Autocrine regulation of proliferation of cerebellar granule neurons by nerve growth factor

Premigratory cerebellar granule neurons, which highly express nerve growth factor (NGF), low (gp75^NGFR) and high (gp140^trkA) affinity NGF receptors, were used as a physiological model to investigate the effects of NGF on neuronal replication. Studies in vivo and on cultures showed that NGF stimulates DNA synthesis, mitotic activity and related cell acquisition by initiating the entry of cells into the S phase and regulating their time in the G1 and S phases. The NGF-induced effects were blocked in vivo and in vitro by both monoclonal anti-NGF and anti-gp75^NGFR antibodies. These results clearly demonstrate that NGF is essential for the crucial first step of cerebellar ontogenesis and support the idea that low affinity receptors are involved in the biological response, possibly by interacting with gp140^trkA. By comparison with a number of well known mitogens, the high affinity form could be the main transducer of the mitogenic signal pathway. The early developing cerebellum appears therefore to be the first autocrine (and/or paracrine) model of NGF action on neurogenesis in the CNS. © 1994 Wiley-Liss, Inc.     

Neuronal age influences the response to neurite outgrowth inhibitory activity in the central and peripheral nervous systems.

Axonal regeneration is abortive in the central nervous system (CNS) of adult mammals, but readily occurs in the injured peripheral nervous system (PNS). Recent experiments indicate an important role for both intrinsic neuronal features and extrinsic substrate properties in determining the propensity for axonal regrowth. In particular, certain components of adult mammalian CNS myelin have been shown to exert a strong inhibitory influence on neurite outgrowth. To determine whether the potent neurite outgrowth inhibitory activity found in CNS myelin may also be present in PNS myelin and to study the influence of neuronal age on neurite outgrowth, we used a cryoculture assay in which dissociated rat dorsal root ganglion (DRG) neurons of different ages were challenged to extend neurites on fractionated myelin and cryostat sections from the PNS (sciatic nerve and myelin-free degenerated sciatic nerve) and CNS (optic nerve) of adult rats. The CNS environment of the optic nerve did not support E17 to P8 DRG neurite adhesion or outgrowth. E17 DRG neurons, unlike their older counterparts, however, were able to attach and extend neurites onto normal sciatic nerve and onto purified PNS myelin. In contrast, a vigorous neurite outgrowth response from all the ages tested was observed on the myelin-free degenerated sciatic nerve. These results indicate that PNS myelin is a potent inhibitor of neurite outgrowth and that DRG neuronal age plays an important role in determining the propensity for neurite outgrowth and regenerative response on inhibitory PNS and CNS substrata.     

Correlation of gp140 expression and NGF-induced neuroblast chemotaxis in the embryonic rat spinal cord

During rat embryogenesis, fibers containing nerve growth factor (NGF) are present near the target destinations of migratory spinal neuroblasts, suggesting that diffusible gradients of NGF provide signals to newly generated neurons in the developing cord. In vitro, pM concentrations of NGF induce neuroblast chemotaxis (directed migration along a chemical gradient), indicating evoked motility is mediated by high-affinity receptors. Binding of ¹²⁵I-labelled NGF to fetal cord cells provides additional evidence that rat spinal neuroblasts express the high-affinity receptors; however, their presence has not been directly demonstrated. In the present study, we used immunocytochemistry to show that the high-affinity NGF receptor protein, gp140^trk (trk) is detectable in embryonic spinal tissue sections and in cord dissociates. Correlation of trk expression with NGF-induced chemotaxis revealed that both the receptor protein expression and functional responses to NGF develop along a ventro-dorsal gradient that parallels the in vivo pattern of neurogenesis and migration. Analysis of the temporal changes in trk immunoreactivity demonstrated that expression of gp140^trk is bimodal, possibly reflecting multiple effects of NGF during development. Chemotaxis to NGF was blocked by nM concentrations of the kinase inhibitor, K252a, suggesting that NGF stimulates motility via high-affinity receptors coupled to kinase activity. Elevated 3′,5′-cyclic adenosine monophosphate (cAMP) also attenuated NGF-induced chemotaxis, presenting preliminary evidence that protein kinase A (PKA) may regulate motility responses to NGF.     

