The cortistatin gene PSS2 rather than the somatostatin gene PSS1 is strongly expressed in developing avian autonomic neurons

Somatostatin and cortistatin are neuromodulators with divergent expression patterns and biological roles. Whereas expression and function of genes encoding somatostatin (PSS1) and the related peptide cortistatin (PSS2) have been studied in detail for the central nervous system (CNS) and immune system, relatively little is known about their expression patterns in the peripheral nervous system (PNS). We compare the expression patterns of PSS1 and PSS2 in chicken embryos. At E14, PSS1 is higher in the CNS versus PNS, whereas PSS2 is higher in the PNS. During early development, PSS1 is transiently expressed in lumbar sympathetic ganglia and is detectable at low levels throughout the development of dorsal root and ciliary ganglia. In contrast, PSS2 expression increases as development progresses in sympathetic and dorsal root ganglia, whereas levels in ciliary ganglia by E8 are more than 100‐fold higher than in sympathetic ganglia. Activin, which induces somatostatin‐like immunoreactivity in ciliary ganglion neurons in vivo and in vitro, controls PSS2 expression by stabilizing PSS2 but not PSS1 mRNA. We conclude that much of the somatostatin‐like immunoreactivity in the developing avian peripheral nervous system is actually cortistatin, the PSS2 product, as opposed to true somatostatin, which is the PSS1 product. The identification of PSS2 as the predominantly expressed somatostatin gene family member in avian autonomic neurons provides a molecular basis for further functional and pharmacological studies. J. Comp. Neurol. 518:839–850, 2010. © 2009 Wiley‐Liss, Inc.     

Identification of cerebellin2 in chick and its preferential expression by subsets of developing sensory neurons and their targets in the dorsal horn

The cerebellins are a family of four secreted proteins, two of which, Cbln1 and Cbln3, play an important role in the formation and maintenance of parallel fiber‐Purkinje cell synapses. We have identified the chicken homologue of Cbln2 and, through the use of in situ hybridization, shown that it is expressed by specific subsets of neurons in the dorsal root ganglia (DRGs) and spinal cord starting shortly after those neurons are generated. In the developing spinal cord, Cbln2 is highly expressed by dI1, dI3, dI5, and dILB dorsal interneurons and to a lesser extent by dI2, dI4, dI6, and dILA dorsal interneurons, but not by ventral (v0–v3) interneurons. After the spinal cord has matured and neurons have migrated to their final destinations, Cbln2 is abundant in the dorsal horn. In the DRGs, Cbln2 is expressed by TrkB+ and TrkC+ sensory neurons, but not by TrkA+ sensory neurons. Interestingly, regions of the spinal cord where TrkB+ and TrkC+ afferents terminate (i.e., laminae II, III, IV, and VI) exhibit the highest levels of Cbln2 expression. Cbln2 is also expressed by preganglionic sympathetic neurons and their targets in the sympathetic chain ganglia. Thus, the results show that Cbln2 is frequently expressed by synaptically connected neuronal populations. This, in turn, raises the possibility that if Cbln2, like Cbln1, plays a role in the formation and maintenance of synapses, it may somehow mediate bi‐directional communication between discrete populations of neurons and their appropriate neuronal targets. J. Comp. Neurol. 518:2818–2840, 2010. © 2010 Wiley‐Liss, Inc.     

Neuronal and glial expression of the adhesion molecule TAG-1 is regulated after peripheral nerve lesion or central neurodegeneration of adult nervous system

Expression of the cell adhesion molecule TAG-1 is down-regulated in adult brain, with the exception of certain areas exhibiting structural plasticity. Here, we present evidence that TAG-1 expression persists also in adult rat spinal cord and dorsal root ganglia (DRG), and can be up-regulated after injury. On Western blots of adult tissue, TAG-1 is detected as a 135-kDa band, with an additional specific 90-kDa band, not present in developing tissue. TAG-1 expression is found both in DRG neurons and in Schwann cells, particularly those associated with the peripherally projecting DRG processes. Quantitative in situ hybridization revealed that TAG-1 expression is significantly higher in small neurons that give rise to unmyelinated fibers, than in large DRG neurons. The regulation of TAG-1 was then examined in two different lesion paradigms. After a sciatic nerve lesion, TAG-1 expression is not up-regulated in DRG neurons, but decreases with time. At the lesion site, reactive Schwann cells up-regulate TAG-1, as demonstrated by both immunohistochemistry and in situ hybridization. In a second paradigm, we injected kainic acid into the spinal cord that kills neurons but spares glia and axons. TAG-1 is up-regulated in the spinal neuron-depleted area as well as in the corresponding dorsal and ventral roots, associated with both target-deprived afferent fibers and with the non-neuronal cells that invade the lesion site. These results demonstrate a local up-regulation of TAG-1 in the adult that is induced in response to injury, suggesting its involvement in axonal re-modelling, neuron-glia interactions, and glial cell migration.     

