We found that both excitatory and inhibitory optogentic tools when expressed in first and second order olfactory neurons proved ineffective in blocking intensity dependent odor response.
We attempted to develop novel tools based current optogentic tools as well as on modulation of presynaptic proteins to manipulate temporal action potential dynamics of post synaptic neurons (albeit in a less precise manner).
To modulate temporal dynamics using presynaptic proteins we chose in addition to the classical release related proteins also presynaptic cholinergic muscarinic G-protein coupled receptor (GPCRs) since they were demonstrated to control the time course of release by a fast voltage dependent process.
We reasoned that modulating these GPCRs voltage sensor could interfere with time course of release and as a result with the post-synaptic neurons temporal dynamics.
Modulating presynaptic proteins gave the required result. We used the ORN-PN synapse to show that the reliability of the neural code is sensitive to perturbations of specific presynaptic proteins controlling distinct stages of transmitter release. Notably, coding reliability of postsynaptic neurons decreases only at high odor intensity. We further showed that while the reduced temporal code reliability arose from monosynaptic effects, the reduced rate code reliability arose from circuit effects, which included the recruitment of iLNs. Finally, we found that reducing neural coding reliability decreases behavioral reliability of olfactory stimulus classification.
For the cholinergic GPCRs little was known about their expression and function in Drosophila. The Drosophila olfactory system is mostly cholinergic and expresses two muscarinic GPCRs: type A and B (mAChR-A and mAChR-B). We showed that mAChR-A/B are voltage dependent and found means to abolish their voltage dependency. We generated genetically edited flies (using CRISPR technology) with a voltage independent mAChR-A (we are in the process of generating also a voltage independent mAChR-B). We then mapped using anatomy, genetics and pharmacology which neurons in the Drosophila olfactory system express mAChR-A and -B. We found that in the antennal lobe (AL), the region where olfactory receptor neurons, ORNs, synapse onto second order projection neurons (PNs), mAChR-A is expressed in inhibitory local neurons (iLNs), and mAChR-B in ORNs and PNs. We showed that mAChRs-A shapes AL output and affects behavior by a dual role: direct excitation of iLNs and stabilization of the ORN-iLN synapse. We also found that the third order olfactory neurons, Kenyon cells (KCs), also express mAChR-A and -B and that both are required for learning and memory. We localized mAChR-A/B to KC dendrites and axons respectively. Both mAChR-A and -B affect long-term depression (LTD) in KCs that is required for learning and memory, but in different mechanisms and play different roles. Since mAChR-A had no effect on ORNs or KCs presynaptic terminals we did not pursue this course of action.
