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Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain. J. Comp. Neurol. 453:292–304, 2002. © 2002 Wiley-Liss, Inc.

Glial cell line-derived neurotrophic factor (GDNF) is a survival factor for several types of neurons, including dopaminergic (DAergic) neurons. GDNF binds with high affinity to the GDNF family receptor alpha-1 (GFRalpha-1), which is highly expressed in the midbrain. Using anatomical and lesion techniques, we demonstrated that GFRalpha-1 was expressed in DAergic and non-DAergic neurons in the rat midbrain. Immunohistochemical characterization of GFRalpha-1-expressing neurons indicated that most of the neurons that were immunopositive for the DAergic marker tyrosine hydroxylase (TH) expressed GFRalpha-1 in the substantia nigra pars compacta (SNC). In contrast, fewer TH-containing neurons expressed GFRalpha-1 in the substantia nigra pars reticulata (SNR) and the ventral tegmental area (VTA). Depletion of GFRalpha-1/TH neurons was observed in the SNC following treatment with the neurotoxin 6-hydroxydopamine (6-OHDA); however, GFRalpha-1 expression remained in some neurons located in the SNR. The gamma-aminobutyric acid (GABA)ergic nature of GFRalpha-1-expressing neurons located in the SNR, which were resistant to (6-hydroxydopamine) 6-OHDA, was established by their expression of glutamic acid decarboxylase (GAD; the synthesizing enzyme for GABA). Further analysis indicated that coexpression of GFRalpha-1 and GAD varied in a rostrocaudal gradient in the SNR, substantia nigra pars lateralis (SNL), and VTA. Midbrain DAergic and GABAergic neurons have been previously classified according to their Ca(2+) binding protein (CaBP) content; thus, we also sought to investigate the proportion of midbrain GFRalpha-1-expressing neurons containing parvalbumin (PV), calbindin (CB), and calretinin (CR) in the midbrain. Although GFRalpha-1 expression was found mainly in CB- and CR-immunoreactive neurons, it was rarely observed in PV-immunolabeled neurons. Analysis of the proportion of GFRalpha-1-expressing neurons for each CaBP subpopulation indicated the coexistence of GFRalpha-1 with CR in the VTA and all subdivisions of the SN; double-labeled GFRalpha-1/CR neurons were distributed in the SNC, SNR, SNL, and VTA. GFRalpha-1/CB neurons were also detected in the SNC, SNL, and VTA. Expression of GFRalpha-1 in DAergic and non-DAergic neurons in the rat SN and VTA suggests that GDNF, via GFRalpha-1, might modulate DAergic and GABAergic functions in the nigrostriatal, mesolimbic, and nigrothalamic circuits of the adult rat.

Although many studies have demonstrated the dependency of taste bud function and/or survival on intact innervation, relatively few have dealt with the development of taste bud innervation. Using in situ hybridization histochemistry, we show that brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) mRNA are expressed in a specific pattern in the taste buds, tongue papillae, and lingual epithelium during development and that expression persists into adulthood. BDNF mRNA is expressed in a fraction of the taste cells of the developing and adult taste buds in rats, showing different labeling intensities among the labeled cells. NT3 mRNA seems to be located in areas other than those where BDNF mRNA is expressed, mainly in the superior epithelial surfaces of circumvallate papillae, the outer surface epithelium of foliate papilae, the superior surface and the lateral epithelium of the fungiform papillae, and the epithelium of the filiform papillae. NT3 mRNA labeling is also observed among muscle and connective tissue of the tongue. The morphological appearance, expression of NT3 mRNA, and ramification of nerve fibers in defined epithelial structures in the posterior wall of the anterior filiform papillae suggest the existence of a mechanosensory apparatus in these papillae. Nerve growth factor and neurotrophin 4 probes did not give rise to selective labeling in tongue, although their presence cannot be totally excluded. Based on present and prior studies, we suggest that BDNF is needed during initiation and for maintenance of gustatory innervation of taste buds and gustatory papillae and that NT3 is mainly needed for somatosensory innervation of the tongue. © 1996 Wiley-Liss, Inc.

Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are expressed in many areas of the nervous system and its target tissues. Using in situ hybridization we have investigated the possible presence of NGF mRNA and BDNF mRNA in the developing fungiform and circumvallate papillae of the rat tongue. BDNF mRNA is present in the epithelium of the developing fungiform papillae in E15, E16, and E17 rat embryos with peak concentration at E16. It starts to diminish after E17 and is almost absent at E21. There is a specific temporospatial change in the expression of BDNF mRNA in developing circumvallate papillae. It is expressed in the epithelium of the superior and posterior surfaces of the papillae at E15, E16, and E17. Already at E17 the BDNF mRNA labeling has started to decrease in the superior epithelium. At E19 and E21, BDNF mRNA is exclusively present in the epithelium of the inner and outer walls of the trench, surrounding the papilla at the posterior and lateral surfaces where the taste buds are located later in life. BDNF mRNA was also detected in the developing palatal taste buds. NGF mRNA was below detection level in the developing papillae. The highly localized expression of BDNF mRNA in areas where taste buds are to be formed suggests that BDNF may be one crucial factor in the formation of the epithelial innervation prior to taste bud formation. It might also participate in the formation and/or maintenance of the papillary and/or taste bud innervation apparatus. We conclude that the neurotrophin BDNF is expressed in early development of taste bud-bearing papillae in the rat tongue in a temporally and spatially controlled manner, presumably to act as a target-derived chemoat tractant for the early nerve fibers. © 1995 Wiley-Liss, Inc.

