The ins and outs of spinal cholinergic interneurons: a keystone for cholinergic analgesia – ACh-Spinal-Pain

Cholinergic neurons are identified by the expression of a fluorescent protein in transgenic mice. They are studied in vitro and in vivo. The electrophysiological recordings enable to study the synaptic inputs of these neurons, which informs us on their recruitment conditions. The anatomical study using transynaptic tools enables to identify the position of these neurons in the spinal nociceptive network. Finally optogenetics is used to specifically activate these neurons in order to study the consequence of their activation on the downstream network.

We have progressed on the first part of the project, consisting in the characterization of synaptic inputs onto cholinergic interneurons. Our data suggest that these neurons have a unique position in the network because they do not receive the same inputs that surrounding neurons. Our recordings give us indications about the possible localization of neurons presynaptic to the cholinergic neurons. We will now explore these leads.

An improved understanding of neuronal networks involved in the integration and control of sensory information flux in the spinal cord is essential for the development of alternative therapies to alleviate pain. In this context, we focus on a new type of control of this information by a sparse and understudied neuronal population, whose behavioral role is robust. It is conceivable that their manipulation might light to therapeutic tools.

At this stage, preliminary results have been presented in two international meetings.

Chronic pain is a devastating and widespread problem, and the existing treatments, including opioids, have limited efficacy (especially in the long term). In the look for alternative therapies, our project focuses on cholinergic analgesia.
In rodents, endogenous acetylcholine (ACh) is an important modulator of sensory processing, also implicated in the analgesic effects of clonidine and morphine, especially at the spinal level. This region is crucial as it is the place where nociceptive (pain-related) stimuli enter the central nervous system and are integrated before being relayed to the brain. We have demonstrated that, in mice, spinal ACh arises from a sparse population of dorsal horn cholinergic interneurons (Mesnage et al., 2011).
Clinical evidence suggests that spinal ACh also modulates pain behavior in humans. Indeed, acetylcholinesterase (AChE) inhibitors, such as neostigmine, are analgesic in postoperative and labor patients following epidural injection. Our latest data, now in press in the Journal of Neuroscience, demonstrates for the first time that a population of cholinergic interneurons exists in the dorsal horn of macaque monkeys, with similar density with the one observed in mice, and is the probable source of ACh involved in the analgesic properties of epidural AChE inhibitors (Pawlowski et al., In press). The similarities in the synaptic organization of this cholinergic population in monkey and rat, as well as the similar analgesic properties AChE inhibitors in rodents and humans supports the idea that the dorsal horn cholinergic system has similar functions in primates and rodents and validates our mouse model.
Understanding how such a sparse population achieves a major control of nociceptive processing is an ambitious challenge that we propose to address in rodents using a combination of in vitro and in vivo electrophysiology, optogenetics and transynaptic labeling. By analogy with other populations of exceedingly rare but intensely connected neurons that serve as hubs or orchestral directors for large networks, our working hypothesis is that a unique positioning within the spinal network makes cholinergic neurons a keystone of sensory processing. Indeed, we have shown that these neurons have extremely large dendritic and axonal territories.
We will now elucidate their interplay with the spinal nociceptive network by identifying (i) the stimuli that trigger the release of ACh, (ii) the neurons that are pre- and postsynaptic to them, and (iii) the consequences of the stimulation of these neurons on the spinal sensory network.
We were very encouraged by the previous reviews and by seeing that the project was sufficiently highly ranked to make it to the complementary list in the last competition. Since then we have completed a study validating the mouse models by confirming homology of this population of cholinergic interneurons in primates (J.Neurosci., In Press) and gathered a fair amount of additional preliminary data. This revised version draws from these recent advances and includes modifications that take into account the reviewers comments as well as pushes further some of our proposed experiments given the new data obtained
Beyond enhancing our understanding of cholinergic analgesia, our project will elucidate part of the microcircuitry of the sensory dorsal horn, and shed new light on the mode of action of classical analgesics. It is therefore expected to help identify new therapeutic targets for the treatment of chronic pain.