions of the components. Response intensities predicted by a noncompetitive model, which is most representative of these olfactory receptor neurons, were improved when the model included a term for empirical measurements of inhibitory binding interactions, suggesting that inhibitory binding interactions are one mechanism contributing to mixture suppression. This model's predictions were accurate for binary mixtures but not for larger mixtures, suggesting that additional inhibitory mechanisms are needed to account for mixture interactions in complex mixtures.

Mechanisms of afterhyperpolarization in lobster olfactory receptor neurons.

In lobster olfactory receptor neurons (ORNs), depolarizing responses to odorants and current injection are accompanied by the development of an afterhyperpolarization (AHP) that likely contributes to spike-frequency adaptation and that persists for several seconds after termination of the response. A portion of the AHP can be blocked by extracellular application of 5 mM CsCl. At this concentration, CsCl specifically blocks the hyperpolarization-activated cation current (Ih) in lobster ORNs. This current is likely to be active at rest, where it provides a constant, depolarizing influence. Further depolarization deactivates Ih, thus allowing the cell to be briefly hyperpolarized when that depolarizing influence is removed, thus generating an AHP. Reactivation of Ih would terminate the AHP. The component of the AHP that could not be blocked by Cs+ (the Cs(+)-insensitive AHP) was accompanied by decreased input resistance, suggesting that this component is generated by increased conductance to an ion with an equilibrium potential more negative than the resting potential. The Cs(+)-insensitive AHP in current clamp and the underlying current in voltage clamp displayed a reversal potential of approximately -75 mV. Both EK and ECl are predicted to be in this range. Similar results were obtained with the use of a high Cl- pipette solution, although that shifted ECl from -72 mV to -13 mV. However, when EK was shifted to more positive or negative values, the reversal potential also shifted accordingly. A role for the Ca(2+)-mediated K+ current in generating the Cs(+)-independent AHP was explored by testing cells in current and voltage clamp while blocking IK(Ca) with Cs+/Co(2+)-saline. In some cells, the Cs(+)-independent AHP and its underlying current could be completely and reversibly blocked by Cs+/Co2+ saline, whereas in other cells some fraction of it remained. This indicates that the Cs(+)-independent AHP results from two K+ currents, one that requires an influx of extracellular Ca2+ and one that does not. Collectively, these findings indicate that AHPs result from three phenomena that occur when lobster ORNs are depolarized: 1) inactivation of the hyperpolarization-activated cation current, 2) activation of a Ca(2+)-mediated K+ current, and 3) activation of a K+ current that does not require influx of extracellular Ca2+. Roles of these processes in modulating the output of lobster ORNs are discussed.