Inflammation-induced GluA1 trafficking and membrane insertion of Ca2+ permeable AMPA receptors in dorsal horn neurons is dependent on spinal tumor necrosis factor, PI3 kinase and protein kinase A.

Peripheral inflammation induces sensitization of nociceptive spinal cord neurons. Both spinal tumor necrosis factor (TNF) and neuronal membrane insertion of Ca2+ permeable AMPA receptor (AMPAr) contribute to spinal sensitization and resultant pain behavior, molecular mechanisms connecting these two events have not been studied in detail. Intrathecal (i.t.) injection of TNF-blockers attenuated paw carrageenan-induced mechanical and thermal hypersensitivity. Levels of GluA1 and GluA4 from dorsal spinal membrane fractions increased in carrageenan-injected rats compared to controls. In the same tissue, GluA2 levels were not altered. Inflammation-induced increases in membrane GluA1 were prevented by i.t. pre-treatment with antagonists to TNF, PI3K, PKA and NMDA. Interestingly, administration of TNF or PI3K inhibitors followed by carrageenan caused a marked reduction in plasma membrane GluA2 levels, despite the fact that membrane GluA2 levels were stable following inhibitor administration in the absence of carrageenan. TNF pre-incubation induced increased numbers of Co2+ labeled dorsal horn neurons, indicating more neurons with Ca2+ permeable AMPAr. In parallel to Western blot results, this increase was blocked by antagonism of PI3K and PKA. In addition, spinal slices from GluA1 transgenic mice, which had a single alanine replacement at GluA1 ser 845 or ser 831 that prevented phosphorylation, were resistant to TNF-induced increases in Co2+ labeling. However, behavioral responses following intraplantar carrageenan and formalin in the mutant mice were no different from littermate controls, suggesting a more complex regulation of nociception. Co-localization of GluA1, GluA2 and GluA4 with synaptophysin on identified spinoparabrachial neurons and their relative ratios were used to assess inflammation-induced trafficking of AMPAr to synapses. Inflammation induced an increase in synaptic GluA1, but not GluA2. Although total GluA4 also increased with inflammation, co-localization of GluA4 with synaptophysin, fell short of significance. Taken together these data suggest that peripheral inflammation induces a PI3K and PKA dependent TNFR1 activated pathway that culminates with trafficking of calcium permeable AMPAr into synapses of nociceptive dorsal horn projection neurons.

Improved resolution of fibronectin mRNA expression in the inner ear using laser scanning confocal microscopy.

We describe a modified in situ hybridization protocol for localizing and quantifying fibronectin gene expression at the cellular level in paraffin sections of rat temporal bone. When combined with a novel analytical approach using laser scanning confocal microscopy (LSCM), this protocol significantly improved the resolution, sensitivity, and specificity of existing procedures for evaluating fibronectin synthesis in developing inner ear. For simultaneous viewing of cochlear anatomy and the autoradiographic signal, transmitted light images of the cochlea were collected separately from LSCM reflected light images of the autoradiographic silver grains and then the two images were electronically merged. Within the first 2 microns below the surface of the emulsion, silver grains were clustered specifically over hybridized cells. In contrast, nonspecific silver grain development (i.e., background noise) was confined primarily to the lower 5 microns of the emulsion adjacent to the tissue section. Limiting the volume of the emulsion examined in the LSCM analysis, i.e., restricting the range of optical sectioning to the first 2 micron below the surface of the emulsion, effectively minimized nonspecific background noise and maximized the specificity of the hybridization signal. The improvements offered by the described methodological approaches are equally appropriate for non-calcified tissues.

Immunohistochemical localization of fibronectin-like protein in the inner ear of the developing gerbil and rat.

Immunohistochemistry was used to demonstrate the distribution of fibronectin-like protein within the developing inner ear of two species of altricial rodents: gerbils and rats. While there were temporal differences between the two species, the developmental sequence of immunostaining was virtually identical. Most notably, in rats from embryonic day 18 through day 1 postpartum, and in gerbils from birth through day 4 postpartum, intense, discrete fibronectin-like immunoreactivity was observed in the cochlea immediately beneath the inner and outer hair cells, sites of active auditory nerve fiber growth and nerve-hair cell synaptogenesis at these ages. The results suggest that fibronectin is appropriately positioned spatially and temporally to play a significant role in promoting, guiding and/or maintaining neural innervation within the developing organ of Corti. The temporo-spatial pattern of immunostaining in Schwann cells and auditory (VIIIth cranial) nerve neurons implies that fibronectin also plays a significant role in the early formation of myelin. In non-neural elements of the cochlea, fibronectin is a major structural component within the basilar membrane at all of the developmental stages investigated.

Congenital cytomegalovirus labyrinthitis and sensorineural hearing loss in guinea pigs.

Cytomegalovirus (CMV) is the leading cause of human nonhereditary congenital deafness. The pathogenesis of congenital CMV infection in the auditory system is poorly understood and no suitable animal model is currently recognized. In this study primary maternal CMV infection in guinea pigs during the first or second trimester of pregnancy resulted in congenital infection in 64% of the offspring. Of the congenitally infected neonates, 28% had significant auditory deficits. Within the inner ear, CMV infection was localized in auditory nerve spiral ganglion cells. These findings indicate that congenital CMV infection of the guinea pig results in physiologic and anatomic neuropathology similar to that seen in human infection and provide the first experimental model for congenital CMV-induced sensorineural hearing loss.