A critical period for the influence of peripheral targets on the central projections of developing sensory neurons

During development, the projections that sensory neurons make within the spinal cord are influenced by the specific targets they contact in the periphery. If sensory ganglia normally supplying principally cutaneous targets are forced to grow into limb muscles, in early stage tadpoles, many sensory neurons within these ganglia innervate limb muscles and subsequently develop spinal projections appropriate for muscle spindle afferents. If the same procedure is performed with adult frogs, however, these novel projections do not form. In this study, we have determined the developmental stages at which this sensitivity to peripheral targets exists. Axons from sensory neurons in thoracic (largely cutaneous) dorsal root ganglia were re-routed into the front leg at various stages through metamorphosis, and the central spinal projections of these re-routed fibers were assessed with HRP labeling. We found that thoracic sensory axons could be made to project to limb muscles throughout development, but that the central projections of these neurons were only appropriate for spindle afferents if the fibers were re-routed before stage XVIII, shortly before metamorphic climax. Because sensory neurons can regenerate specifically into the appropriate spinal laminae even in adult frogs, these results suggest that changes in either the DRG or the arm musculature occur by stage XVII so that DRG neurons cannot respond to novel peripheral targets.     

Expression of capsaicin receptor immunoreactivity in human peripheral nervous system and in painful neuropathies

We describe the expression of the capsaicin receptor (TRPV1) in human peripheral nervous system (PNS) and its changes in sural nerve and skin nerve fibers of patients with painful neuropathy. Dorsal root ganglion (DRG), root, and spinal cord autopsy specimens from subjects without PNS diseases were immunoassayed with anti-TRPV1 antibodies. Bright-field and confocal microscope studies using anti-TRPV1, protein gene product 9.5 (PGP 9.5), and unique-beta-tubulin (TuJ1) antibodies were performed in skin biopsies from 15 healthy subjects and 10 painful neuropathies. The density of intraepidermal nerve fiber (IENF) labeled by each antibody was quantified. Sural nerve biopsies from three patients with painful, one patient with nonpainful diabetic neuropathy, and two patients with multifocal motor neuropathy used as controls were immunoassayed with anti-TRPV1 antibodies and investigated by immunoelectron microscopy. TRPV1 strongly labeled laminae I and II of dorsal horns, most small-size and some medium-size DRG neurons, and small-diameter axons of dorsal roots. In sural nerve, TRPV1 was expressed within the cytoplasm of most unmyelinated and some small myelinated axons, in the muscular lamina of epineural vessels, and in the endothelium of endoneurial vessels. The density of IENF labeled by TRPV1, PGP 9.5, and TuJ1 did not differ. TRPV1 colocalized with TuJ1 in all IENF and dermal nerve bundles. Painful neuropathies showed a diffuse loss of TRPV1-positive axons both in the sural nerve and in the skin. Our findings demonstrated that TRPV1 is normally expressed throughout the nociceptive pathway of PNS and that TRPV1-positive peripheral nerve fibers degenerate in painful neuropathies.     

