Do Rorb/calretinin interneurons (CR islet cells) gate spinal nociceptive inputs?

Nerve fibres that enter the spinal cord (primary afferent fibres) carry various types of sensory information. Some of these fibres (nociceptors) respond to tissue-damaging stimuli that are normally perceived as pain. Although nociceptors are found in tissues throughout the body, one particular class, known as C-MrgD afferents, exclusively supplies the skin. Selective destruction of the C-MrgD afferents in mice leads to reduced pain behaviour following mechanical stimuli, but not hot or cold stimuli. This suggests that these afferents are required for the normal perception of mechanical pain. Primary afferent fibres activate a variety of nerve cells in the spinal cord. Most of these cells are interneurons, which give rise to local circuits that process and modify the incoming sensory information before it is conveyed to the brain for conscious perception. Around a third of these interneurons release chemical messengers (neurotransmitters) that reduce the activity of other nerve cells, and therefore have an inhibitory function. These inhibitory interneurons use two basic mechanisms: postsynaptic and presynaptic inhibition. Postsynaptic inhibition involves suppressing activity of nearby nerve cells, while presynaptic inhibition operates directly on the incoming sensory nerve fibres by reducing their ability to activate their target cells. This has the advantage of providing a highly selective inhibition of specific types of sensory information.
Presynaptic inhibition is known to operate on C-MrgD afferents, and this will presumably suppress pain. Until recently nothing was known about the inhibitory interneurons responsible for this, but we have now identified a population of cells that presynaptically inhibit the C-MrgD afferents. These cells can be recognised because they contain two proteins: calretinin (CR) and Rorb. These proteins are only co-localised in this population, and because of their morphology we have called them CR islet cells. These findings are important because they provide a way of investigating the role of presynaptic inhibition of nociceptors in pain mechanisms.
In this project, we will use a multi-disciplinary approach to test a set of hypotheses concerning the CR islet cells. We will use genetically altered mice in which specific populations of nerve cells are labelled with fluorescent markers and/or contain proteins that allow their functions to be manipulated, either by application of light pulses or through the administration of highly selective drugs. We will initially carry out physiological studies to test the hypothesis that the CR islet cells are the major source of presynaptic inhibition of the C-MrgD afferents and that they can block the transmission of activity evoked by these afferents in spinal cord nerve cells. We know that the C-MrgD afferents are not the only target for the CR islet cells, and we will therefore test whether they also inhibit excitatory nerve cells that are activated by these afferents. If so, this would allow the CR islet cells to generate a powerful inhibition of the transmission of pain information from nociceptive primary afferents to the projection cells that convey this information to the brain, and represent the main output for spinal cord pain circuits. Finally, we will use genetically altered mice to activate the CR islet cells selectively in vivo. We predict that this will alleviate the hypersensitivity to mechanical stimuli that occurs in pathological pain states, and we will use behavioural testing in standard models of inflammatory and nerve injury-evoked pain to determine whether this is the case.
These experiments will greatly improve our understanding of the nerve circuits in the spinal cord that are responsible for controlling pain. This information is important in the search for new drugs to treat pain, and also for future studies to investigate changes in the spinal cord that underlie chronic pain states.

