Spinal inhibitory interneurons that suppress itch

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Chronic itch is a distressing feature of many diseases, including conditions affecting the skin, kidneys and blood, as well as certain forms of cancer. It is also a side-effect of certain drugs, such as morphine. Although some types of itch respond to antihistamines, in many cases there are no satisfactory treatments available. At present we have very limited knowledge of the nerve circuits in the spinal cord and brain that transmit itch.

Pain can powerfully inhibit itch, which is why scratching produces temporary relief. This inhibition, which occurs in the spinal cord, involves a type of local nerve cell (interneuron) that is activated by painful stimuli and inhibits transmission of the itch signal to cells (known as projection neurons) that convey sensory information to the brain. A recently developed mutant mouse, which lacks a protein called bhlhb5, shows dramatically increased itching, resulting from loss of inhibitory interneurons in the spinal cord. Interestingly, these "bhlhb5 knockout" mice respond normally to most types of painful stimulus, which suggests that the missing interneurons selectively inhibit itch, but not pain. However, because the knockout mouse loses these cells before birth, there may have been compensatory changes, leading to relatively normal pain sensation. One of the main problems with understanding how sensory information is processed in the spinal cord is that there are many different populations of inhibitory interneurons, each with different functions. We have recently found that several populations can be recognised on the basis of the different substances that they contain. In preliminary studies, we have identified two populations that are largely absent from the Bhlhb5 knockout mouse, and it is likely that one or both of these is responsible for the inhibition of itch by painful stimuli.

In this project we will test whether one of these populations is responsible for the suppression of itch by pain, and attempt to identify the pathway that underlies this phenomenon. We will initially determine whether the loss of nerve cells is restricted to the two populations we have identified by staining spinal cords from normal and bhlhb5 knockout mice with markers to reveal these cells and all other types of inhibitory interneuron. If these interneurons are responsible for preventing itch, then blocking their activity in normal adult mice should increase itch. We will use two different approaches to test this. First, we will inject a chemical to destroy a relatively large number of inhibitory interneurons, including all of those that are absent in the knockout mice. We will then use a more selective genetic method to destroy or inactivate one of these populations. By examining the responses of these mice to stimuli that normally cause itch or pain, we will improve our understanding of the roles played by these different populations of inhibitory interneurons. We will then investigate the underlying nerve circuits by identifying projection neurons that are activated by itch-inducing stimuli in normal and Bhlhb5 knockout mice. We will test the prediction that one of the populations of inhibitory interneurons that is lost in the knockout mice acts directly on projection neurons that signal itch. Although most pain tests are normal in the knockout mouse, these animals do show an increased response following injection of formalin into the skin. We have identified a type of projection neuron that loses much of its normal inhibitory input in the bhlhb5 knockout mouse, and we will test whether these cells are involved in the abnormal response to formalin.

The project will provide valuable insight into the organisation of nerve pathways in the spinal cord that transmit and modify sensory information. This will improve our understanding of pathological itch and pain states, and should help towards the identification of targets for new treatments for these conditions.

Technical Summary

Itch, a distressing symptom of many diseases, is often difficult to treat. Noxious stimuli suppress itch through GABAergic and glycinergic inhibition in the spinal cord. A major breakthrough in our understanding of itch came from the Bhlhb5 knockout mouse, which shows exaggerated itch and sensitivity to formalin (but otherwise normal pain responses), resulting from loss of dorsal horn inhibitory interneurons. It is therefore important to identify these interneurons and the circuits through which they act. We have recently shown that four non-overlapping neurochemical populations of GABAergic neurons can be identified, and in pilot studies we find that two of these: cells expressing nNOS or galanin, are substantially depleted in the Bhlhb5 knockout.

Since nNOS (but not galanin) neurons are thought to use GABA and glycine as co-transmitters, we predict that these cells inhibit itch. We will test this hypothesis and investigate the underlying neuronal circuitry. We will first check whether loss of inhibitory interneurons in the knockout is restricted to nNOS and galanin cells. We will then use two approaches to kill or inactivate inhibitory interneuron populations in normal animals and test the effect on itch and pain. We will target either sst2A-expressing inhibitory interneurons (which include all nNOS and galanin cells) or only the nNOS cells. We will then test whether the inhibition acts directly on projection neurons by looking for loss of inhibitory synapses on itch-activated cells in knockout mice. Finally, we will test the prediction that giant cells in lamina I (which lose inhibitory synapses in the knockout) are involved in the increased response to formalin.

The project will provide important information about the organisation of somatosensory pathways in spinal cord. This will improve our understanding of pathological itch and pain, and should assist in the identification of targets for new antipruritic and analgesic drugs.

