Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Journal of Neuroimmunology

Capsaicin-mediated depletion of neuropeptides in the skin was previously shown to abolish a dinitrocholorobenzene (DNCB)-induced contact sensitivity (CS) response. To understand the basis for this disruption, we explored whether nerve fibres innervating the draining lymph node (LN) could be involved. As expected, removal of the draining LN after DNCB sensitisation abolished the CS response. Furthermore, the CS response could be abolished by destroying the nerve fibres in the draining LN and could be restored by providing the LN with the neuropeptide substance P. The size of the CS response restored by substance P was dose dependent. The response was also inhibited by exposing the lymph node to a neurokinin-1 receptor antagonist which blocks binding of substance P. The results suggest that an afferent signal from the skin via the sympathetic arm of the central nervous system evokes an efferent signal to the LN which combines to regulate the CS response. The efferent signal may serve to control or release from the LN primed effector lymphocytes into the circulation.

Immunology

Interest in the interactions between nervous and immune systems involved in both pathological and homeostatic mechanisms of host defence has prompted studies of neuroendocrine immune modulation and cytokine involvement in neuropathologies. In this review we concentrate on a distinct area of homeostatic control of both normal and abnormal host defence activity involving the network of peripheral c-fibre nerve fibres. These nerve fibres have long been recognized by dermatologists and gastroenterologists as key players in abnormal inflammatory processes, such as dermatitis and eczema. However, the involvement of nerves can all too easily be regarded as that of isolated elements in a local phenomenon. On the contrary, it is becoming increasingly clear that neural monitoring of host defence activities takes place, and that involvement of central/spinal mechanisms are crucial in the co-ordination of the adaptive response to host challenge. We describe studies demonstrating neural control of host defence and use the specific examples of bone marrow haemopoiesis and contact sensitivity to highlight the role of direct nerve fibre connections in these activities. We propose a host monitoring system that requires interaction between specialized immune cells and nerve fibres distributed throughout the body and that gives rise to both neural and immune memories of prior challenge. While immunological mechanisms alone may be sufficient for local responsiveness to subsequent challenge, data are discussed that implicate the neural memory in co-ordination of host defence across the body, at distinct sites not served by the same nerve fibres, consistent with central nervous mediation.

The Journal of Biological Chemistry

γ-Aminobutyric acid (GABA) release from inhibitory interneurons located within the cerebellar cortex limits the extent of neuronal excitation in part through activation of metabotropic GABA(B) receptors. Stimulation of these receptors triggers a number of downstream signaling events, including activation of GIRK channels by the Gβγ dimer resulting in membrane hyperpolarization and inhibition of neurotransmitter release from presynaptic sites. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key regulator of GABA(B)R signaling in cerebellum. RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners Gβ(5) and R7BP. Mice lacking RGS6 exhibit abnormal gait and ataxia characterized by impaired rotarod performance improved by treatment with a GABA(B)R antagonist. RGS6(-/-) mice administered baclofen also showed exaggerated motor coordination deficits compared with their wild-type counterparts. Isolated cerebellar neurons natively expressed RGS6, GABA(B)R, and GIRK channel subunits, and cerebellar granule neurons from RGS6(-/-) mice showed a significant delay in the deactivation kinetics of baclofen-induced GIRK channel currents. These results establish RGS6 as a key component of GABA(B)R signaling and represent the first demonstration of an essential role for modulatory actions of RGS proteins in adult cerebellum. Dysregulation of RGS6 expression in human patients could potentially contribute to loss of motor coordination and, thus, pharmacological manipulation of RGS6 levels might represent a viable means to treat patients with ataxias of cerebellar origin.