Cellular localization of pan-trk immunoreactivity and trkC mRNA in the enteric nervous system

The members of the trk family of tyrosine receptor kinases, trk_A, trk_B, and trk_C, are the functional receptors for neurotrophins, a family of related neurotrophic factors. In this study, we investigated 1) the distribution of neurotrophin receptors in the developing and adult rat digestive tract with a pan-trk antibody that recognizes all known trks and 2) the cellular localization of trk-encoding mRNAs in the adult gut with single-stranded RNA probes specific for trk_A, trk_B, and trk_C. In the developing myenteric plexus, trk immunoreactivity was present at embryonic day (ED) 14. Cells and fibers immunoreactive for trk could be visualized in the myenteric plexus at ED 16. At this age, dense staining was found in thick bundles of fibers in proximity to the myenteric plexus in the longitudinal muscle and in association with blood vessels in the mesentery. At ED 18, trk immunoreactivity was also seen in thin processes running from the myenteric plexus into the circular muscle, and in fibers and cells in intrapancreatic ganglia. By ED 20, immunoreactive staining was quite dense in both the myenteric and submucosal plexuses. At birth, virtually all enteric ganglia displayed strong trk immunoreactivity; the intensity of the staining at this age made it difficult to discern individual cells. During postnatal development, there was a decrease in cell body staining and an increase in the density of trk-containing fibers that became widely distributed to the gut wall and pancreas. The adult pattern of trk immunoreactivity was established between postnatal days 5 and 10. In adults, trk immunoreactivity was found in numerous enteric and intrapancreatic ganglion cells and in dense networks of fibers innervating all the layers of the gut, the pancreas, and vasculature. The trk_C mRNA was expressed in adult enteric ganglion cells of both the myenteric and submucous plexus. By contrast, the trk_A and trk_B mRNAs could not be detected in enteric ganglia. All three trk mRNAs were expressed in dorsal root ganglia, which were used as positive controls. The density and wide distribution of trk immunoreactivity together with its persistence in adulthood support the concept that neurotrophins play a broad role in the digestive system from development through adult life, perhaps being involved in differentiation, phenotypic expression, and tissue maintenance. The presence of trk_C mRNA in enteric neurons along with recent evidence that neurotrophin-3 plays a role in the development of the enteric nervous system suggest that trk_C and neurotrophin-3 are a major neurotrophin system in the gastrointestinal tract. © 1996 Wiley-Liss, Inc.     

The combined effects of trkB and trkC mutations on the innervation of the inner ear

Previous research has demonstrated that only the two neurotrophins and their cognate receptors are necessary for the support of the inner ear innervation. However, detailed analyses of patterns of innervation in various combinations of neurotrophin receptor mutants are lacking. We provide here such an analysis of the distribution of afferent and efferent fibers to the ear in various combinations of neurotrophin receptor mutants using the lipophilic tracer DiI. In the vestibular system, trkC^+/− heterozygosity aggravates the trkB^−/− mutation effect and causes almost complete loss of vestibular neurons. In the cochlea innervation, various mutations are each characterized by specific topological absence of spiral neurons in Rosenthals canal of the cochlea. trkC^−/− mutation alone or in combination with trkB^+/− heterozygosity causes absence of all basal turn spiral neurons and afferent fibers extend from the middle turn to the basal turn along inner hair cells with little or no contribution to outer hair cells. Both types of basal turn spiral neurons appear to develop and project via radial fibers to inner and, more sparingly, outer hair cells. Simple trkB^−/− mutations show a reduction of fibers to outer hair cells in the apex and, less obvious, in the basal turn. Basal turn spiral neurons may be the only neurons present at birth in the cochlea of a trkB^−/− mutant mouse combined with trkC^+/− heterozygosity. In addition, the trkB^−/− mutation combined with trkC^+/− heterozygosity has a patchy and variable loss of middle turn spiral neurons in mice of different litters. Comparisons of patterns of innervation of afferent and efferent fibers show a striking similarity of absence of fibers to topologically corresponding areas. For example, in trkC^−/− mutants afferents reach the basal turn, spiraling along the cochlea, rather than through radial fibers and efferent fibers follow the same pathway rather than emanating from intraganglionic spiral fibers. The data presented suggest that there are regional specific effects with some bias towards a specific spiral ganglion type : trkC is essential for support of basal turn spiral neurons whereas trkB appears to be more important for middle and apical turn spiral neurons.     

Cell death of adult pyramidal CA1 neurons after intraventricular injection of a novel peptide derived from trkA