Planned Impact

Itch is a symptom of many diseases and can occur in otherwise healthy individuals. Chronic pruritus (itching that lasts more than 6 weeks) is very common, for example a recent study reported a point prevalence of 16% in a large sample of the working age population in Germany, with a quarter of those affected having suffered from itching that had lasted for more than 5 years. Chronic pruritus can also have a detrimental effect on sleep, leading to reduced ability to work. Because of the lack of adequate treatment options for many types of itch, it represents a major unmet clinical need. Until recently there has been relatively little research on the CNS mechanisms that are responsible for the perception of itch. Further insight into the organisation of inhibitory interneurons in the dorsal horn will also improve our understanding of pain mechanisms. 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 also often poorly treated, due to the lack of suitable medications.

Who will benefit from this research?
Those who will benefit directly include scientists from other disciplines (e.g. pharmacology, molecular genetics, developmental biology) who are working on the somatosensory system, as well as the pharmaceutical industry, in particular in relation to the development of new antipruritic and analgesic drugs. In the longer term, beneficiaries include human patients suffering from chronic pruritus and pain, and the clinicians responsible for their treatment. This work should therefore have an impact on health and well-being. Improved treatments for pain and itch would be of considerable economic benefit to society due to the reduction in time lost from work.

How will they benefit from this research?
Improved understanding of the roles of different interneuron populations in neuronal circuits that regulate somatosensory transmission through the dorsal horn will facilitate advances in other disciplines, for example by providing a framework for defining the location and function of receptors, ion channels and transcription factors within these circuits.
This understanding will also be important for pharmaceutical industry, for the development of new drugs. The dorsal horn of the spinal cord contains a complex set of inhibitory interneuron populations that can powerfully and selectively suppress itch and pain, as well as a large array of molecules that are expressed by specific populations of neurons. Identifying the inhibitory interneuron populations involved in these different somatosensory pathways will allow us to determine which cells can be targetted in the development of new antipruritic or analgesic drugs. The project will provide training in in vivo experimental approaches, which have been identified as a priority for the pharmaceutical industry.
In addition, greater knowledge of dorsal horn neuronal circuits is important for our understanding of the changes that can occur in chronic pain states. 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 in the dorsal horn) are not necessary for the development of chronic pain after peripheral nerve injury, thus directing research away from these areas.
The named researcher on this project will increase her skill set, by acquiring novel techniques that can be used to silence/ablate specific neuronal populations and investigate their function. This can subsequently be applied to different neurochemically-identified classes, thus opening up the possibility of determining the roles of other neuronal populations in sensory processing.

Publications

10 25 50

 
Description Contribution to "Masterclass" on Spinal Cord Stimulation (Saluda Medical)
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Neuronal circuits for itch in the spinal dorsal horn
Amount £790,021 (GBP)
Funding ID MR/S002987/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2019 
End 12/2021
 
Description The role of NPY-containing inhibitory interneurons in spinal pain pathways
Amount £680,496 (GBP)
Funding ID BB/N006119/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2016 
End 03/2019
 
Description Wellcome Trust Strategic Award
Amount £5,287,909 (GBP)
Funding ID 102645 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2014 
End 02/2019
 
Title Refinement of intraspinal injection technique to deliver viral vectors to genetically modified mice 
Description Intraspinal injection of viral vectors coding for recombinase-dependent constructs is important for assessing the function of different populations of spinal neurons, defined by their adult expression of specific genes. We developed a technique for making intraspinal injections that target mid-lumbar spinal segments (relevant for behavioural testing of the hindpaw) that avoids the need for laminectomy (which can result in swelling and distortion of the spinal cord). The method involves injecting on either side of the T13 vertebra, and through a small hole drilled in the lamina of this vertebra. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? Yes  
Impact We have used this method to target several different populations of inhibitory and excitatory interneurons 
 
Description Collaboration with Dr Mark Hoon 
Organisation National Institutes of Health (NIH)
Country United States 
Sector Public 
PI Contribution Research findings contributing to a paper in Nature Neuroscience (DOI: 10.1038/s41593-018-0119-z ).
Collaborator Contribution Research findings contributing to a paper in Nature Neuroscience (DOI: 10.1038/s41593-018-0119-z ).
Impact Huang et al (2018) Nature Neuroscience 21:707-16 (DOI: 10.1038/s41593-018-0119-z ). Collaboration is multi-disciplinary: anatomy, behaviour, pharmacology
Start Year 2016
 
Description Collaboration with laboratory of Prof Uli Zeilhofer 
Organisation University of Zurich
Country Switzerland 
Sector Academic/University 
PI Contribution Research findings contributing to a paper in Nature Neuroscience (DOI: 10.1038/s41593-018-0119-z )
Collaborator Contribution Training in intraspinal injection technique provided to Research Assistant funded on this grant
Impact Huang et al (2018) Nature Neuroscience 21:707-16 (DOI: 10.1038/s41593-018-0119-z ). Collaboration is multi-disciplinary: anatomy, behaviour, pharmacology
Start Year 2014
 
Description Nuffield Research Placement Scotland 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact School pupil hosted for summer project placement under the Nuffield Research Placements Scotland scheme on several occasions
Year(s) Of Engagement Activity 2013,2014,2015,2017,2018,2019
URL http://www.nuffieldresearchplacements.org/