Spinal cord injury induces a complex cascade of degenerative and remodeling events evolving over time. The possible roles of changed intercellular communication via gap junctions after spinal cord injury (SCI) have remained relatively unexplored. We investigated the temporospatial expression patterns of gap junctional genes and proteins, connexin 43 (Cx43), Cx36, and Cx32, by in situ hybridization and immunohistochemistry in the rat neonatal, adult normal, and adult injured spinal cord. Cx36 was strongly expressed in immature neurons, and levels declined markedly during development, whereas Cx43 and Cx32 persisted throughout adulthood. After a complete transection of the adult spinal cord, the levels of Cx43 mRNA and protein were up-regulated within hours, especially in gray matter rostral to the lesion, reaching over three times normal levels at 4 weeks postinjury. Cx43 immunoreactivity was seen primarily in astrocytes and rarely in microglia. In contrast, Cx36 and Cx32 mRNA and proteins were relatively sparse and unchanged after spinal cord injury along the entire axis of the spinal cord. Cx43 is the most abundant gap junctional protein in the adult CNS and has been shown to form channels between astrocytes as well as between astrocytes and oligodendrocytes. Long-term up-regulation of Cx43 in reactive astrocytes may be one critical component in the rearrangement of the local astroglial network following SCI.

Inhibition of RhoA has been shown to enhance axonal regeneration following spinal cord injury. Here we mapped mRNA expression patterns of RhoA, B, and C, Rac1, Cdc42, and Tc10 in spinal cord, sensory ganglia, and sensorimotor cortex in uninjured rats, and following spinal cord injury or sham laminectomy. In the intact spinal cord, neurons displayed high levels of Rac1, Cdc42, and Tc10 mRNA hybridization signal. GFAP-immunoreactive astrocytes expressed primarily RhoB and Rac1, while oligodendrocyte-like cells expressed RhoA, Rac1, and Cdc42. Injury caused profound, long-lasting upregulation of RhoA, Rac1, Cdc42, and Tc10 mRNA in the spinal cord, while RhoB was modestly increased and RhoC did not change. GFAP-immunoreactive reactive astrocytes exhibited a dramatic increase of RhoA mRNA expression along with increases of Rac1 and Cdc42. Injury also led to elevation of RhoA, Cdc42, and Tc10 in neurons and modest increases of RhoA, Rac1, and Tc10 in oligodendrocyte-like cells. Laminectomy caused similar, but less pronounced alterations of investigated mRNA species. In dorsal root ganglia neuronal RhoA, Rac1, Cdc42, and Tc10 mRNA levels were increased similarly by spinal cord injury and sham surgery. The CST pyramidal cells expressed Tc10 mRNA and the CST itself was Tc10-immunoreactive. Tc10-immunoreactivity disappeared distal to injury. We conclude that there are gene-specific patterns of expression of the six different Rho-GTPases in normal spinal cord and dorsal root ganglia, and that specific changes of temporal and spatial expression patterns occur in response to spinal cord injury, suggesting different roles of these GTPases in the cellular sequelae of CNS injury.

We describe the expression of mRNA encoding ligands and receptors of members of the GDNF family and members of the neurotrophin family in the adult human spinal cord and dorsal root ganglia (DRG). Fetal human spinal cord and ganglia were investigated for the presence of ligands and receptors of the neurotrophin family. Tissues were collected from human organ donors and after routine elective abortions. Messenger RNA was found encoding RET, GFR alpha-1, BDNF, trkB, and trkC in the adult human spinal cord and BDNF, NT-3, p75, trkB, and trkC in the fetal human spinal cord. The percentage of adult human DRG cells expressing p75, trkA, trkB, or trkC was 57, 46, 29, and 24%, respectively, and that of DRG cells expressing RET, GFR alpha-1, GFR alpha-2, or GFR alpha-3 was 79, 20, 51, and 32%, respectively. GFR alpha-2 was expressed selectively in small, GFR alpha-3 principally in small and GFR alpha-1 and RET in both large and small adult human DRG neurons. p75 and trkB were expressed by a wide range of DRG neurons while trkA was expressed in most small diameter and trkC primarily in large DRG neurons. Fetal DRG cells were positive for the same probes as adult DRG cells except for NT-3, which was only found in fetal DRG cells. Messenger RNA species only expressed at detectable levels in fetal but not adult spinal cord tissues included GDNF, GFR alpha-2, NT-3, and p75. Notably, GFR alpha-2, which is expressed in the adult rat spinal cord, was not found in the adult human spinal cord.

