The composition of Na+ currents in dorsal root ganglia (DRG) neurons depends on their neuronal phenotype and innervation target. Two TTX-resistant (TTX-R) Na+ currents [voltage-gated Na channels (Nav)] have been described in small DRG neurons; one with slow inactivation kinetics (Nav1.8) and the other with persistent kinetics (Nav1.9), and their modulation has been implicated in inflammatory pain. This has not been studied in neurons projecting to the colon. This study examined the relative importance of these currents in inflammation-induced changes in a mouse model of inflammatory bowel disease. Colonic sensory neurons were retrogradely labeled, and colitis was induced by instillation of trinitrobenzenesulfonic acid (TNBS) into the lumen of the distal colon. Seven to ten days later, immunohistochemical properties were characterized in controls, and whole cell recordings were obtained from small (<40 pF) labeled DRG neurons from control and TNBS animals. Most neurons exhibited both fast TTX-sensitive (TTX-S)- and slow TTX-R-inactivating Na+ currents, but persistent TTX-R currents were uncommon (<15%). Most labeled neurons were CGRP (79%), tyrosine kinase A (trkA) (84%) immunoreactive, but only a small minority bind IB4 (14%). TNBS-colitis caused ulceration, thickening of the colon and significantly increased neuronal excitability. The slow TTX-R-inactivating Na current density (Nav1.8) was significantly increased, but other Na currents were unaffected. Most small mouse colonic sensory neurons are CGRP, trkA immunoreactive, but not isolectin B4 reactive and exhibit fast TTX-S, slow TTX-R, but not persistent TTX-R Na+ currents. Colitis-induced hyperexcitability is associated with increased slow TTX-R (Nav1.8) Na+ current. Together, these findings suggest that colitis alters trkA-positive neurons to preferentially increase slow TTX-R Na+ (Nav1.8) currents.

Sympathetic axons invade the trigeminal ganglia of mice overexpressing nerve growth factor (NGF) (NGF/p75(+/+) mice) and surround sensory neurons having intense NGF immunolabeling; the growth of these axons appears to be directional and specific (). In this investigation, we provide new insight into the neurochemical features and receptor requirements of this sympathosensory sprouting. Using double-antigen immunohistochemistry, we demonstrate that virtually all (98%) trigeminal neurons that exhibit a sympathetic plexus are trk tyrosine kinase receptor (trkA)-positive. In addition, the majority (86%) of those neurons enveloped by sympathetic fibers is also calcitonin gene-related peptide (CGRP)-positive; a smaller number of plexuses (14%) surrounded other somata lacking this neuropeptide. Our results show that sympathosensory interactions form primarily between noradrenergic sympathetic efferents and the trkA/CGRP-expressing sensory somata. To assess the contribution of the p75 neurotrophin receptor (p75(NTR)) in sympathosensory sprouting, a hybrid strain of mice was used that overexpresses NGF but lacks p75(NTR) expression (NGF/p75(-/-) mice). The trigeminal ganglia of NGF/p75(-/-) mice, like those of NGF/p75(+/+) mice, have increased levels of NGF protein and display a concomitant ingrowth of sympathetic axons. In contrast to the precise pattern of sprouting seen in the ganglia of NGF/p75(+/+) mice, sympathetic axons course randomly throughout the ganglionic neuropil of NGF/p75(-/-) mice, forming few perineuronal plexuses. Our results indicate that p75(NTR) is not required to initiate or sustain the growth of sympathetic axons into the NGF-rich trigeminal ganglia but rather plays a role in regulating the directional patterns of axon growth.

Axonal growth in the adult mammalian CNS is limited because of inhibitory influences of the glial environment and/or a lack of growth-promoting molecules. Here, we investigate whether supplementation of nerve growth factor (NGF) to the CNS during postnatal development and into adulthood can support the growth of sympathetic axons within myelinated portions of the maturing brain. We have also asked whether p75(NTR) plays a role in this NGF-induced axon growth. To address these questions we used two lines of transgenic mice overexpressing NGF centrally, with or without functional expression of p75(NTR) (NGF/p75(+/+) and NGF/p75(-/-) mice, respectively). Sympathetic axons invade the myelinated portions of the cerebellum, beginning shortly before the second week of postnatal life, in both lines of NGF transgenic mice. Despite the presence of central myelin, these sympathetic axons continue to sprout and increase in density between postnatal days 14 and 100, resulting in a dense plexus of sympathetic fibers within this myelinated environment. Surprisingly, the growth response of sympathetic fibers into the cerebellar white matter of NGF/p75(-/-) mice is enhanced, such that both the density and extent of axon ingrowth are increased, compared with age-matched NGF/p75(+/+) mice. These dissimilar growth responses cannot be attributed to differences in cerebellar levels of NGF protein or sympathetic neuron numbers between NGF/p75(+/+) and NGF/p75(-/-) mice. Our data provide evidence demonstrating that growth factors are capable of overcoming the inhibitory influences of central myelin in the adult CNS and that neutralization of the p75(NTR) may further enhance this growth response.

Human clinical trials have begun worldwide that use olfactory ensheathing cells (OECs) to ameliorate the functional deficits following spinal cord injury. These trials have been initiated largely because numerous studies have reported that OECs transform into Schwann Cell (SC)-like cells that myelinate axons and support new growth in adult rats with spinal injury. This phenomenon is remarkable because OECs do not myelinate olfactory axons in their native environment. Furthermore, these myelinating OECs are morphologically identical to SCs, which can invade the spinal cord after injury. One factor that has contributed to a possible confusion in the identification of these cells is the lack of phenotypic markers to distinguish unequivocally between OECs and SCs. Such markers are required to first assess the degree of SC contamination in OEC cultures before intraspinal implantation, and then to accurately identify grafted OECs and invading SCs in the injured spinal cord. Using two-dimensional gel electrophoresis, we have identified calponin, an actin binding protein, as the first definitive phenotypic marker that distinguishes between OECs and SCs in vitro and in vivo. We have also provided ultrastructural evidence that calponin-immunopositive OECs do not transform into myelinating SC-like cells after intraspinal implantation. Rather, the grafted OECs retain their morphological and neurochemical features. These data yield new insight into the phenotypic characteristics of OECs, which together with invading SCs can enhance regeneration of the injured spinal cord.

No abstract