Nerve cells communicate along the length of their axons by means of action potentials, or transient reversals (depolarisations) of the voltage across the cell membrane. When a sensory stimulus impinges on the skin surface it must, in order to be detected, elicit action potentials in the sensory nerve, or neurone, innervating the body surface at the point of contact. If the stimulus is sustained then it will often elicit a train of action potentials whose frequency encodes the intensity of the stimulus, with higher frequencies signalling a more intense stimulus. Each action potential in a train is followed by a return of the membrane potential to its former negative level (a repolarisation), and in order to elicit the next action potential in the train the membrane voltage must be depolarised again to threshold. The processes which mediate the rate of this depolarisation between action potentials is thus a crucial determinant of the action potential frequency and therefore of the perceived intensity of the stimulus. One important determinant of this rate is the strength of the stimulus, but it is not the only one. In many sensory neurones repolarisation switches on an inward current which then aids the depolarisation to threshold of the next action potential. This current, the hyperpolarisation-activated inward current, or Ih, is the subject of this grant application. Ih is interesting because it can be enhanced by many mediators which promote a sensation of pain, and it therefore may be important in hyperalgesia, or the enhanced pain which follows injury, and in neuropathic pain, an anomalous pain state characterised by ongoing pain and hypersensitivity to even moderate tactile and thermal stimuli. Neuropathic pain is not well understood and is poorly treated by currently available drugs. The condition is often life-long and causes a substantial reduction in quality of life for those who suffer from it. Ih ion channels are made up from various combinations of four different subunits, HCN1-4. Preliminary evidence leading up to this application has shown that there is a segregation in expression of these subunits, with the fast HCN1 subunit expressed in large neurones sensing light touch, and the slower HCN2 in small neurones, most of which sense painful stimuli of various kinds. Why is this? A possible reason is that HCN2 is enhanced by various mediators known to enhance pain, while HCN1 is unaffected. Thus this segregation may provide at least a partial explanation for the increase in pain following injury. We aim to find out more about which subunits are expressed in which types of sensory neurones, and how their behaviour is modulated by inflammatory mediators. There is also evidence from work in other labs that Ih is involved in neuropathic pain, but which subunit is important here and how it enhances neuropathic pain is unknown. We will tackle these and other questions by the use of mice in which each HCN subunits has been genetically deleted (knocked out). We will use a range of techniques to study these mice and to compare them with their wild-type littermates. One major technique will be to record the electrical responses from neurones, both in cell culture, where their behaviour can more readily be investigated, and in an isolated preparation of neurones in situ in skin, which has the advantage that the neurones are in their natural environment. In addition, we will study the behaviour of wild-type and HCN knockout mice in response to a mild painful stimulus, from which they are free with withdraw when it begins to hurt. These studies will advance our understanding of the role of HCN subunits in pain, and if particular subunits have crucial roles in some aspects of pain (e.g. in neuropathic pain) the work will act as a stimulus to the development of novel drugs aimed at specifically blocking those subunits.

Technical Abstract:
The hyperpolarization-activated membrane current, Ih, is an inward current which is activated by the membrane hyperpolarization following an action potential. This current plays an important role in many excitable tissues in bringing the membrane potential up to the level at which the following action potential is initiated, and is therefore an important determinant of action potential frequency. Ih ion channels are composed of Hyperpolarisation activated Cyclic Nucleotide gated (HCN) subunits 1-4. Ih is known to be present in primary sensory neurons but its importance is not clear. Ih seems to be a significant player in neuropathic pain but it is not clear which isoform is implicated. Our own preliminary studies show that HCN1 is expressed in large non-nociceptive neurons, while a slower cAMP-sensitive Ih, probably mediated by HCN2 and/or HCN4, is present in small nociceptive neurons. An underlying assumption of the proposed study is that this segregation will prove to play an important role in non-noxious somatosensation, which is mediated by large neurons, and in pain sensation, which is mainly mediated by small neurons, but with an important role for input from large neurons in the allodynia associated with neuropathic pain. The proposed study will determine the isoforms underlying Ih in various subclasses of sensory neurons. A second major objective will be to elucidate the role of Ih, and of the HCN isoforms which underlie it, in somatic sensation and in both inflammatory and neuropathic pain. We will tackle the project by implementing a broad range of techniques, from single-cell electrophysiology to whole-animal behavioural studies, and by correlating the results obtained by different techniques throughout the project. We will use global and nociceptor-specific HCN isoform mouse knockout models to elucidate the roles played by each HCN isoform, both at the single-cell level and in the pain behaviour of the whole animal.