Unravelling the Physiological Roles of GPCR Voltage Dependence

Project Information

GPCR Volt Dep Role

Grant agreement ID: 101085605

DOI

10.3030/101085605

EC signature date 27 June 2023

Start date 1 October 2023

End date 30 September 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 992 500,00

EU contribution

€ 1 992 500,00

Coordinated by

TEL AVIV UNIVERSITY
Israel

Periodic Reporting for period 1 - GPCR Volt Dep Role (Unravelling the Physiological Roles of GPCR Voltage Dependence)

Reporting period: 2023-10-01 to 2026-03-31

Summary of the context and overall objectives of the project

G protein coupled receptors (GPCRs) are broadly expressed in the brain, mediate responses to many molecules, and are crucial for normal brain function and therapeutic intervention.
About 20 years ago, it was shown that the activity of many GPCRs is regulated by membrane potential. For example, cholinergic muscarinic M2R and metabotropic glutamate mGluR3 receptors are reduced by depolarization, while M1R and mGluR1a are increased. However, due to technical challenges, the physiological roles of this voltage dependency, its effect on neural activity, and its behavioral relevance remain unclear.
We recently showed that M1R voltage dependence is crucial for its recruitment. Under physiological conditions, M1R cannot be activated without depolarization, and depolarization alone is sufficient to activate it. Flies with voltage-independent M1R show increased odor habituation, indicating major behavioral effects. These findings redefine how we think about GPCR recruitment and activity. To make a paradigm shift, we must determine whether GPCR voltage dependence has roles in other GPCR types and neuronal processes.
The fly is an ideal model to explore GPCR voltage dependence roles because it has a low variety of receptors with minimal overlap. Drosophila dopaminergic, muscarinic, and serotonergic receptors, highly expressed in the olfactory system, are ideal candidates.
We combine electrophysiology, two-photon imaging, genetics, and behavior to examine GPCR voltage dependency, manipulate it, and determine its physiological and behavioral roles.
Recognizing a “voltage rheostat” controlling GPCR activity will open new research fields and therapeutic avenues.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During this grant period, we addressed three topics:
I. The role of muscarinic receptor mAChR-B voltage dependence in learning and memory.
II. Whether Drosophila dopaminergic and serotonergic receptors are voltage dependent.
III. Other GPCR roles in other brain regions and behaviors, to show GPCR voltage dependence as a fundamental process in brain function.

I. mAChR-B voltage dependence in learning and memory
Learning and memory are constrained by opposing demands: efficiency (detect weak signals, minimize false negatives) versus specificity (avoid false positives). In Drosophila olfactory learning, sensory inputs and reinforcement converge in mushroom body (MB) lobes, where Kenyon cells (KCs) connect to dopaminergic neurons (DANs). Simultaneous KC and DAN activation induces long-term depression (LTD) at KC-MB output neuron (MBON) synapses. We recently showed that KC-KC lateral neuromodulation via mAChR-B opposes signals required for KC-MBON plasticity, preventing plasticity where it should not occur. However, this interference could also impair plasticity where it should occur.
We show that KC lateral neuromodulation indeed hampers efficient learning. mAChR-B is voltage dependent - highly active at rest and with reduced activity upon depolarization. This voltage shift silences mAChR-B in active KCs, allowing proper plasticity and efficient learning. Thus, voltage dependence of mAChR-B enables both specific and efficient learning by minimizing both type of errors.

II. Voltage dependence of dopaminergic and serotonergic receptors
We demonstrated that Drosophila dopaminergic receptors Dop1R2 and Dop2R, and serotonergic receptors 5HT1A and 5HT1B, are voltage dependent. For serotonergic receptors, we identified one mutation (of 12 tested) that made the receptor voltage independent while maintaining normal activity, suitable for in vivo study.

III. GPCR roles in other brain regions and behaviors
We found that mAChR-B contributes to odor discrimination in olfactory receptor neurons (ORNs) via intraglomerular axo-axonal connections. Contrary to its usual inhibitory role, mAChR-B mediates excitation at high ORN firing rates. This nonlinear excitation decorrelates ORN activity patterns and improves odor classification, revealing a novel mechanism for neuronal pattern decorrelation.

In another project, we uncovered the mechanism of operant memory in flies with surprising results. Animals form classical or operant cue-outcome associations, traditionally thought to coexist and be additive. However, they can drive opposing behavioral responses. We showed that Drosophila classical and operant olfactory conditioning rely on distinct neuronal pathways leading to different behavioral responses. Plasticity in both pathways cannot be formed simultaneously. If plasticity occurs at both pathways, an interference between them occurs and learning is disrupted. Importantly, activity of the navigation center is required to prevent plasticity in the classical pathway and enable it in the operant pathway. These findings fundamentally challenge hierarchical views of operant and classical learning and show that active processes prevent coexistence of the two memories.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Major breakthroughs
1. We are the first to demonstrate a behavioral role for GPCR voltage dependence in vivo. Specifically, we show it is essential for efficient yet specific learning and memory. Efficient learning requires high sensitivity, while specific learning requires low sensitivity. We show neurons can toggle GPCRs on or off via membrane potential, generating variable sensitivity - high where learning should occur, low where it should not. Thus, neurons can tune how they “listen” to others, changing how we view neural circuit communication.

2. We also challenge the dogma on how different learning forms interact. Classical conditioning links cues to outcomes independent of behavior; operant conditioning links behavior to outcomes. Although traditionally viewed as additive, these pathways can drive conflicting behaviors. We show that classical and operant memories are not additive and cannot form simultaneously, rather they interfere with one another. Moreover, we identified the mechanism determining which memory is formed. These findings redefine how distinct learning systems interact and open a new research field.