Shaking B Mediates Synaptic Coupling between Auditory Sensory Neurons and the Giant Fiber of Drosophila melanogaster.

The Johnston's Organ neurons (JONs) form chemical and electrical synapses onto the giant fiber neuron (GF), as part of the neuronal circuit that mediates the GF escape response in Drosophila melanogaster. The purpose of this study was to identify which of the 8 Drosophila innexins (invertebrate gap junction proteins) mediates the electrical connection at this synapse. The GF is known to express Shaking B (ShakB), specifically the ShakB(N+16) isoform only, at its output synapses in the thorax. The shakB2 mutation disrupts these GF outputs and also abolishes JON-GF synaptic transmission. However, the identity of the innexin that forms the presynaptic hemichannels in the JONs remains unknown. We used electrophysiology, immunocytochemistry and dye injection, along with presynaptically-driven RNA interference, to investigate this question. The amplitude of the compound action potential recorded in response to sound from the base of the antenna (sound-evoked potential, or SEP) was reduced by RNAi of the innexins Ogre, Inx3, Inx6 and, to a lesser extent Inx2, suggesting that they could be required in JONs for proper development, excitability, or synchronization of action potentials. The strength of the JON-GF connection itself was reduced to background levels only by RNAi of shakB, not of the other seven innexins. ShakB knockdown prevented Neurobiotin coupling between GF and JONs and removed the plaques of ShakB protein immunoreactivity that are present at the region of contact. Specific shakB RNAi lines that are predicted to target the ShakB(L) or ShakB(N) isoforms alone did not reduce the synaptic strength, implying that it is ShakB(N+16) that is required in the presynaptic neurons. Overexpression of ShakB(N+16) in JONs caused the formation of ectopic dye coupling, whereas ShakB(N) prevented it altogether, supporting this conclusion and also suggesting that gap junction proteins may have an instructive role in synaptic target choice.

Engrailed alters the specificity of synaptic connections of Drosophila auditory neurons with the giant fiber.

We show that a subset of sound-detecting Johnston's Organ neurons (JONs) in Drosophila melanogaster, which express the transcription factors Engrailed (En) and Invected (Inv), form mixed electrical and chemical synaptic inputs onto the giant fiber (GF) dendrite. These synaptic connections are detected by trans-synaptic Neurobiotin (NB) transfer and by colocalization of Bruchpilot-short puncta. We then show that misexpressing En postmitotically in a second subset of sound-responsive JONs causes them to form ectopic electrical and chemical synapses with the GF, in turn causing that postsynaptic neuron to redistribute its dendritic branches into the vicinity of these afferents. We also introduce a simple electrophysiological recording paradigm for quantifying the presynaptic and postsynaptic electrical activity at this synapse, by measuring the extracellular sound-evoked potentials (SEPs) from the antennal nerve while monitoring the likelihood of the GF firing an action potential in response to simultaneous subthreshold sound and voltage stimuli. Ectopic presynaptic expression of En strengthens the synaptic connection, consistent with there being more synaptic contacts formed. Finally, RNAi-mediated knockdown of En and Inv in postmitotic neurons reduces SEP amplitude but also reduces synaptic strength at the JON-GF synapse. Overall, these results suggest that En and Inv in JONs regulate both neuronal excitability and synaptic connectivity.

Auditory responses of engrailed and invected-expressing Johnston's Organ neurons in Drosophila melanogaster.

The roles of the transcription factor Engrailed (En), and its paralogue Invected (Inv), in adult Drosophila Johnston's Organ sensory neurons are unknown. We used en-GAL4 driven CD8-GFP and antibody staining to characterize these neurons in the pedicel (second antennal segment). The majority of En and Inv-expressing Johnston's Organ neurons (En-JONs) are located in the ventral part of the posterior group of JONs, with only a few in the medial group. Anatomical classification of En-JON axon projections shows they are mainly type A and E, with a few type B. Extracellular recording of sound-evoked potentials (SEPs) from the antennal nerve was used along with Kir2.1 silencing to assess the contribution that En-JONs make to the auditory response to pure-tone sound stimuli. Silencing En-JONs reduces the SEP amplitude at the onset of the stimulus by about half at 100, 200 and 400 Hz, and also reduces the steady-state response to 200 Hz. En-JONs respond to 82 dB and 92 dB sounds but not 98 dB. Despite their asymmetrical distribution in the Johnston's Organ they respond equally strongly to both directions of movement of the arista. This implies that individual neurons are excited in both directions, a conclusion supported by reanalysis of the morphology of the pedicel-funicular joint. Other methods of silencing the JONs were also used: RNAi against the voltage-gated Na⁺ channel encoded by the para gene, expression of attenuated diphtheria toxin, and expression of a modified influenza toxin M2(H37A). Only the latter was found to be more effective than Kir2.1. Three additional JON subsets were characterized using Flylight GAL4 lines. inv-GAL4 88B12 and Gycß100B-GAL4 12G03 express in different subsets of A group neurons and CG12484-GAL4 91G04 is expressed in B neurons. All three contribute to the auditory response to 200 Hz tones.

