Populations of inhibitory interneurons in the dorsal horn of the spinal cord

Nerve fibres that enter the spinal cord carry various types of sensory information. Some of these fibres (nociceptors) respond selectively to tissue damaging stimuli. The signals conveyed by nociceptors are transmitted to local nerve circuits in the spinal cord that are responsible for withdrawal reflexes, and also to a group of nerve cells (projection neurons) that carry the information to the brain, where it is perceived as pain. The great majority of nerve cells in the spinal cord are only involved in local circuits, and these are defined as interneurons. Many of these cells release chemical messengers that reduce the activity of other nerve cells, and therefore have an inhibitory function. There are complex nerve circuits within the spinal cord that connect incoming sensory fibres with interneurons and projection neurons. These circuits play an important part in modulating the flow of sensory information and regulating the intensity of pain. For example it has been shown that blocking the function of inhibitory interneurons in the spinal cord causes excessive pain, and disorders affecting these cells can lead to chronic pain states. However, despite their great importance, we still know little about the organisation of the nerve circuits that process pain signals within the spinal cord. A major reason for this has been that spinal cord interneurons are very diverse in their appearance, and it has therefore been difficult to classify them into distinct functional populations. Until we can do this, it will not be possible to unravel the complex connections between the different types of nerve cell, and therefore to understand their roles in pain processing. We have found that several groups of inhibitory interneurons in the spinal cord can be recognised on the basis of different substances that they contain. The main aim of this project is to test whether these groups differ in their connections with other nerve cells, and therefore in their function. We will perform experiments on genetically altered mice in which a naturally fluorescent protein is present in these different groups of inhibitory interneurons. This will allow us to record the activity of these cells and to label them with another fluorescent dye, so that the structure and connections of each cell can be investigated. We have preliminary evidence that cells belonging to one of these groups are not activated by painful stimuli, and we will therefore test whether cells in this group receive fewer connections from nociceptors. We will use a powerful technique known as 'cluster analysis' to look at a large sample of cells pooled from each of these groups. Cluster analysis compares a wide range of measures obtained for each cell, and uses these to provide an objective assessment of the number of different populations within the sample. This technique has been used to define different functional populations of interneurons in several brain regions, but has not yet been applied to pain pathways in the spinal cord. We have also found that cells belonging to two of these chemically-defined groups provide a powerful inhibitory input to two different types of pain-activated projection neuron. We will therefore test whether these cells are activated by nociceptors, and whether they correspond to populations identified by the cluster analysis. The project will provide important information about the different types of inhibitory interneuron that are involved in regulating pain. By revealing their connections with incoming sensory fibres and projection neurons, it will add a great deal to our knowledge of how the nerve circuits in the spinal cord are organised. Identifying the interneurons that directly inhibit projection neurons may reveal new targets for the development of drugs designed to treat pain.

GABAergic interneurons in the dorsal horn control nociceptive inputs to reflex pathways and to the projection neurons reponsible for pain perception. However, because of their morphological diversity and the resulting difficulty of identifying distinct functional populations among these cells, we still know little about their role in neuronal circuits. We have found that neuronal nitric oxide synthase (nNOS), galanin and neuropeptide Y (NPY) are present in non-overlapping groups that constitute over half of the GABAergic cells in laminae I-II in the rat. These groups also differ in their post-synaptic targets, and in their expression of activity-dependent markers following noxious stimulation. In this project we will perform patch-clamp recordings from GABAergic interneurons in each of these groups by using spinal cord slices from mice in which the cells contain green fluorescent protein. Recorded cells will be revealed with fluorescent dye to allow subsequent morphological and immunocytochemical analysis. We will test the hypothesis that NPY and galanin cells have a greater input from nociceptive primary afferents than nNOS cells. Cluster analysis has been used to define interneuron populations elsewhere in the CNS, and we will apply this to a wide range of anatomical and physiological parameters obtained from a large sample of cells. We will look for specific populations of GABAergic neurons and determine whether these map onto the neurochemical groups. Some NPY and nNOS cells innervate specific types of nociceptive projection neuron. We will test the hypothesis that these represent distinct subsets within the corresponding neurochemical groups as defined by cluster analysis, and will characterise them with respect to their morphology, laminar location and pattern of primary afferent input. The project will help to clarify the neuronal circuitry involving inhibitory interneurons and will identify those responsible for inhibiting nociceptive projection neurons.

Pain represents a major cause of suffering for both humans and animals. For example, it has been estimated that 20% of adults in Europe suffer from chronic pain of moderate to severe intensity, and this proportion is likely to increase as the population ages. Chronic pain is often poorly treated and the main reason for this is the lack of suitable medications. This in turn results from 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 also has a massive societal and economic impact, since a substantial proportion of sufferers are unable to work, while many report a direct effect on their employment prospects. Who will benefit from this research? Those who will benefit indirectly include scientists in the pharmaceutical industry who are involved in the development of analgesics, human patients and animals suffering from chronic pain, and the clinicians responsible for their treatment. Improved treatments for chronic pain will also be of enormous economic benefit to society. How will they benefit from this research? Development of new drugs to treat chronic pain will depend to a large extent on our understanding of the neuronal pathways that underlie pain perception, from the peripheral receptors through to the various cortical areas involved. The dorsal horn of the spinal cord contains intrinsic inhibitory circuits that can powerfully suppress pain, and therefore provides important potential sites of action for new analgesics. However, the organisation of these circuits is still poorly understood. Identifying and characterising the different components in dorsal horn pain pathways is likely to lead to the discovery of novel targets for analgesics. For example, certain ion channels and receptors are selectively expressed by subpopulations of neurons within the dorsal horn. Determining the neuronal types that express these channels/receptors will indicate whether activating or inhibiting them is likely to be anti-nociceptive (e.g. by increasing excitability of inhibitory interneurons, or by suppressing the activity of excitatory interneurons or projection cells). In addition, identifying changes in neuronal function and circuitry that occur in chronic pain states will be important for the development of new treatments, and this will require a greatly improved understanding of the normal organisation of pain pathways. Work from our laboratory has already generated important insights into the mechanisms underlying neuropathic pain following peripheral nerve injury, by demonstrating that several mechanisms that had been proposed (dorsal sprouting of low-threshold mechanoreceptive A-beta afferents, up-regulation of substance P in the central terminals of these afferents and death of inhibitory interneurons) are not necessary for the development of neuropathic pain. These results have shown that these proposed mechanisms are unlikely to provide useful targets for developing treatments for neuropathic pain. 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.