Immediate-early gene expression and intracellular signalling in the trigeminal system

Lead Research Organisation: University of Sheffield
Department Name: Clinical Dentistry

Abstract

Within the nervous system, communication between neurones requires transmission of messages across synapses. Anatomically, synapses consists of the membranes of two neurones which are separated by a microscopic fluid-filled space. The mechanism which allows communication across this space is called synaptic transmission. This process consists of release of a chemical from one neurone (the presynaptic neurone) which crosses the space where it activates very specific receptors on a second neurone (the postsynaptic neurone). This may produce an excitatory effect in the second neurone increasing its activity, or it may inhibit its activity. The balance between these excitatory and inhibitory inputs determines whether or not the message is successfully transmitted across the synapse. This complex process is not static and the sensitivity of synapses can be altered by a wide range of mechanisms. Changes in the sensitivity of a synaptic transmission are known as synaptic plasticity. One form of synaptic plasticity is known as use-dependent synaptic plasticity. This occurs as a consequence of prolonged activity in the presynaptic neurone and it leads to increased sensitivity of the synapse, such that incoming messages are more likely to be relayed to the postsynaptic neurone. This process is fundamental to many neurological functions, including memory and pain. This study will examine the mechanisms which lead to synaptic plasticity following the type of stimulation of sensory nerves that would cause pain, such as repetition of a painful stimulus, a nerve injury or tissue injury and inflammation. Under these conditions, synapses become hyperexcitable, and this may lead to spontaneous pain, exaggerated pain, or pain in response to stimuli that would not normally cause pain. The mechanisms causing this are known to be complex and diverse, but they involve increased activity in chemical pathways inside the neuronal cells that initiate change in these cells. These changes are believed to be significant in the development of chronic pain. The aim of this study is to identify signalling molecules within neuronal cells that are important in increasing excitability under conditions of inflammation. We will also identify which specific neuronal receptors are involved in this process. The results from this work will improve our understanding of the mechanisms leading to the development of chronic pain. Information from this type of study is vital in developing new pain-relieving drugs for conditions that currently have no reliable treatment, and many of the therapies that are available have unacceptable or life-threatening side effects.

Technical Summary

The overall aim of this project is to investigate use-dependent synaptic plasticity that occurs as a consequence of prolonged neuronal activation and is fundamental to many neurological functions, including memory and pain. This proposal focuses on stimulation-transcription coupling and the signalling cascades that are activated following prolonged input from nociceptive afferents, eg repetition of acute peripheral stimuli or peripheral nerve injury and inflammation. We will examine these mechanisms using Fos, pERK, pCREB and p38 as molecular markers of neuronal activation and plasticity. The proposal will focus on mechanisms of central sensitisation within the trigeminal nucleus. This nucleus is situated within the brainstem and receives input from the trigeminal nerve, a cranial nerve supplying the oro-facial region. We have developed an in-vivo model and a trigeminal slice preparation to investigate mechanisms of Fos expression and central sensitisation in the trigeminal nucleus. These models enable us to monitor activation of trigeminal brainstem neurones following stimulation of normal and inflamed peripheral tissues (in-vivo model) or following the application of agonists to specific receptor subtypes (slice preparation). Data from our models and other studies indicate that whilst mechanistic parallels can be drawn, the trigeminal system displays some distinctive elements that require further elucidation. In the last decade studies of spinal cord systems have identified a number of intracellular signal transduction cascades that play a role in long-term changes in dorsal horn neurones. However, to date very little is known about the role of these cascades in central sensitisation in the trigeminal system. Our in-vivo model provides an excellent opportunity to identify central changes upstream to Fos expression that may play a role in central sensitisation within this system. In addition, it provides a means for determining the ability of compounds to reduce neuronal activation and we have shown that this correlates with analgesic efficacy. Our slice preparation allows us to directly link activation of ERK, p38, CREB and Fos in the trigeminal nucleus with specific glutamatergic or peptidergic receptor classes previously implicated in sensitisation phenomena. The proposed work involves 3 different studies. The main objective of Study 1 is to investigate a role for MAP kinase cascade activation in sensitisation induced by tooth pulp inflammation, and establish whether activation occurs in neurones and/or glia. Using our in-vivo model and immunohistochemical techniques we will examine the levels of pERK, P-p38, pCREB and Fos in the trigeminal nucleus under 4 conditions: 1) control; 2) following stimulation of normal pulp; 3) following induction of pulpal inflammation; 4) following stimulation of the inflamed pulp. The main objective of Study 2 is to determine the role of pERK and P-p38 in Fos expression and determine potential analgesic efficacy of MAP kinase inhibitors in inflammatory trigeminal pain. We will use our in-vivo model and immunohistochemistry to examine the effect of inhibitors of ERK and p38 on Fos and pCREB expression following stimulation of normal and inflamed pulp. The main objective of Study 3 is to identify which specific classes of endogenous receptors are responsible for activation of MAP kinase-dependent cascades and expression of pERK, pCREB and Fos in neurons and P-p38 in glia. We will exploit the advantages of an in-vitro trigeminal slice preparation that we have developed. Under conditions of TTX block, indirect neuronal activation is eliminated and therefore expression of intracellular molecular markers can be associated directly with identified glutamatergic or peptidergic receptor classes. Protocols that utilise selective receptor agonists and kinase inhibitors/activators will be applied to trigeminal slices obtained from control groups and following induction of pulpal inflammation.