Differential binding of antibodies against the neurofilament triplet proteins in different avian neurons

Using monospecific antisera against each of the three chicken neurofilament (NF) proteins, NF70, NF160 and NF180, the distribution of each of these proteins in several types of neurons was examined by immunohistochemistry. Striking differences were observed in the relative staining by the three antibodies when the soma of different types of neurons were compared, and also when the soma of some neurons were compared with their axons. Both the soma and axons of dorsal root sensory neurons were brightly stained by each of the antisera. The soma of spinal cord ventral horn neurons, however, were stained only by A-NF70 and A-NF180, not by A-NF160. The axons of these neurons were uniformly stained by A-NF70 and A-NF180, while only gradually becoming NF160-positive over the first several hundred microns. The lack of staining by A-NF160 was also observed in many neuronal soma in cultures of dissociated spinal cord cells. The soma and dendrites of adult cerebellar Purkinje cells were weakly stained by A-NF70 and A-NF180 and not at all by A-NF160, but both A-NF70 and A-NF180 yielded prominent staining of immature Purkinje cells and dendrites. These results suggest that the three NF proteins may be unequally distributed within the soma and processes of different types of neurons and/or may be subject to regionally selective modification.     

Neurogenesis in the adult peripheral nervous system

Most researchers believe that neurogenesis in mature mammals is restricted only to the subgranular zone of the dentate gyrus and the subventricular zone of the lateral ventricle in the central nervous system. In the peripheral nervous system, neurogenesis is thought to be active only during prenatal development, with the exception of the olfactory neuroepithelium. However, sensory ganglia in the adult peripheral nervous system have been reported to contain precursor cells that can proliferate in vitro and be induced to differentiate into neurons. The occurrence ofinsult-induced neurogenesis, which has been reported by several investigators in the brain, is limited to a few recent reports for the peripheral nervous system. These reports suggest that damage to the adult nervous system induces mechanisms similar to those that control the generation of new neurons during prenatal development. Understanding conditions under which neurogenesis can be induced in physiologically non-neurogenic regions in adults is one of the major challenges for developing therapeutic strategies to repair neurological damage. However, the induced neurogenesis in the peripheral nervous system is still largely unexplored. This review presents the history of research on adult neurogenesis in the peripheral nervous system, which dates back more than 100 years and reveals the evidence on the under estimated potential for generation of new neurons in the adult peripheral nervous system.     

Prolonged sympathetic innervation of sensory neurons in rat thoracolumbar dorsal root ganglia during chronic colitis

Background Peripheral irritation‐induced sensory plasticity may involve catecholaminergic innervation of sensory neurons in the dorsal root ganglia (DRG). Methods Catecholaminergic fiber outgrowth in the thoracolumbar DRG (T13‐L2) was examined by tyrosine hydroxylase (TH) immunostaining, or by sucrose‐potassium phosphate‐glyoxylic acid histofluorescence method. TH level was examined by Western blot. Colonic afferent neurons were labeled by retrograde neuronal tracing. Colitis was induced by intracolonic instillation of tri‐nitrobenzene sulfonic acid (TNBS). Key Results The catecholaminergic fibers formed ‘basket‐like’ structures around the DRG cells. At 7 days following TNBS treatment, the number of DRG neurons surrounded by TH‐immunoreactive fibers and the protein levels of TH were significantly increased in T13, L1, and L2 DRGs (two‐ to threefold, P < 0.05). The DRG neurons that were surrounded by TH immunoreactivity were 200 kDa neurofilament‐positive, but not isolectin IB4‐positve or calcitonin gene‐related peptide‐positive. The TH‐immunoreactive fibers did not surround but adjoin the specifically labeled colonic afferent neurons, and was co‐localized with glial marker S‐100. Comparison of the level of TH and the severity of colonic inflammation showed that following TNBS treatment, the degree of colonic inflammation was most severe at day 3, subsided at day 7, and significantly recovered by day 21. However, the levels of TH in T13‐L2 DRGs were increased at both 3 days and 7 days post TNBS treatment and persisted up to 21 days (two‐ to fivefold increase, P < 0.05) as examined. Conclusions & Inferences Colonic inflammation induced prolonged catecholaminergic innervation of sensory neurons, which may have relevance to colitis‐induced chronic visceral hypersensitivity and/or referred pain.     