Members of the nerve growth factor (NGF) family of neurotrophins bind to the second leucine-rich motif (LRM₂) within the extracellular domains of their respective receptors (trkA, trkB, trkC). Small LRM₂ peptides have been recently demonstrated to selectively bind the neurotrophins revealing similar complex binding characteristics as full-length receptors. We extend our recent findings, showing that the peptides (A and C) do not block nerve fiber outgrowth through high affinity trk receptors in a ganglia bioassay. Since the highest concentration of neurotrophins [NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3)] is found in the hippocampus, the peptides were injected into the 3rd ventricle of anesthetized adult rats. The (NGF binding) LRM₂-A peptide, but not the (BDNF binding) LRM₂-B or the (NT-3 binding) LRM₂-C peptides, caused severe apoptotic neurodegeneration of hippocampal pyramidal CA1 neurons as revealed by cresyl violet staining and the TUNEL reaction. The degeneration was protected by intrahippocampal injection of NGF-beta and by the non-N-methyl-D-aspartate (NMDA) antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), indicating a glutamatergic mechanism. In situ hybridization revealed that pyramidal CA1 neurons did not express trkA and p75 receptor mRNA in sham and LRM₂-A-lesioned animals. It is concluded that the LRM₂-A peptide represents a novel peptide with properties to induce apoptotic cell death of pyramidal CA1 neurons and may be useful as an experimental agent. J. Neurosci. Res. 50:402–412, 1997. © 1997 Wiley-Liss, Inc.     

Axonal injury and peripheral nerve grafting in the thalamus and cerebellum of the adult rat: upregulation of c-jun and correlation with regenerative potential

The protooncogene c-jun is highly expressed for long periods in axotomized PNS neurons. This may be related to their growth and regeneration. In contrast, axotomized CNS neurons show only a small and transient upregulation of c-jun. It has been suggested that there may be a correlation between this failure to maintain high levels of c-jun expression after axotomy and abortive CNS axonal regeneration. We have studied, by in situ hybridization and immunohistochemistry, the c-jun response after stab wound lesion, and after peripheral nerve grafting in the thalamus and cerebellum of the adult rat. A lesion elicits upregulation of c-jun in thalamic neurons ipsilateral to the lesion. This is most evident and prolonged in neurons such as those of the thalamic reticular nucleus, which have an established propensity to regenerate. After peripheral nerve grafting, the c-jun response in thalamic neurons is enhanced, mostly in neurons which have axons regenerating along the grafts. These neurons also upregulate growth-associated protein 43 (GAP-43). By comparison, injured Purkinje cells of the cerebellum which do not regenerate their axons along a graft, do not upregulate either c-jun or GAP-43, although they increase their expression of p75. Thus CNS neurons able to regenerate their axons along a peripheral nerve graft are those in which c-jun is induced after injury, and c-jun may play a critical role in the control of gene programs for axonal regeneration. Moreover, the observed differences in the ability of CNS neurons to regenerate their axons may relate to a difference in their intrinsic molecular response to axotomy.     

Nuclear and cytoplasmic Ca signals in developing rat dorsal root ganglion neurons studied in excised tissue

Confocal microscopy and the Ca²⁺-sensitive fluorescent dye fluo-3 were used to study subcellular Ca²⁺ signals in embryonic, neonatal, and adult dorsal root ganglion (DRG) neurons in excised dorsal rooot ganglia. Optical images obtained from isolated' whole embryonic and neonatal ganglia revealed a marked variability in the resting Ca²⁺ signals of different neurons as compared to signals in adult neurons which were uniformly faint. Many of the embryonic and neonatal neurons displayed nuclear Ca²⁺ signals at rest which were larger than those in the cytoplasm. Embryonic DRG neurons showed a significant increase in nuclear and cytoplasmic fluorescence in response to depolarization with elevated extracellular potassium or electrical stimulation. A single brief electrical stimulus was sufficient to elicit nuclear Ca²⁺ signals in a subset of the embryonic neurons. The depolarization-induced Ca²⁺ signals were blocked by removal of extracellular Ca²⁺, but not by treatment with 2,5-di (tert-butyl)- 1,4 benzohydroquinone (DTBHQ), a compound which depletes intracellular Ca²⁺ stores. The intensity of the depolarization-induced Ca²⁺ signals declined significantly between the late embryonic (E18–E20) and early postnatal time periods (P0–P1). The nuclear and cytoplasmic Ca²⁺ signals of the embryonic DRG neurons in the excised tissue preparation occur at a time of intense target innervation, suggesting a role for Ca²⁺ signals in the development and maturation of rat DRG neurons.     