Non-peptidergic (C-MrgD) nociceptors innervate the skin and are required for perception of mechanical pain. They project to lamina II, forming central axons of synaptic glomeruli. These receive axoaxonic synapses (the substrate for GABAergic presynaptic inhibition), but until now the source of these synapses was unknown. We have identified a population of inhibitory interneurons in lamina II, defined by co-expression of calretinin (CR) and Rorb, and provide evidence that these are islet cells that are innervated by the C-MrgD afferents, and give rise to axoaxonic synapses on them. This provides a way of investigating the function of the neurons that generate presynaptic inhibition of nociceptors.
We will initially use optogenetics in a Rorb-Cre mouse to activate CR islet cells selectively, test their involvement in presynaptic inhibition of C-MrgD input, and investigate the pharmacology of this effect. We will use this approach to determine whether the CR islet cells are able to block transmission of C fibre-evoked action potentials.
Not all axonal boutons from CR islet cells contact C-MrgD terminals, indicating that they must have other postsynaptic targets and we will identify these with optogenetics. Specifically, we will test whether they target excitatory interneurons that receive C-MrgD input and innervate lamina I, and whether these include vertical cells. If so, this would mean that the CR islet cells are well placed to inhibit transmission from C-MrgD nociceptors to lamina I projection neurons.
Finally, we will use an intersectional chemogenetic strategy that will selectively activate CR islet cells in vivo and test the prediction that this will reduce mechanical hyperalgesia in inflammatory and neuropathic pain models.
The findings of this project will provide important insight into the organisation of dorsal horn inhibitory interneurons, and the role of cells that generate presynaptic inhibition in controlling nociceptive transmission.

Pain is a major cause of suffering for both humans and animals. It has been estimated that over 10% of the adult population in the UK live with chronic pain, and this proportion is likely to increase as the population ages. Two-thirds of those with chronic pain report difficulty sleeping, half suffer from depression, and for ~15% "the pain is so bad that they want to die". Only around two-thirds of those with pain respond to currently available treatments, and chronic pain therefore represents a major unmet clinical need. One of the main reasons for the lack of effective treatments for pain is the limited information about the underlying mechanisms, and this is particularly significant for neuropathic pain, which commonly occurs following injuries to peripheral nerves or the spinal cord. Chronic pain also has major societal and economic impact, because in many cases, sufferers are unable to work and report a direct effect on their employment prospects.

Who will benefit from this research?
Those benefitting directly will include scientists working on pain mechanisms, those from other disciplines (e.g. developmental biologists, molecular geneticists) who work on the somatosensory system, as well as those in the pharmaceutical industry, particularly in relation to development of new analgesic drugs. In the longer term, beneficiaries will include human patients and animals suffering from chronic pain, and the clinicians responsible for their treatment. Improved treatments for chronic pain would have a major impact on the nation's health and economic prosperity. The project will also benefit neuroscientists other fields, since the tools that we develop to manipulate the function of specific neuronal populations in vivo can be applied throughout the CNS.

How will they benefit?
The development of new treatments for pain, in particular neuropathic pain, is a high priority for the pharmaceutical industry. This is likely to depend to a large extent on our knowledge of the neuronal pathways that underlie pain perception, from the peripheral receptors right through to the cortical areas that are responsible for different aspects of pain. The spinal dorsal horn contains inhibitory circuits that can powerfully suppress nociceptive inputs, but the organisation of these circuits is poorly understood. Identifying the different inhibitory interneuron populations, and defining their roles in pain mechanisms should lead to the discovery of novel targets for analgesics. In particular, the discovery and characterisation of the CR islet population, which appears to be responsible for selective presynaptic inhibition of mechanical nociceptors, may reveal signalling mechanisms that would allow these cells to be targeted. In addition, recognising changes in the dorsal horn that result from nerve injury will be important for development of new treatments for neuropathic pain, and this requires a greatly improved understanding of the normal circuitry in spinal pain pathways. Work from our laboratories has already generated important insights into neuropathic pain, by demonstrating that certain proposed mechanisms (dorsal sprouting or de novo peptide expression affecting tactile afferents, and significant loss of inhibitory interneurons) are not necessary for the development of allodynia and hyperalgesia after peripheral nerve injury, thus directing research away from these areas.
The named RA will increase his skill set by acquiring expertise in the use of intraspinal injection for delivery of AAVs and nerve injury models of neuropathic pain. Training in in vivo experimental approaches has been identified as a priority for the pharmaceutical industry.
The proposal is in line with BBSRC policy, since "mechanisms underlying pain" is identified as an important area within the Welfare of Managed Animals priority. By contributing to our understanding of chronic pain, it is also highly relevant to "Healthy ageing across the life course".