Inhibitory effects of CB1 and CB2 receptor agonists on responses of DRG neurons and dorsal horn neurons in neuropathic rats

Cannabinoid 2 (CB2) receptor mediated antinociception and increased levels of spinal CB2 receptor mRNA are reported in neuropathic Sprague-Dawley rats. The aim of this study was to provide functional evidence for a role of peripheral, vs. spinal, CB2 and cannabinoid 1 (CB1) receptors in neuropathic rats. Effects of the CB2 receptor agonist, JWH-133, and the CB1 receptor agonist, arachidonyl-2-chloroethylamide (ACEA), on primary afferent fibres were determined by calcium imaging studies of adult dorsal root ganglion (DRG) neurons taken from neuropathic and sham-operated rats. Capsaicin (100 nm) increased [Ca2+]i in DRG neurons from sham and neuropathic rats. JWH-133 (3 microm) or ACEA (1 microm) significantly (P<0.001) attenuated capsaicin-evoked calcium responses in DRG neurons in neuropathic and sham-operated rats. The CB2 receptor antagonist, SR144528, (1 microm) significantly inhibited the effects of JWH-133. Effects of ACEA were significantly inhibited by the CB1 receptor antagonist SR141716A (1 microm). In vivo experiments evaluated the effects of spinal administration of JWH-133 (8-486 ng/50 microL) and ACEA (0.005-500 ng/50 microL) on mechanically evoked responses of neuropathic and sham-operated rats. Spinal JWH-133 attenuated mechanically evoked responses of spinal neurons in neuropathic, but not sham-operated rats. These inhibitory effects were blocked by SR144528 (0.001 microg/50 microL). Spinal ACEA inhibited mechanically evoked responses of neuropathic and sham-operated rats, these effects were blocked by SR141716A (0.01 microg/50 microL). Our data provide evidence for a functional role of CB2, as well as CB1 receptors on DRG neurons in sham and neuropathic rats. At the level of the spinal cord, CB2 receptors have inhibitory effects in neuropathic, but not sham-operated rats suggesting that spinal CB2 may be an important analgesic target.     

Loss of primary sensory neurons in the very old rat: Neuron number estimates using the disector method and confocal optical sectioning

Loss of neurons has been considered to be a prime cause of nervous disturbances that occur with advancing age. However, the notion of a constitutive aging-related loss of neurons has been challenged recently in several studies that used up-to-date methods for counting neurons. In this study, we have applied stereological techniques with the objective of obtaining quantitative data on total neuron numbers and the distribution of neuron cross-sectional areas in the fifth cervical (C₅) and fourth lumbar (L₄) dorsal root ganglion (DRG) of 3- and 30-month-old Sprague-Dawley rats. Tissue data were recorded on a confocal laser-scanning microscope with the use of the optical-disector technique and random, systematic sampling. Aged rats of both sexes disclosed only a small decrease (≈12%) in the number of cervical and lumbar DRG neurons. Furthermore, there was no significant correlation between the degree of neuron loss and the extent of behavioral deficits among the aged individuals. The DRG neurons of aged rats had a smaller mean cross-sectional area (≈15%; P < 0.001) at both DRG levels. Further analysis of the male cohorts was carried out by using isolectin B4 and neurofilament subunit (phosphorylated 200 kDa; RT97) immunoreactivity (IR) as selective markers for unmyelinated and myelinated axons, respectively, and disclosed no significant change in the relative frequencies of immunoreactive neuron profiles in the old rats. However, RT97-IR DRG neurons of the aged rats had significantly smaller cross-sectional areas (≈9% in C₅; ≈16% in L₄; P < 0.001) than the young adult rats, indicating a selective cell body atrophy among myelinated primary afferents during aging. The results indicate that loss of primary sensory neurons cannot exclusively explain the functional deficits in sensory perception among senescent individuals. It seems likely that other factors at the subcellular level and/or target interaction(s) contribute substantially to the sensory impairments observed with advancing age. J. Comp. Neurol. 396:211–222, 1998. © 1998 Wiley-Liss, Inc.     

Embryonic development of choline acetyltransferase in thoracic spinal motor neurons: somatic and autonomic neurons may be derived from a common cellular group.

