Spinal modulation of non-peptidergic C-nociceptor input: A role for inhibitory calretinin interneurons

Pain and itch are major clinical problems that not only cause great personal suffering, but also have a substantial economic impact since many sufferers are unable to work. It is estimated that chronic pain affects ~20% of the population, but only 1 in 3 patients respond to currently available treatments. To improve treatment for these conditions, we need to find out more about the complex circuits through which the nervous system processes pain and itch information. This information is initially transmitted to the spinal cord by specialised populations of nerve cells, including a type known as non-peptidergic nociceptors, and it has been shown that these can be divided into three functional groups based on the different types of proteins that they contain. These nociceptors are responsible for the sensation of itch, as well as for certain types of pain such as that resulting from pinching of the skin or a pinprick. They are also thought to contribute to the chronic pain that results from inflammation in different parts of the body, for example the pain after surgical operations. We have recently identified a distinct population of nerve cells in the spinal cord that are likely to block the passage of information from these nociceptors. These cells contain a protein called calretinin, and we refer to them as "inhibitory calretinin cells" (iCRs). Our preliminary findings suggest that the iCRs may be activated by all three types of non-peptidergic nociceptors. We also find that the iCRs are ideally positioned to prevent the nociceptors from activating other nerve cells, thus blocking pain and itch signals. We propose that under normal circumstances, these cells play an important role in switching off pain and itch input at the point of entry into the central nervous system. In this project, we will use several different experimental approaches to investigate the role of iCRs in pain and itch processing. Our studies will involve genetically-modified mice, as these will allow us to target the iCRs selectively. We will initially use a microscope that allows us to examine the connections (synapses) between these cells at very high magnification. In particular, we will ask whether iCRs are the only type of nerve cell that make the types of synapse that can block activity in the non-peptidergic nociceptors. We will then use an approach that allows us to study the activity of the iCRs, and test whether they are activated by all three of the nociceptor classes. In order to assess how effectively iCRs block the transmission of signals from the nociceptors, we will record the activity of a specific population of spinal cord nerve cells, known as projection neurons. These cells form the major route through which sensory information is transmitted from the spinal cord to the brain, allowing us to feel pain and itch. We will test whether activating the iCRs prevents sensory information carried by the nociceptors from reaching the projection neurons, and then assess the impact that this has on the activity of the projection neurons when the skin is stimulated. We will go on to use a recently developed technique that allows us to selectively alter the activity of the iCRs in mice. We will first determine whether activating these cells reduces both the pain that results from inflammation and the itch that is seen when certain chemicals are injected into the skin. We will then use a similar approach to inactivate the iCRs and see whether this leads to spontaneous pain or itch behaviours. If so, this would indicate that activity of the iCRs blocks these sensations under normal circumstances. This project will provide valuable information about the nerve circuits within the spinal cord that control the incoming sensory information that results in pain and itch. Importantly, it will also reveal whether the iCRs represent a target for new treatments that could be used to alleviate chronic pain and itch.

Non-peptidergic C nociceptors, which also function as pruritoceptors, can be assigned to 3 distinct transcriptomic populations (NP1-3). These afferents underlie itch and mechanical nocifensive reflexes, and have also been implicated in inflammatory pain states. Our recent findings suggest that all 3 classes innervate a population of spinal inhibitory interneurons that express calretinin (iCRs), and that they receive axoaxonic synapses (the substrate for presynaptic inhibition) from these cells. In this project, we will use a multidisciplinary approach based on mouse genetics to target iCRs selectively and reveal their roles in sensory processing. We will use electron microscopy to determine whether iCRs are the sole source of axoaxonic synapses onto NP1-3 afferents, and optogenetics to demonstrate functional synaptic input to iCRs from each afferent class. Lamina I projection neurons (PNs) form a major output from the dorsal horn, and we will therefore use these as a readout to assess the inhibition generated by the iCRs. We will achieve this by testing whether optogenetically activating iCRs suppresses synaptic transmission from non-peptidergic nociceptors to the PNs, and by examining the effect that this has on their responses to different types of noxious stimulus applied to the skin. We will use an intersectional chemogenetic approach to activate iCRs and test the prediction that this will suppress both the mechanical hypersensitivity in inflammatory pain states and the itch behaviour evoked by pruritogens. Finally, we will use chemogenetic inhibition or toxin-mediated silencing to establish whether the iCRs generate ongoing inhibition of pain and/or itch behaviours. The project will provide important information about spinal cord circuits that modulate noxious mechanical and pruritic input, and reveal whether the iCRs represent a potential therapeutic target for the treatment of inflammatory pain and itch.