We used the intraocular transplantation technique to test the developmental potential of isolated, fetal area dentata, and to help define the determinants of such critical aspects of brain development as cell arrangements, dendritic arborization, axon growth, synaptogenesis, and connectional specificity. Fetal hippocampal formations were removed from embryos of 19 to 20 days of embryonic age. A region, termed area dentata, was dissected from the hippocampal formation. This region included the anlage of the dentate gyrus, the hilus, and the medial regio inferior. Areae dentatae were transplanted into the anterior eye chamber of isogenic, adult rats. The growth and vascularization of area dentata transplants were monitored over the first 2 months in oculo. From 2 to 7 months after transplantation host animals were killed and the area dentata transplants were examined with a variety of histological techniques. The cytoarchitectonics of a great majority of the transplants were highly reminiscent of area dentata in situ. A typical morphogenetic outcome included tightly packed, continuous, C-shaped sheets of granule cells with mossy fibers which made contact with the proximal dendrites of a segregated group of dispersed hilar cells and a loosely packed layer of pyramidal cells. The axons of pyramidal and hilar cells, in turn, provided the major innervation of the granule cell dendrites. The granule cell dendritic spine density was nearly normal, and synapses were common in the granule cell neuropil. Very little innervation of the transplant was found to originate from the axons of the ciliary or trigeminal ganglia which innervate the host iris and enter the transplant. Sympathetic fibers from the host iris innervate the transplant in a reproducible and organotypic fashion although the degree of innervation is less than the in situ noradrenergic innervation of area dentata. Few Timm-positive fibers of the transplant were found to leave the transplant and enter the host iris. Area dentata transplants had several features which were different from their in situ counterparts. These differences included less expansive granule cell dendrites, extensive presence of fibrous astrocytes, pyramidal cells with reduced kainic acid sensitivity, hypervascularized neuropil, and transformation of peripheral adrenergic fiber morphology to one of central characteristics. Thus, pieces of isolated area dentata display a great self-developing capacity, in terms of cellular and afferent organization, both of which follow “rules” present in situ. A powerful capacity for intrinsic and appropriate innervation of area dentata is apparent in the face of a total lack of normal extrinsic afferents and an availability of extrinsic but inappropriate sources of innervation. This initial description of isolated area dentata organization in oculo is meant to lay the groundwork for experimental investigations into the regulatory mechanisms governing morphogenesis and afferent organization.

A number of recent experiments suggest that grafted dopaminergic neurons provide functional input to a host caudoputamen which previously had been deprived of its dopaminergic input. The purpose of the present study was to determine whether tyrosine hydroxylase immunoreactive processes which originate in the graft participate in morphologically identifiable synapses in the host neuropil. Prior to transplantation, adult Sprague-Dawley rats received unilateral injections of 6-hydroxydopamine into the medial forebrain bundle. Animals were screened for the success of striatal denervation by a test of apomorphine-induced rotation. Transplants of fetal substantia nigra then were placed into cavities in the caudoputamen. After a 6–8 month survival period, animals were perfused and prepared for tyrosine hydroxylase immunocytochemistry. No evidence of sprouting of the host catecholoaminergic system was observed, even after long survival times. Both pre- and postsynaptic immunoreactive elements were clearly present in the host caudoputamen. Immunoreactive axons made synaptic contact with unlabeled dendrites; immunoreactive dendrites were postsynaptic to unlabeled axon terminals. The present results suggest that both host-to-graft and graft-to-host synapses are present in the host caudoputamen.

The anterior eye chamber was used as a model environment to study, in isolation, the interaction of embryonic area dentata transplants with transplants of one of three important sources of in situ innervation: entorhinal cortex, locus coeruleus or septal nuclei. None of these brain regions significantly affected the morphogenesis or in oculo growth of area dentata transplants. All three brain regions innervated the area dentata transplant. Entorhinal cortical transplants sent nerve fibers into a limited, and apparently specific, region of area dentata that was adjacent to the entorhinal transplant. This light innervation contrasts to the predominant innervation of area dentata by entorhinal cortex in situ. The fluorescent, noradrenergic neurons of locus coeruleus provided the area dentata transplant with an abundance of fine varicose nerve fibers. Given about 100 noradrenergic neurons in the locus coeruleus transplant and 4 to 6 months joint survival, the area dentata transplant was noradrenergically hyperinnervated. The cholinergic neurons of the septal nuclei transplant had a prolific ingrowth of acetylcholinesterase (AChE)-positive nerve fibers to the area dentata transplant. There appeared to be a mutual exclusion between the extrinsic AChE-positive fibers and the intrinsic Timm's-positive granule cell mossy fibers in the area dentata transplant. We conclude that isolated replicas of the coeruleo-, septo-, and entorhinal cortico-dentate pathways can be made through sequential intraocular double grafting. The nature of the in oculo connectivity between these replicates offers clues as to the mechanisms that might account for the regulation of nerve growth.