The role of NPY-containing inhibitory interneurons in spinal pain pathways

Nerve fibres entering the spinal cord carry various types of sensory information to a region called the dorsal horn. Incoming sensory information is transmitted to a class of nerve cells called projection neurons, which convey it to the brain for conscious perception. However, the vast majority of nerve cells in the spinal cord are interneurons, which are responsible for local processing and modulation of sensory information before it reaches the projection neurons. Around a third of the interneurons release chemical messengers (neurotransmitters) that reduce the activity of other nerve cells, and therefore have an inhibitory function. These inhibitory interneurons control the flow of sensory information, including that which is perceived as pain. It is thought that a reduction of their activity contributes to some types of chronic pain, for example the pain that can occur after nerve injury.
Despite the importance of the dorsal horn in pain mechanisms, we still know relatively little about the organisation of its nerve cells and circuits, or about how they process the incoming sensory information. This is largely because of the difficulty in defining specific functional populations among the interneurons. Work from our laboratory has shown that several different types of inhibitory interneuron can be recognised, based on the presence of specific chemical markers. One group consists of cells that make a substance called neuropeptide Y (NPY), and these account for around 15% of inhibitory interneurons in the superficial part of the dorsal horn and are scattered through its deeper part. Recent studies have selectively silenced or activated two other populations of inhibitory interneurons, and shown that these have different roles in reducing pain.
From what we already know about the connections of the NPY cells, we predict that they will inhibit several different types of acute pain (but not itch), and that they will suppress chronic pain caused by inflammation or nerve injury. The main aim of this project is to test this hypothesis, and we will do this by using a strain of genetically altered mouse in which the NPY cells can be specifically targeted. We will make injections into the spinal cords of these mice of viruses that will either silence or activate these cells, while having no effect on other neurons. We will then test the prediction that inhibiting the NPY cells causes a reduction of pain thresholds, while activating them results in less pain following inflammation or nerve injury. We will verify that only NPY cells have been affected, and that we can therefore attribute any behavioural changes to an effect on these cells.
Two different inhibitory neurotransmitters, GABA and glycine, are used by spinal cord interneurons, and we will test the predictions that the NPY cells in the superficial part of the dorsal horn use only GABA, and that they make synaptic connections with many other types of neuron in this region. This will be achieved with "optogenetics", in which light is used to activate specific populations of nerve cells. In addition, we will test whether the NPY cells in the deep part of the dorsal horn are also inhibitory interneurons, and find out which neurotransmitter they use.
We will also use viral injections to visualise the NPY cells without altering their activity. In this way, we will find out more about the functions of a specific subset of these neurons, which strongly inhibit a particular group of projection neurons that respond to painful stimuli. We will test the prediction that they are activated by both tactile and painful stimuli, and therefore contribute to suppression of pain by touch, as well as setting a level of pain that is appropriate to the strength of the stimulus.
These experiments will provide important insight into how inhibitory nerve cells in the spinal cord are involved in suppressing pain. This type of information is essential in the search for new analgesic drugs.

Inhibitory interneurons in the dorsal horn play an important role in controlling transmission of nociceptive information, and their dysfunction contributes to pathological pain. One of the main limitations to our understanding of sensory processing has been the difficulty of identifying functional populations among these cells. We have shown that those in laminae I-III can be divided into several non-overlapping populations, one of which is defined by expression of neuropeptide Y (NPY). Recent studies have suggested different anti-nociceptive roles for two other classes of inhibitory interneurons: those that express dynorphin, and glycinergic neurons, which make up the majority of those in deeper laminae.
However, virtually nothing is known about the role of the NPY-expressing cells, which account for ~15% of inhibitory interneurons in laminae I-III and are scattered through the deeper laminae. In this project we will investigate the functions of these cells by using intraspinal injections of viral vectors carrying cre-dependent proteins into transgenic mice that express cre recombinase under control of the NPY promoter. We will selectively silence or activate the NPY cells to test the prediction that they have diverse roles in suppressing several forms of acute and chronic pain, but are not involved in reducing itch evoked by pruritogens. We will use optogenetics to determine whether NPY cells in laminae I-III use GABA, rather than glycine, as their fast transmitter, and identify the types of dorsal horn interneuron that they innervate. We will also test whether those in deeper laminae are inhibitory interneurons. We have identified a specific class of NPY cell that selectively inhibits nociceptive projection neurons in lamina III, and we will investigate the primary afferent input to these cells.
The study will provide important insights into the organisation and functions of inhibitory interneurons in the dorsal horn, and their roles in suppressing pain.

Pain is a major cause of suffering for both humans and animals, and represents an important unmet clinical need. It has been estimated that 7.8 million people in the UK live with chronic pain, and that only two-thirds of these will respond to currently available treatments. One of the main reasons for the lack of effective treatments is our limited understanding of the underlying mechanisms, particularly in the case of the neuropathic pain that results from peripheral nerve damage or spinal cord injury. Chronic pain has massive societal and economic impact, since many sufferers are unable to work, and many report a direct effect on their employment prospects.

Who will benefit from this research?
Those who will benefit directly include scientists working on spinal pain mechanisms, those from other disciplines (e.g. pharmacology, molecular genetics, developmental biology) who are working on the somatosensory system, as well as scientists in the pharmaceutical industry who are involved in the development of analgesics. The ultimate beneficiaries of this project would be human patients and animals suffering from chronic pain, and the clinicians responsible for their treatment. Improved treatments for chronic pain would impact greatly on the nation's health and economic prosperity.

How will they benefit from this research?
Development of new treatments for chronic pain, in particular neuropathic pain, will depend to a large extent on our understanding of the neuronal pathways that underlie pain perception, from peripheral receptors, through to the cortical areas involved in perception. The spinal dorsal horn contains inhibitory circuits that can powerfully suppress nociceptive inputs and prevent the cross-talk between modalities that underlies tactile allodynia. However, the organisation of these circuits is still poorly understood. Identifying and characterising their different components should lead to the discovery of novel targets for analgesics. Certain ion channels and receptors are selectively expressed by subpopulations of dorsal horn neurons. Determining the neuronal types that express these channels/receptors will indicate whether activating or inhibiting them is likely to be anti-nociceptive, for example by increasing excitability of specific inhibitory interneurons. In addition, identifying changes in neuronal function and circuitry following damage to peripheral nerves or spinal cord will be important for the development of new treatments for neuropathic pain, and this requires a greatly improved understanding of the normal organisation of pain pathways.

Work from our laboratory has already generated important insights into neuropathic pain, by demonstrating that several previously proposed mechanisms (e.g. dorsal sprouting of tactile afferents, death of inhibitory interneurons) are not required for the development of chronic pain after peripheral nerve injury, thus directing research away from these areas. This project will determine the anti-nociceptive role of NPY-expressing cells, which represent a large subset of dorsal horn inhibitory interneurons. If, as expected, activation of these cells reduces inflammatory and neuropathic pain, this will demonstrate that they are an important potential target for the development of new analgesics.

The Research Assistants working on the project will increase their skill sets by acquiring training in key techniques, including intraspinal injection of AAVs to manipulate genetically-defined neuronal populations, neuropathic/inflammatory/pruritic models and the associated behavioural testing, optogenetics and DREADD ("designer receptors exclusively activated by designer drugs") technology. Training in in vivo experimental approaches has been identified as a priority for the pharmaceutical industry.

This proposal is in line with BBSRC policy, since "mechanisms underlying pain" is identified as an important area within the "Welfare of managed animals" priority.