Dissecting the relative contributions of injured and intact primary afferents to neuronal plasticity and neuropathic pain

Normal (nociceptive) pain is generated when specialised nerves (nociceptors) detect noxious stimuli and is a vital warning system that helps us to prevent or limit injury. Nociceptors send signals about the noxious stimuli to the spinal cord. Here, the signal is heavily modified before being transmitted to the brain, where the perception of pain is generated. Unlike nociceptive pain, neuropathic pain, which results from injury or disease of the nervous system, is a condition for which there is no known purpose. It affects over five million people in the UK alone and the majority of these patients are failed by current treatments and live in disabling pain.

Neuropathic pain represents a dysfunction of pain transmission that can include spontaneous pain (stabbing or burning pain) and/or enhanced pain perception to both noxious and non-noxious stimuli (termed hyperalgesia and allodynia respectively). Changes in the way nociceptors and spinal circuits function are known to contribute to neuropathic pain. However, we know little about the precise nerves involved in different aspects of neuropathic pain, with the underlying causes of spontaneous pain a particularly understudied example. This is an incredibly important research area for two reasons. Firstly, because spontaneous pain is the major complaint from patients with neuropathic pain and, secondly, because knowing which nerves to target and where to do so, will be vital for the rational design of new drugs capable of ameliorating pain.

It is clear from studies in patients that excessive electrical activity in nociceptors generates pain. Following injury to a nerve, injured nerves and intact neighbouring nerves develop spontaneous electrical activity; raising the question of how these two groups contribute to pain. This will be the central question of my research programme. To investigate, I will use a genetically altered mouse in which damaged and intact nerves are targeted separately with a protein capable of turning off electrical activity when a drug is given. I will use new behavioural methods to assess spontaneous pain in mouse models of neuropathic pain. The level of spontaneous pain when injured or intact nerves are switched off will be compared to give an insight into their respective contributions.

While electrical activity in nociceptors is vital for spontaneous pain, the way the signal is processed in the spinal cord likely enhances or prolongs the pain experienced. Therefore, to study the changes that occur in the spinal cord, I will measure and compare the relative ease with which injured and intact nociceptors can activate spinal circuits. To visualise whether there are changes in spinal cord circuit structure following nerve injury, I will use genetically altered mice to label injured and intact nociceptors with different colours of fluorescent protein. In this way, I will be able to compare where these nerves go in the spinal cord and whether their connections with spinal nerves change after injury. These studies will be supported by recording from spinal nerves in tissue preparations while activating injured or intact nociceptors. By doing so, I will answer whether either pathway is strengthened following injury. Finally, I will study whether electrical activity in injured or intact nerves activates immune cells in the spinal cord, which are known to contribute to chronic pain. This will give us important information on whether activation of the immune system, independent of nociceptor electrical activity, should be an important consideration for drug development.

This work will provide important information on the nerves involved in generating pain and how their activity can result in long-lasting and enhanced pain. In doing so, I will define new therapeutic targets that allow us to better design novel drugs for the treatment of neuropathic pain.

Following peripheral nerve injury, aberrant primary afferent (PA) activity and altered spinal processing are key drivers of neuropathic pain. Ectopic activity occurs in PAs directly injured by the insult as well as in intact neighboring afferents; raising the question of how these two populations contribute to the different types of pain experienced. I will use genetic targeting of chemo- and optogenetic tools in rodent nerve injury models to address this central question. Adeno-associated viral (AAV) delivery of a modified glutamate-gated chloride channel (GluCl) to PAs allows for long-term silencing upon treatment with Ivermectin (channel agonist), making this approach ideal to study the effect of long-term neuronal activity to chronic pain. I will use Cre-ON and Cre-OFF GluCl AAV vectors in transgenic mice in which Cre is expressed de novo in injured afferents (ATF3-Cre and NPY-Cre). This approach will allow separate silencing of injured and intact PAs while pain behaviour is assessed. Condition place preference paradigms will be used to assess spontaneous pain and assays of reflex withdrawal will be measured (Von Fey and Cold Plantar) as secondary outcomes. PA activity is necessary for pain, however it also sensitizes spinal processing. We do not know whether changes occur in the central terminals of injured or intact PAs that facilitates pain transmission. Projection neurons (PNs) are directly innervated by nociceptors and relay nociceptive signals to the brain. I will record from PNs in ex vivo spinal slice preparations while injured/intact PA terminals are optogenetically activated and test whether synaptic transmission changes following nerve injury. Injured/intact PA synapse size and number on PNs will be contrasted in the same tissue by delivering a FLEX-switch reporter AAV to PAs of injured ATF3Cre mice. This work will gain important insights into the drivers of neuropathic pain and will define key neurons to target for effective therapeutic treatment.

Fundamental biology: Academics benefiting from this will include pain scientists and molecular neuroscientists. Understanding the contribution of injured and intact afferent activity to pain following nerve injury is of considerable interest to the pain field and has been a matter of contentious debate for several decades. Information on this topic will guide researchers as to which neurons should be studied to understand the pathological drivers of neuropathic pain. Relatively little is known about the contribution that different afferents make to spontaneous pain and the proposed research will enhance this knowledge. Findings derived from Objective 2 will be of interest for researchers interested in neural plasticity and neuro-immune coupling, two extremely important topics in wider neuroscience.

The pharmaceutical industry: The pharmaceutical industry has a major interest in developing novel analgesics to treat painful conditions. Pain is an extremely prevalent burden and is becoming more common as the population ages and prevalence of diseases such as diabetes rise. The analgesic drug market is therefore very large. A major barrier for the rational design of novel treatments is our current lack of understanding of the neural pathways that underlie the development of pathological pain. This includes the identity of the neurons, which underlie neuropathic pain. Information on the afferent sub-types that should be targeted for treatment (e.g. intact or injured) will be helpful in identifying new drug targets. Findings on the contribution of peripheral and central drivers/modifiers of spontaneous pain will be particularly relevant for the design of new drugs and their requirement to cross the blood brain barrier.

Chemogenetics to understand neural circuitry: Chemogenetic approaches offer long-term neuronal silencing suitable for addressing the functional relevance of the neuronal population under study. To date, chemogenetic silencing of sensory neurons has been challenging using traditional means such as DREADDs. My method of using a modified glutamate-gated chloride channel (GluCl) has proved effective when targeted to a broad range of sensory neurons. Expanding the toolkit of GluCl vector systems, including the generation of cre-dependent and cre-inactivated forms will be very helpful for the pain field and wider neuroscience community. These tools will be useful for researchers interested in studying the effects of long-term silencing of activity in genetically identified neuronal populations. All vectors will be deposited in repositories so that they are available to researchers worldwide.

Society: Patients and the wider society may benefit from this study. Pain resulting from injury or disease of the somatosensory nervous system (neuropathic pain) is estimated to affect over 5 million people in the UK, with a major impact on quality of life and workdays lost. Unfortunately, treatment options are sorely lacking; efficacy of current treatments is low and hampered by dose-limiting off-target effects. Research findings of this proposal will enhance the development of new and better drug therapies, particularly in relation to neuropathic pain.

Personal development: The Fellowship will develop my research skills and as such contribute significantly to my career progression. The CDA will allow me to establish my independence and build my own research team. This will put me in an extremely strong position to successfully attain future funding and senior level research positions. Glasgow University have committed to considering a salaried position following successful completion of a CDA. By employing/supervising a research group, I will be able to pass the skills that I have learned onto the next generation of pain scientists.