No abstract

Dramatically up-regulated in the dorsal horn of the mammalian spinal cord following inflammation or nerve injury, neuropeptide Y (NPY) is poised to regulate the transmission of sensory signals. We found that doxycycline-induced conditional in vivo (Npy tet/tet ) knockdown of NPY produced rapid, reversible, and repeatable increases in the intensity and duration of tactile and thermal hypersensitivity. Remarkably, when allowed to resolve for several weeks, behavioral hypersensitivity could be dramatically reinstated with NPY knockdown or intrathecal administration of Y1 or Y2 receptor antagonists. In addition, Y2 antagonism increased dorsal horn expression of Fos and phosphorylated form of extracellular signal-related kinase. Taken together, these data establish spinal NPY receptor systems as an endogenous braking mechanism that exerts a tonic, long-lasting, broad-spectrum inhibitory control of spinal nociceptive transmission, thus impeding the transition from acute to chronic pain. NPY and its receptors appear to be part of a mechanism whereby mammals naturally recover from the hyperalgesia associated with inflammation or nerve injury.N europeptide Y (NPY) is a 36-aa peptide that acts at Gprotein-coupled Y receptors to initiate cellular signaling. NPY systems modulate a variety of physiological processes, including somatosensation (1). Expressed in neurons and terminals of the spinal cord dorsal horn (2), and up-regulated by peripheral inflammation or nerve injury, NPY is poised to inhibit the spinal transmission of sensory signals. For example, nerve injury elicits a profound de novo synthesis of NPY in large-diameter neurons of dorsal root ganglion (DRG) and their terminal regions in the dorsal horn (3-7), perhaps serving as an adaptive compensatory response to increased excitatory signaling (8). Increases in NPY last at least 24 wk, indicating that it is temporally available to confer long-lasting inhibition of pronociceptive neurotransmission (9), but this hypothesis has not been tested.Of the five NPY receptors cloned in the mouse, only Y1 and Y2 are expressed in the adult DRG (they colocalize with peptides that are predominantly found in unmyelinated to thinly myelinated neurons) and dorsal horn (10-12). Y1-ir is found in cells and terminals of lamina II i , whereas Y2-ir is found in terminals of I-II o (13-15). Numerous studies using an intrathecal delivery approach have demonstrated that exogenous delivery of NPY reduces hypersensitivity to tactile and thermal stimulation in models of chronic pain, including the spared nerve injury (SNI) model of neuropathic pain; Y1 and Y2 receptor antagonists reverse these antiallodynic actions (16)(17)(18)(19)(20). However, the use of receptor-selective antagonists to evaluate the intrinsic actions of NPY is uncommon, perhaps because behavioral thresholds demonstrate a floor effect in major animal models. For example, although the Y1 receptor antagonist BIBO3304 slightly but significantly increased heat hyperalgesia when administered during the peak of complete Freund'...

In mammals, cilia are critical for development, sensation, cell signaling, sperm motility, and fluid movement. Defects in cilia are causes of several congenital syndromes, providing additional reasons to identify cilia-related genes. We hypothesized that mRNAs selectively abundant in tissues rich in highly ciliated cells encode cilia proteins. Selective abundance in olfactory epithelium, testes, vomeronasal organ, trachea, and lung proved to be an expression pattern uniquely effective in identifying documented cilia-related genes. Known and suspected cilia-related genes were statistically overrepresented among the 99 genes identified, but the majority encoded proteins of unknown function, thereby predicting new cilia-related proteins. Evidence of expression in a highly ciliated cell, the olfactory sensory neuron, exists for 73 of the genes. In situ hybridization for 17 mRNAs confirmed expression of all 17 in olfactory sensory neurons. Most were also detected in vomeronasal sensory neurons and in neighboring tissues rich in ciliated cells such as respiratory epithelium. Immunoreactivity for one of the proteins identified, Spa17, colocalized with acetylated tubulin in the cilia layer of the olfactory epithelium. In contrast, the ciliary rootlet protein, Crocc, was located in discrete structures whose position was consistent with the dendritic knobs of the olfactory sensory neurons. A compilation of >2,000 mouse genes predicted to encode cilia-related proteins revealed a strong correlation (R = 0.99) between the number of studies predicting a gene's involvement in cilia and documented evidence of such involvement, a fact that simplifies the selection of genes for further study of the physiology of cilia.

Olfactory epithelial cells from olfactory marker protein-green fluorescent protein (OMP-GFP) mice were separated by fluorescence-activated cell sorting into a GFP+ sample enriched in mature olfactory sensory neurons (OSNs) and a GFP- sample enriched in all other cells. GeneChip expression profiling of these samples provided a predictive measure of expression in OSNs. Validation tests comparing the ratio of GFP+/GFP- signal intensity against expression patterns from in situ hybridization for 189 mRNAs proved statistically significant and provided probabilities of expression in OSNs scaled according to the signal intensity ratios. These probabilities predict that, among 11,596 mRNAs detected in the GFP+ sample, more than 10,000 are expressed in OSNs. Transcripts and overrepresented categories of mRNAs detected in the GFP+ sample agreed with known properties of OSNs and predict additional properties. For example, ciliogenesis and spermatogenesis were overrepresented, consistent with similarities between OSN cilia and sperm flagella. Chromatin assembly mRNAs were expressed throughout the OSN cell lineage, consistent with the hypothesis that chromatin remodeling plays a role in OSN differentiation. We detected numerous signaling proteins and receptors, such as 30 nonchemosensory G-protein-coupled receptors, including the presynaptic glutamate receptor mGlur4 and the Wnt receptor Fzd3. The largest group of mRNAs, however, was the hundreds of transcriptional regulators that presumably determine the OSN phenotype. The absence of OMP protein in OMP-GFP mice had no detectable effect on mRNA abundance. Within limits prescribed by the nature of microarray data and the in situ hybridization validation, these data should be useful in directing further experiments on OSN function.

In comparing purified mouse olfactory sensory neurons (OSNs) with neighboring cells, we identified 54 differentially expressed transcripts. One-third of the transcripts encode proteins with no known function, but the others have functions that correlate with challenges faced by OSNs. The OSNs expressed a diversity of signaling protein genes, including stomatin (Epb7.2), S100A5, Ddit3, Sirt2, CD81, Sdc2, Omp, and Ptpla. The elaboration of dendrites, cilia, and axons that places OSNs in contact with diverse cell types and signals presumably also requires large investments in cytoskeletal-associated proteins, lipid biosynthesis, and energy production. Several of the genes encode proteins that participate in these biological processes, including ATP5g3, Ndufa9, Sqrdl, Mdh1, Got1, beta-2 tubulin, Capza1, Bin3, Tom1, Acl6, and similar to O-MACS. Three transcripts had restricted expression patterns. Similar to O-MACS and Gstm2 had zonally restricted expression patterns in OSNs and sustentacular cells but not in Bowman's glands, suggesting that zonality can be differentially regulated by cell type. The mosaic expression pattern of S100A5 in approximately 70% of OSNs predicts that it is coexpressed with a subset of odorant receptors. We captured four abundant transcripts, Cyp2a4, similar to Cyp2g1, Gstm2, and Cbr2, that encode xenobiotic metabolizing enzymes expressed by sustentacular cells or Bowman's glands, reinforcing the interpretation that clearance of xenobiotic compounds is a major function of these cells. Within the olfactory epithelium, Cbr2 is a new anatomical marker for sustentacular cells. We also discovered that Reg3g is a marker for respiratory epithelium.