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

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.

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.