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Do behaving animals extract information from precise spike timing? – The use of temporal codes

Periodic Reporting for period 4 - Temporal Coding (Do behaving animals extract information from precise spike timing? – The use of temporal codes)

Reporting period: 2020-07-01 to 2021-06-30

Neural temporal codes have come to dominate our way of thinking on how information is coded in the brain. When precise spike timing is found to carry information, the neural code is defined as a temporal code. In spite of the importance of temporal codes, whether behaving animals actually use this type of coding is still an unresolved question. To date studying temporal codes was technically impossible due to the inability to manipulate spike timing in behaving animals. However, very recent developments in optogenetics solved this problem. Despite these modern tools, this key question is very difficult to resolve in mammals, because the meaning of manipulating a part of a neural circuit without knowledge of the neural activity of all the neurons involved in the coding is unclear
The fly is an ideal model system to study temporal codes because its small number of neurons allows for complete mapping of the neural activity of all the neurons involved. Since temporal codes are suggested to be involved in olfactory intensity coding, we will study this process. We will device a multidisciplinary approach of electrophysiology, two-photon imaging and behavior
We aim to examine for the first time directly whether temporal coding is used by behaving animals and to unravel the circuits and mechanisms that underlie intensity coding. To do so, we will manipulate the temporal codes in behaving animals and examine whether the behavioral responses change accordingly.

Due to technical problems we were unable to directly examine the necessity of temporal codes for behavior. However, an indirect approach of reducing temporal code reliability showed a less reliable behavior suggesting that temporal codes are required for neural coding.
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.
Although unexpected, this study gave rise to two completely novel research projects.
First we were the first to link in vivo the contribution of presynaptic proteins to the reliability of post synaptic neural codes.
This study links two fields together, the field of synaptic release and the field of neural coding.

The second and more exciting unexpected result was the relevance of GPCR voltage dependence to behavior. Although GPCR voltage dependence has been demonstrated in vitro to affect the time course of synaptic release, whether GPCR voltage dependence contributes to neuronal coding and behavioral output under physiological conditions in vivo has never been demonstrated. We showed that mAChR-A mediated neuronal potentiation in vivo is voltage dependent. This potentiation voltage dependency was abolished in mutant animals expressing a voltage independent receptor. We could also show that muscarinic receptor voltage independence caused a strong behavioral effect of increased odor habituation. Together, we provided the very first demonstration of a physiological role for the voltage dependency of GPCRs by demonstrating crucial involvement of GPCR voltage dependence in neuronal plasticity and behavior.