This investigation focused on the relationship between neurotransmitter phenotype expression and rat motor neuron development, as studied with choline acetyltransferase (ChAT) immunocytochemical techniques. The development of two subclasses of motor neurons, somatic and autonomic efferents, was examined in the upper thoracic spinal cord. ChAT was first detected in a few neurons on embryonic day 12 1/2 (E12 1/2), and in numerous cells located in a single, ventrolaterally located column in the intermediate zone on E13. By E14, this group of ChAT-positive neurons was more intensely immunoreactive, and their axons could be traced to appropriate targets in developing somatic muscle and paravertebral sympathetic ganglia. During the E15-16 period, somatic and autonomic motor neurons separated into two distinct subgroups, with the latter cells being observed to translocate dorsally. By E17, these autonomic motor neurons reached their final positions in the midportion of the intermediate zone. The autonomic motor neurons were observed to extend transverse dendritic bundles across the spinal cord between E15-16, but evidence of the longitudinal bundles of sympathetic preganglionic dendrites was not observed until after birth. A recent study of cholinergic thoracic motor neurons found that both somatic and autonomic cells were generated synchronously during the E11-12 period (Barber et al., Soc Neurosci Abstr 15:588, 1989). In combination with the present results, these data indicate that no more than 1 1/2 days are necessary after motor neuron genesis before a few cells begin to express detectable levels of ChAT, and that no more than 2 days are required before large numbers express this marker of the cholinergic phenotype. Further comparisons of the present findings with those of previous investigations of the development of both somatic and autonomic motor neurons (Dennis et al., Dev Biol 81:266, 1981; Rubin, J Neurosci 5:685, 697, 1985) indicate that these cells contain ChAT at the time their axons are growing toward their respective peripheral targets 1 day before the time when physiological evidence of function is manifest. Furthermore, the present results suggest that both subclasses of motor neurons initially migrate together from the ventricular zone into a single motor column within the ventral intermediate zone, and that the autonomic neurons subsequently translocate dorsally. Thus, autonomic motor neurons appear to be an exception to the generalization that postmitotic neurons migrate directly from the germinal zone to their final positions within the central nervous system.     

Effect of reflux-induced inflammation on transient receptor potential vanilloid one (TRPV1) expression in primary sensory neurons innervating the oesophagus of rats

A possible mechanism of oesophageal hypersensitivity is the acid-induced activation of transient receptor potential vanilloid receptor 1 (TRPV1) in the primary sensory neurons. We investigated TRPV1 expression and its colocalization with substance P (SP) and isolectin B4 (IB4)-positive cells in the thoracic dorsal root ganglia (DRGs) and nodose ganglia (NGs) of rats with reflux-induced oesophagitis (RO). RO was developed by fundus ligation and partial obstruction of the pylorus of Sprague-Dawley rats. Four groups of rats were used; fundus ligated acute (RO 48 h), chronic 7 days (RO 7D), RO 7D + omeprazole (7D + Omz, 40 mg kg(-1), i.p.) and sham-operated controls. Immunohistochemical analysis of TRPV1, SP and IB4 expression were carried out in spinal cord (SC), DRGs and NGs. RO rats exhibited significant inflammation and increase in TRPV1-ir and SP-ir expressions in the SC, DRGs and NGs. The maximum colocalization of TRPV1 and SP was observed in RO 7D rats, but Omz prevented inflammation and over expression of TRPV1 and SP. TRPV1-ir significantly increased in IB4-positive cells in DRGs and SC, but not in the NGs. Results document that acid-induced oesophagitis increases TRPV1 expression in both SP- and IB4-positive sensory neurons. The over expression of TRPV1 may contribute to oesophageal hypersensitivity observed in gastro-oesophageal reflux disease (GORD).     

Expression and localization of insulin receptor in rat dorsal root ganglion and spinal cord

The expression and localization of the insulin receptor (IR) was examined in rat dorsal root ganglia (DRG) and spinal cord using Western blotting, in situ hybridization and immunocytochemistry. Western blotting showed that the molecular weight of the IR beta subunit was higher in PNS than that found in CNS. Both IR mRNA and protein expressions were highest in small-sized sensory DRG neurons and myelinated sensory root fibers expressed higher levels of IR protein than myelinated anterior root fibers. In the spinal cord, IR immunoreactive neurons were present in lateral lamina V and in lamina X, suggesting the presence of IR in nociceptive pathways. Electronmicroscopy of DRGs revealed a polarized localization of the IR in abaxonal Schwann cell membranes, outer mesaxons in close vicinity to tight junctions of both myelinating and non-myelinating Schwann cells and to plasma membranes of sensory neurons. From these findings, we speculate that insulin may play a role in sensory fibers involved in nociceptive function often perturbed in diabetic neuropathy. The high expression of IR localizing to tight junctions of dorsal root mesaxons of DRGs may suggest a regulatory role on barrier functions compensating for the lack of a blood-nerve barrier in dorsal root ganglia. This is consistent with the colocalization of IR with tight junctions of the paranodal barrier and endoneurial endothelial cells in peripheral nerve.     