Calcium activates a chloride conductance likely involved in olfactory receptor neuron repolarization in the moth Spodoptera littoralis.

The response of insect olfactory receptor neurons (ORNs) to odorants involves the opening of Ca(2+)-permeable channels, generating an increase in intracellular Ca(2+) concentration. Here, we studied the downstream effect of this Ca(2+) rise in cultured ORNs of the moth Spodoptera littoralis. Intracellular dialysis of Ca(2+) from the patch pipette in whole-cell patch-clamp configuration activated a conductance with a K(1/2) of 2.8 microm. Intracellular and extracellular anionic and cationic substitutions demonstrated that Cl(-) carries this current. The anion permeability sequence I(-) > NO(3)(-) > Br(-) > Cl(-) > CH(3)SO(3)(-) >> gluconate(-) of the Ca(2+)-activated Cl(-) channel suggests a weak electrical field pore of the channel. The Ca(2+)-activated current partly inactivated over time and did not depend on protein kinase C (PKC) and CaMKII activity or on calmodulin. Application of Cl(-) channel blockers, flufenamic acid, 5-nitro-2-(3-phenylpropylamino) benzoic acid, or niflumic acid reversibly blocked the Ca(2+)-activated current. In addition, lowering Cl(-) concentration in the sensillar lymph bathing the ORN outer dendrites caused a significant delay in pheromone response termination in vivo. The present work identifies a new Cl(-) conductance activated by Ca(2+) in insect ORNs presumably required for ORN repolarization.

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.

Ca2+ stabilizes the membrane potential of moth olfactory receptor neurons at rest and is essential for their fast repolarization.

The role of Ca(2+) in insect olfactory transduction was studied in the moth Spodoptera littoralis. Single sensillum recordings were made to investigate in vivo the role of sensillar Ca(2+) on the electrophysiological properties of sex pheromone responsive olfactory receptor neurons (ORNs). Lowering the sensillar Ca(2+) concentration to 2 x 10(-8) M increased ORN spontaneous firing activity and induced long bursts of action potentials (APs) superimposed on spontaneous negative deflections of the transepithelial potential. We inferred that Ca(2+) stabilizes the membrane potential of ORNs, keeping the spontaneous firing activity at a low and regular level. Neither the amplitude and kinetics of the rising phase of sensillar potentials (SPs) recorded in response to pheromone stimuli nor the AP generation during stimulation depended on the extracellular Ca(2+) concentration. Thus, extracellular Ca(2+) is not absolutely necessary for ORN response. Partial inhibition of responses with a calmodulin antagonist, W-7, also indicates that intracellular Ca(2+) contributes to the ORN response and suggests that Ca(2+) release from internal stores is involved. In 2 x 10(-8) M Ca(2+), the repolarization of the SP was delayed when compared with higher Ca(2+) concentrations. Therefore, in contrast to depolarization, ORN repolarization depends on extracellular Ca(2+). Ca(2+)-gated K(+) channels identified from cultured ORNs with whole-cell recordings are good candidates to mediate ORN repolarization.

Male aggregation pheromone of date palm fruit stalk borer Oryctes elegans.

Laboratory and field investigations were carried out to characterize the chemical communication system of the date palm fruit stalk borer, Oryctes elegans, and to develop pheromone-based trapping in Eastern Iran. Adults of both sexes feeding on date palm pieces attracted conspecifics, whereas date palm alone was minimally attractive. Males were twice as attractive as females. More beetles were captured at the palm crown than at ground level. Odors from adults feeding on sugarcane were sampled and analyzed by gas chromatography and mass spectrometry. Whereas females did not emit sex specific volatiles, males emitted a blend of 4-methyloctanoic acid (1: major component) and ethyl 4-methyloctanoate (2), occasionally mixed with minor components: 4-methyloctanyl acetate (3), methyl 4-methyloctanoate (4), 4-methyloctanol (5), and nonanyl acetate (6). Electroantennography and field trapping experiments demonstrated that compound 1 is an essential component of the male aggregation pheromone of O. elegans. It was barely attractive by itself but synergistic with fresh date palm odor. It attracted many more beetles than any of compounds 2-6. The addition of one or several of compounds 2-6 to 1 did not improve trap captures. During the course of 2 years, we captured 4000 beetles, with a weekly average of 6.3 beetles/trap, and were able to monitor the seasonal flight of O. elegans. Our results provide the basis for developing mass trapping for control of this pest.