Publications

10 25 50
 
Description This study provides the first demonstration that chronic inflammation of the tooth pulp induces significant and persistent bilateral activation of pERK and pp38 within the trigeminal nucleus, and that expression of these molecules is further increased by acute stimulation. This altered activity in intracellular signalling is likely to be linked to the increased Fos expression that is seen in the inflamed stimulated animals in this and our previous studies, and is likely to be relevant to the increased sensitivity that is seen in patients with pulpitis. The data from this study also indicate that pERK and pp38 are more accurate markers of central change than Fos expression. In this model of chronic inflammation, colocalisation of pERK and pp38 within specific cell types differs from that reported following the application of acute stimulation. These findings provide further evidence for sequential activation of these molecules in different cell types in models of chronic pain, and may indicate specific roles for different cell types in induction and maintenance of pulpitic and other types of pain.
Exploitation Route They provide further understanding of the mechanisms underlying the development of chronic pain.
Sectors Healthcare

 
Description BBSRC Sparking Impact Award
Amount £7,500 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start  
 
Description BBSRC Sparking Impact Award
Amount £8,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start  
 
Description BBSRC White Rose DTP
Amount £91,452 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description EU Marie Curie Incoming International Fellowship
Amount € 310,000 (EUR)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start  
 
Description MRC Centenary Award
Amount £15,800 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start  
 
Description MRC Confidence in Concept Award
Amount £50,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start  
 
Description MRC Industrial CASE studentship
Amount £50,000 (GBP)
Funding ID G0800133/3-1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2011 
End 09/2014
 
Description University of Sheffield Faculty/Pfizer PhD Studentship
Amount £60,000 (GBP)
Organisation University of Sheffield 
Department School of Clinical Dentistry Sheffield
Sector Academic/University
Country United Kingdom
Start  
 
Description University of Sheffield Festival of the Mind
Amount £5,000 (GBP)
Organisation University of Sheffield 
Sector Academic/University
Country United Kingdom
Start  
 
Description Festival of Medicine, Dentistry and Health 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Interactive activity exploring nerve function stimulated Y6 children's' interest

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Year(s) Of Engagement Activity 2014
 
Description Festival of the Mind 2012 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Talk sparked questions and discussion afterwards

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Year(s) Of Engagement Activity 2012
 
Description Festival of the Mind 2014 - You've Got a Nerve! (Art and Animation Installation) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Art installation ignited interest in nerve injury and repair

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Year(s) Of Engagement Activity 2014
 
Description National Science and Engineering Week - Researchers' Night 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Raised public interest and awareness

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Year(s) Of Engagement Activity 2013,2014
 
Description Researcher's Night. For one night, hundreds of universities across Europe open their doors to the public. Sheffield is one of handful of UK universities taking part. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The University of Sheffield opened its doors to allow visitors to explore the latest research at the University, through talks, exciting demonstrations and hands on activities. Aimed at secondary school students, sixth formers and adults, Researchers' Night enabled visitors to interact with a vast array of research projects from across the University.

The activities related to this research award were:

Eyes, Ears, Mouth, Nose and Fingers A neuroscientist's understanding of the senses.
Our senses are bombarding us with information all the time, observing, listening, smelling and even tasting the world around us, on the look out for danger, sending a powerful 'ouch' response when we encounter something harmful or dangerous. Come find out more about how these amazing senses work, how your brain perceives the heat of a chilli pepper, and how sensitive are you to different stimuli?

Not Just Teeth: Research at Sheffield Dental School.
Researchers from Sheffield Dental School will present an interactive set of exhibits highlighting ground-breaking research and targeted at dispelling any myths you may have that it is 'all about teeth'. Come along and find out about our work on bacteria, nerves, oral cancer, patient experience and tissue engineering, oh, and teeth.
Year(s) Of Engagement Activity 2016
URL http://www.sheffield.ac.uk/researchersnight/programme
 
Description The Mobile University 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Presentation sparked questions and discussion

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Year(s) Of Engagement Activity 2013
 
Description University of Sheffield's Festival of the Mind - A showcase for the University's world-leading research. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The festival, which pairs University academics with experts from Sheffield's cultural and creative industries, attracted 50,000 visitors to its performances, talks, exhibitions, virtual reality experiences and interactive events designed to educate, inform and create debate.

Professor Vanessa Toulmin, Director of City and Cultural Engagement at the University of Sheffield, said: "Festival of the Mind 2016 has captured the spirit of invention and collaboration which makes Sheffield such a great place to live.

"We have had overwhelmingly positive feedback to this year's festival with some wonderful comments from visitors who have been inspired by our world-leading research.

"Ninety-two per cent of visitors rated the festival as excellent and sixty-eight per cent said it had changed their perception of our research in a positive way."

During the festival, footfall in the city centre increased by 23 per cent compared to the same period the previous year. The Moor Market and Winter Garden areas saw an increase of 51 per cent.

Vanessa continued: "Festival of the Mind is an example of what incredible things can be achieved when you bring together some of the world's leading researchers and the best creative minds in the industry.

The exhibit related to this research award consisted of a short film called "13 Teeth", a gothic thriller about a man who thinks the voices in his head are coming from his teeth. His quest to extract them (eek!) leads to a shocking revelation. Based on Gwynne Jones's narrative poem, the film mixes live action and animation to brilliantly unsettling effect. It asks the big question: what is behind consciousness?
In the accompanying documentary, The Making of 13 Teeth, the academics involved in the project (Boissonade, Bird, Birkhead) talk us through some of the film's most striking images, including skeletons, nerves and teeth; and their research related to these.
Year(s) Of Engagement Activity 2016
URL https://www.sheffield.ac.uk/news/nr/university-of-sheffield-festival-of-the-mind-inspires-the-city-1...