Evidence for spinal origin of neurons synapsing with dorsal root ganglion cells of the cat

The visuotopic structure of primary visual cortex was studied in a group of 7 human volunteers using positron emission transaxial tomography (PETT) and 18F-labeled 2-deoxy-2-fluoro-D-glucose ( [18F]DG). A computer animation was constructed with a spatial structure which was matched to estimates of human cortical magnification factor and to striate cortex stimulus preferences. A lateralized cortical 'checker-board' pattern of [18F]DG was stimulated in primary visual cortex by having subjects view this computer animation following i.v. injection of [18F]DG. The spatial structure of the stimulus was designed to produce an easily recognizable 'signature' in a series of 9 serial PETT scans obtained from each of a group of 7 volunteers. The predicted lateralized topographic 'signature' was observed in 6 of 7 subjects. Applications of this method for further PETT studies of human visual cortex are discussed.     

Organization of spinal inputs to the perihypoglossal complex in the cat

First- and second-order spinal afferents to the perihypoglossal complex were sought by using axonal transport of WGA-HRP. Injections in C1, 2, and 3 dorsal root ganglia resulted in axonal labeling in the nucleus intercalatus and the external cuneate nucleus, with a number of retrogradely labeled cells seen as well in the latter. A similar pattern of axonal labeling in the nucleus intercalatus as well as several retrogradely labeled cells were found after spinal cord injections at levels C1, 2, and 3. A prominent field of labeled axons was also present in the rostral main cuneate nucleus. No labeling was seen in the perihypoglossal nuclei after injections in the spinal cord or dorsal root ganglia at levels caudal to C3. After injections of HRP into the perihypoglossal nucleus we were able to identify labeled neurons within Rexed's laminae V-VIII and the central cervical nucleus. Anterograde labeling in the main cuneate nucleus was observed after C1 to C5 ganglion and C1 to C6 cord injections. The pattern and extent of labeling in the perihypoglossal nuclei and adjacent structures seen after cerebellar injections into lobules V and VI were comparable to those previously reported and permitted evaluation of the relay from dorsal root ganglia through the intercalatus to the vermis. Topography of the cervical projections to the nucleus intercalatus is considered with respect to that of the perihypoglossal-collicular projection. A discussion is offered of the apparent importance of nucleus intercalatus as a relay of cervical and vestibular afferent information to premotor structures involved in neck motor control. The perihypoglossal complex is viewed as being organized in such a fashion as to allow the nuclei intercalatus and prepositus hypoglossi to function as key structures in the integration of inputs related to neck and ocular motor control, respectively.     

Retinoic acid responsive gene product,midkine, has neurotrophic functions for mouse spinal cord and dorsal root ganglion neurons in culture

Midkine (MK) is the product of a retinoic acid responsive gene and is a member of a new family of heparin-binding growth factors. Neurotrophic effects of MK were examined using cultured spinal cord and dorsal root ganglion (DRG) neurons derived from fetal mouse. MK, which was added to the culture medium at concentrations of 1–100 ng/ml, promoted survival of both types of neurons approximately 5-fold after 7 days in culture. For spinal cord neurons, the increased survival was reflected in an increase of choline acetyltransferase activity. MK also promoted neurite extension in spinal cord (2-fold) and DRG (1.7-fold) neurons. The survival-promoting activity of MK to these neurons was comparable to that of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF). In spite of its significant effects on fetal neurons, MK was ineffective in sustaining survival of DRG neurons derived from postnatal mice. From these results, we conclude that MK is a neurotrophic factor to embryonic spinal cord and DRG neurons, and we propose that MK plays a significant role in embryogenesis of the nervous system. © 1993 Wiley-Liss, Inc.