The role of NPFF-expressing excitatory interneurons in spinal pain mechanisms

Chronic pain is a major clinical problem that affects ~30% of the UK population and results in significant societal and economic impacts. Currently available treatments often provide inadequate pain relief, and a major reason for the limited success in design of new treatments is lack of knowledge about the neuronal circuits that underlie pain. Primary afferent neurons, many of which signal noxious stimuli, transmit sensory information to the dorsal horn of the spinal cord. The dorsal horn contains projection cells belonging to the anterolateral system (ALS) that convey this information to the brain, and these cells are required for the conscious perception of pain. However, the vast majority of dorsal horn neurons are interneurons, which are involved in local synaptic circuits that modify sensory signals before transmission to ALS cells. Most of the interneurons are excitatory, and these can be assigned to several distinct functional populations that can be distinguished based on morphological and transcriptomic criteria. One group of excitatory interneurons consists of vertical cells, which convey input from various types of primary afferent to ALS cells. Vertical cells are thought to be particularly important, as they contribute to hypersensitivity in pathological pain states. We have recently identified two different populations of vertical cell that are defined by expression of the gastrin releasing-peptide receptor (GRPR) or neuropeptide FF (NPFF), and have shown that these differ in anatomical, physiological and pharmacological properties. The GRPR cells are strongly implicated in the perception of itch, but little is known about the roles of the NPFF-expressing vertical cells in spinal processing of sensory information. This project will use a multidisciplinary approach to investigate the functions of NPFF-expressing vertical cells through five complementary workpackages (WPs). In WP1, we will use anatomical methods to define the excitatory synaptic input to these cells, allowing us compare this with the synaptic input to GRPR cells, which we have recently described. WP2 will involve recording of activity of the NPFF cells with electrophysiology and calcium-imaging, using an ex vivo “semi-intact preparation” in which a piece of skin remains connected to the spinal cord via intact peripheral nerves. This will allow us to determine the responses of the NPFF cells to natural cutaneous stimuli. In WP3 we will use chemogenetics to test the hypothesis that activating NPFF cells in vivo causes pain-like behaviours and is aversive. We will also use this approach to identify ALS neurons that are downstream of the NPFF cells, by revealing the activity marker Fos. WP4 will involve silencing the NPFF cells by means of optogenetics. We will test whether this suppresses responses to acute painful stimuli, and also whether it reduces the hypersensitivity and on-going pain seen in an inflammatory pain model. Finally, in WP5 we will use the semi-intact preparation to characterise a class of ALS projection neurons that receive numerous synapses from the NPFF cells. We will then optogenetically silence the NPFF cells to reveal their contribution to the sensory input to these ALS neurons. This work will provide important fundamental insights into pain neurobiology, by revealing neuronal circuits in the spinal cord that are involved in acute and inflammatory pain. It should also reveal potential targets for the development of new strategies for pain relief.