Regulation of the properties of sensory nerves innervating mammalian bone and joints

In vertebrates, the primary afferent neurons are specialized to detect chemical, mechanical and thermal stimuli. Much of our knowledge about the function of these primary afferent sensory neurons has been obtained by studying the properties of specific neuronal sub-types innervating soft tissues such as skin and muscle. The general properties of sensory neurons have also been investigated using isolated neurons. Little is known about the properties of sensory nerves innervating bone and joints. We have shown that it is possible to identify the cell bodies of neurons innervating either joint tissues or bone by dye (e.g. True Blue) injection into the target tissue (Fernihough et al. 2004, 2005). Back labelled neurons can then be studied immunohistochemically, electrophysiologically and with functional readouts such as imaging changes in intracellular calcium levels in response to various stimuli. Where possible mice will be used in our study as this offers the possibility to use genetically modified animals to probe the function of various receptors and ion channels expressed by sensory neurons as well as molecules expressed by associated cells in the target organ. The phenotype of identified neurons will be studied using markers (e.g. antibodies) that define sub-specific known sub-types of sensory neurons (e.g. neurofilament protein, sensory neuropeptides and neurotrophin receptors). The expression of specific receptors and ion channels, known or postulated to be involved in sensory transduction, will also be studied using immunolabelling and in situ hydridization of back-labelled neurons in sections of lumbar DRG. The types of receptors and ion channels studied will include those that define nociceptive neurons (e.g. TRPV1, TRPA1, Nav1.8), which signal noxious stimuli. The expression of molecules implicated in mechanotransduction will be explored. The functional properties of enzymatically isolated, identified joint neurons will be studied electrophysiologically (whole cell recording) or using calcium imaging methods. One area of specific interest is the mechanical sensitivity of the neurons. Low and high threshhold mechanically evoked responses have been reported in sensory neurons and we will identify which types of mechanosensors are associated with joint sensory neurons. These combined studies will provide key information about the normal joint sensory neuron phenotype. Sensory neuron properties are influenced by the environment. Increased mechanical sensitivity is seen with joint inflammation. The effects of inflammatory mediators such as TNF-alpha on mechanotransduction are poorly understood. Neurons from animals in which the knee has been experimentally inflamed by intra-articular injection of Complete Freund's Adjuvant will be studied. This results in increased responses to mechanical stimulation of the joint. We will determine if the increased sensitivity is due to a hypersensitivity of the primary transduction process. Identified joint neurons from normal animals will also be exposed to inflammatory mediators and growth factors in vitro (acutely or chronically) to determine if mechanosensitivity is increased. If so we can investigate the molecular processes that underlie changes in sensitivity. The cellular assays will be complemented by behavioural studies that will be carried out in the research organization. Wyeth has expertise in behavioural models that measure responses to mechanical stimulation of the knee. We will exploit information about the types of neurons innervating the joint and bone and use ablation methods to remove sub-types of neurons. Tracers which selectively bind to sub-populations of sensory nerves will be conjugated to saporin (a ribotoxin) and administered into the knee joint. The behavioural consequence of the treatments will be studied and correlated with histological examination to determine the degree and selectivity of nerve ablation.