Optogenetic pharmacology for the precise, biochemical control of cholinergic neuromodulation – Nicopto

In order to implement optogenetic pharmacology in vivo, we ween to fulfill certain criteria. First we need to bring the gene for the modified receptor. This is classically done using viruses. Second we need to deliver the small chemical photoswitch, which will photosensitize the modified receptor. This is done by locally delivering the chemical into the brain. Finally we need to deliver light and an electrode for simultaneously measuring the activity of the cell and photocontroling the receptor. All these techniques are currently being developed in the lab.

Neurons that synthesize dopamine (or dopamine neurons) are implicated in the response to natural rewards, while abused drugs, such as nicotine, hijack them. Nicotine acts on nicotinic receptors located on these cells. Light stimulation enables selective inhibition of the nicotinic receptors located on dopamine neurons, without affecting the other receptors in the brain. Using this precise optical stimulus we could show that the electrical activity of dopamine neurons is affected by the activity of nicotinic receptors. In addition, we could photo-inhibit the effect of nicotine on these neurons. These experiments were performed on anesthetized animals. Our ultimate goal would be to optically control nicotinic receptor in the behaving mouse, to optically reverse the reinforcing effects of nicotine.

Controling endogenous neuronal receptors with light is a technical challenge that we are trying to overcome in this project. Achieving this new perspective could help us have a better understanding of the molecular mechanisms of brain function, noth in physiological and pathological states. We will focus here on nicotinic receptors and their roles in nicotine addiction. However, we believe the strategies developed here should be applicable by neuroscientists and chemists to most receptors, cells, circuits and animal models.

We have just published a method paper on the development of light-controlled nicotinic receptors. The first author is the post-doc paid by the ANR, while the second author is the PhD student working on the project:
Lemoine D, Durand-de Cuttoli R, Mourot A. «Optogenetic Control of Mammalian Ion Channels with Chemical Photoswitches.» Methods Mol Biol. 2016;1408:177-93.
Romain Durand de Cuttoli presented is research project at the Society for neuroscience meeting in Chicago, October 2015.
In addition I just co-authored an article on the role of the nicotinic receptors in modulating curiosity in mice (Naudé et al., Nat Neurosci. 2016 Mar;19(3):471-8.). CNRS press release: www2.cnrs.fr/en/2683.htm

The brain delivers neuromodulatory signals such as dopamine (DA), serotonine, or acetylcholine (ACh) in spatially and temporally precise, pulsed, phasic or tonic patterns depending on the situation (1, 2). These signals are fundamental to the development and adaptation of the nervous system, and are believed to be the basis of such higher functions as learning and memory. Most neuromodulators act on multiple receptor classes, with different subtypes able to exert diverse physiological effects through different intracellular signaling pathways (2, 3). Hence neuromodulation depends not so much on the neurotransmitter released at the synapse as on the receptor subtype to which it binds. There is currently no method for acute, rapid, targeted activation/inactivation of specific receptors in defined cells within living animals. Traditional pharmacological agents show some receptor-subtype selectivity but, even when locally microinjected into brain nuclei, activate or inhibit a large, heterogeneous group of cells. Genetic techniques produce animal phenotypes where possible developmental alterations and compensatory changes are superimposed on the true effects of receptor removal. Currently available optogenetic tools (4), such as microbial opsins, enable precise remote control of fast electrical events, but cannot replicate the full complexity of slower neuromodulatory events. Remote-controlling specific receptor subtypes with millisecond precision in defined neuronal types and in living animals is unprecedented, yet it would be extremely valuable produce a clearer picture of the function of neuromodulatory-driven signals in neural information processing and plasticity.

To achieve this aim, we developed a versatile opto-chemical genetic strategy that enables mammalian receptors to be repeatedly turned off and on with great temporal precision, at a time scale compatible with fast synaptic transmission (5). The idea is to attach a synthetic photosensitive ligand onto a genetically engineered protein to allow activation or inhibition of only that specific protein with light. In this project we will implement this technique in the mouse brain in vivo, focusing on the neuromodulator ACh and its ionotropic receptor (nAChR). Brain nAChRs influence a large number of physiological functions. Furthermore, perturbation of cholinergic nicotinic neurotransmission can lead to various diseases including schizophrenia, Alzheimer's or Parkinson’s disease, or addiction to nicotine. Because neuromodulators such as ACh have long-lasting and often long-ranged, diffuse effects, it is of utmost importance to study cholinergic neuromodulation in an intact system, that is in vivo. We will first molecularly engineer novel light-regulated nAChRs (LinAChRs), and optimize these tools for in vivo utilization (Aim 1). We will then aim at simultaneously recording and photomodulating the excitability of a single DA neuron in a living animal. For this, we will develop strategies for in vivo delivery of the gene to specific locations in specific cell types, for bio-conjugation of the chemical photoswitch to the engineered receptor, for delivery of light into the living animal and for simultaneously recording action potentials from single neurons (Aim 2). Finally, we will use these tools in vivo to understand how a specific nAChR subtype, the ß2-containing (ß2*) one, finely modulates the DA system in the first steps of nicotine addiction (Aim 3).

We will focus on the nAChR, and its role in the modulation of the ventral tegmental area (VTA) DA system. However, we believe the strategies developed here should be applicable by neuroscientists and chemists to most receptors (e.g. muscarinic, DA, serotonine etc…), cells, circuits and animal models. Achieving this new perspective could help change the landscape of our understanding of normal physiology and neuroplasticity, and help move toward insights into the etiology and treatment of neuropsychiatric disease.