Ionotropic crustacean olfactory receptors

Abstract

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.

Figure 1. Alignment of spiny lobster IR25a and IR8a S1 and S2 ligand binding domains.

Spiny lobster and Drosophila S1 and S2 ligand binding domains were manually aligned. Putative ligand binding residues are highlighted in red and bolded.

Figure 2. Spiny lobster IR25a and IR93a transcripts can be localized to the ORNs.

In situ hybridization of vibratome sections of gelatin embedded lobster olfactory tissue. Antisense probes for PargIR25a and PargIR93a label most, if not all, mature ORNs. In contrast, no labeling was detectable with probes for other IRs. Specifically labeled cell bodies are indicated with an arrow in the first panel. Representative sections labeled with antisense probes for PargIR8a and PargIR9 are shown. No specific labeling was detected with the sense probes for the same gene regions. The cuticle in each section is non-specifically labeled.

Figure 3. Spiny lobster IR transcripts can be detected in single ORN clusters.

RT-PCR detection of IRs in RNA prepared from (top panel) total olfactory tissue and (bottom panel) a single ORN cluster. While all of the IRs tested could be detected in the total RNA sample, only a limited number could be detected in the single cluster. No amplification was detected in RNA samples in the absence of reverse transcription or template (not shown). Numbers above panels indicate the IR amplified in the wells below. X indicates empty well.

Figure 4. Spiny lobster IR25a can be immunolocalized by the transduction compartment (outer dendrites) of ORNs.

(A) Western blot detection of IR25a in the proteins from the outer dendrites. Detergent lysates were prepared from the outer 50% of aesthetasc hairs after manual removal of the guard hairs from the lobster olfactory organ and ORN cell bodies collected from the same region. PargIR25a was detected with an anti-iGluR1 (anti-IR25a) antibody (generously provided by Dr. Timothy McClintock) after SDS-PAGE and transfer of proteins to a nitrocellulose membrane. The IR25a protein band is indicated with an arrow. (B) Immunolocalization of IR25a to the outer dendrite tissue within sections of the aesthetasc hairs of the lobster olfactory organ. Autofluorescent cuticle surrounds the outer dendrite tissue (white arrow). Controls included labeling with an anti-I(h) channel antibody and no primary antibody. Cryosections were prepared from the outer 50% of the aethetasc hairs. (C) Immunolocalization to the cell bodies and inner dendrites. In the first panel, cell bodies and inner dendrites of ORNs are indicated with arrows. Axons are indicated with an asterisk. The cuticle is indicated with a white C. The bottom two panels are diagrams showing the orientation of the sections directly above them. Cryosections were prepared from 8 annuli segments of the olfactory organ. Immunolocalization in both the outer dendrites and tissue cross sections was performed with the same antibody used in (A).

Figure 5. Lobster IRs can be detected in non-olfactory chemosensory tissues.

(A) Western blot detection of PargIR25a in detergent lysates of Panulirus argus olfactory, foot and mouth tissues. No expression could be detected in lysates from the eye or non-olfactory second antenna. (B) RT-PCR detection of PargIR25a, PargIR93a, PargIR8a, and beta-tubulin (btub) gene expression in Panulirus argus eye, foot and mouth tissues. No amplification was detected in RNA samples in the absence of reverse transcription (data not shown) or template (X).

Figure 6. Single odorants specifically activate a restricted number of lobster ORNs.

ORN ensemble activity from single neuronal cluster (A) evoked by both complex odor mixture and single odorants (B). A – Map of individual cell regions analyzed. Position of every ORN region was carefully selected and corrected, if necessary, during the recording course to maximally avoid overlapping of optical signals. B – Fluorescence intensity traces from indexed ORN regions (A, bottom) were color coded using the intensity range characterizing individual ORN. Color gradient code applied to all data points changes from blue (minimum value) to yellow (maximum fluorescent intensity value). Each prospective odor, except TET, was applied 3 times. Stimulus pulse duration was 1s in all cases. Time between successive sweeps, 60 sec. Delay between successive trials, ∼120 s (not shown). Note, while complex odor mixture would activate majority of ORNs (first sweep), single odorants evoke calcium responses in a restricted number of ORNs. In some cases, ORNs are predominantly sensitive to single odorants (e.g. ORN30 demonstrates robust responses exclusively to acetylcholine). All stimuli were used at concentration 1 mM except an aqueous extract of TET (∼0.2 mg/ml). C – Incidence histogram of the effects of L- (left column) and D- amino acid isomers (right column). Bars represent a number of ORNs sensitive to a particular amino acid expressed as percentage of a number of TET activated ORNs. Overall 10.3+−1% of ORNs are sensitive to L-AAs while in average only 2.3+−0.3% responded to D- isomers. Amino acids are grouped and color coded based on their side chains properties. D-Isoleucine and D-Asparagine were not tested.