Partial deletion of p75NTR in large-diameter DRG neurons exerts no influence upon the survival of peripheral sensory neurons in vivo

The p75 neurotrophin receptor (p75^NTR) is required for maintaining peripheral sensory neuron survival and function; however, the underlying cellular mechanism remains unclear. The general view is that expression of p75^NTR by the neuron itself is required for maintaining sensory neuron survival and myelination in the peripheral nervous system (PNS). Adopting a neuronal-specific conditional knockout strategy, we demonstrate the partial depletion of p75^NTR in neurons exerts little influence upon maintaining sensory neuron survival and peripheral nerve myelination in health and after demyelinating neuropathy. Our data show that the density and total number of dorsal root ganglion (DRG) neurons in 2-month-old mice is not affected following the deletion of p75^NTR in large-diameter myelinating neurons, as assessed by stereology. Adopting experimental autoimmune neuritis induced in adult male mice, an animal model of demyelinating peripheral neuropathy, we identify that deleting p75^NTR in myelinating neurons exerts no influence upon the disease progression, the total number of DRG neurons, and the extent of myelin damage in the sciatic nerve, indicating that the expression of neuronal p75^NTR is not essential for maintaining peripheral neuron survival and myelination after a demyelinating insult in vivo. Together, results of this study suggest that the survival and myelination of peripheral sensory neurons is independent of p75^NTR expressed by a subtype of neurons in vivo. Thus, our findings provide new insights into the mechanism underpinning p75^NTR-mediated neuronal survival in the PNS.     

Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: A double-label in situ hybridization study

The documented trophic actions of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) upon ventral mesencephalic dopamine neurons in vitro and in vivo are presumed to be mediated through interactions with their high-affinity receptors TrkB (for BDNF and NT-4/5) and TrkC (for NT-3). Although both neurotrophin receptor mRNAs have been detected within the rat ventral midbrain, their specific association with mesencephalic dopaminergic cell bodies remains to be elucidated. The present study was performed to determine the precise organization of trkB and trkC mRNAs within rat ventral midbrain and to discern whether the neurotrophin receptor mRNAs are expressed specifically by dopaminergic neurons. In situ hybridization with isotopically labeled cRNA probes showed that trkB and trkC mRNAs were expressed in all mesencephalic dopamine cell groups, including all subdivisions of the substantia nigra and ventral tegmental area, and in the retrorubral field, rostral and caudal linear raphe nuclei, interfascicular nucleus, and supramammillary region. Combined isotopic/nonisotopic double-labeling in situ hybridization demonstrated that virtually all of the tyrosine hydroxylase (the catecholamine biosynthetic enzyme) mRNA-containing neurons in the ventral midbrain also expressed trkB or trkC mRNAs. Additional perikarya within these regions expressed the neurotrophin receptor mRNAs but were not dopaminergic. The present results demonstrate that essentially all mesencephalic dopaminergic neurons synthesize the neurotrophin receptors TrkB and TrkC and thus exhibit the capacity to respond directly to BDNF and NT-3 in the adult midbrain in vivo. Moreover, because BDNF and NT-3 are produced locally by subpopulations of the dopaminergic cells, the present data support the notion that the neurotrophins can influence the dopaminergic neurons through autocrine or paracrine mechanisms. J. Comp. Neurol. 403:295–308, 1999. © 1999 Wiley-Liss, Inc.     

Reelin activates the small GTPase TC10 and VAMP7 to promote neurite outgrowth and regeneration of dorsal root ganglia (DRG) neurons

Axonal outgrowth is a fundamental process during the development of central (CNS) and peripheral (PNS) nervous system as well as in nerve regeneration and requires accurate axonal navigation and extension to the correct target. These events need proper coordination between membrane trafficking and cytoskeletal rearrangements and are under the control of the small GTPases of the Rho family, among other molecules. Reelin, a relevant protein for CNS development and synaptic function in the adult, is also present in the PNS. Upon sciatic nerve damage, Reelin expression increases and, on the other hand, mice deficient in Reelin exhibit an impaired nerve regeneration. However, the mechanism(s) involved the Reelin‐dependent axonal growth is still poorly understood. In this work, we present evidence showing that Reelin stimulates dorsal root ganglia (DRG) regeneration after axotomy. Moreover, dissociated DRG neurons express the Reelin receptor Apolipoprotein E‐receptor 2 and also require the presence of TC10 to develop their axons. TC10 is a Rho GTPase that promotes neurite outgrowth through the exocytic fusion of vesicles at the growth cone. Here, we demonstrate for the first time that Reelin controls TC10 activation in DRG neurons. Besides, we confirmed that the known CNS Reelin target Cdc42 is also activated in DRG and controls TC10 activity. Finally, in the process of membrane addition, we found that Reelin stimulates the fusion of membrane carriers containing the v‐SNARE protein VAMP7 in vesicles that contain TC10. Altogether, our work shows a new role of Reelin in PNS, opening the option of therapeutic interventions to improve the regeneration process.     

Strongly GD3+ cells in the developing and adult rat cerebellum belong to the microglial lineage rather than to the oligodendrocyte lineage

A recent study has shown that ramified microglia in the adult rat optic nerve express the ganglioside G_D3[Wolswijk Glia 10:244–249, 1994], thereby raising the possibility that some G_D3⁺ in the developing rat central nervous system (CNS) belong to the microglial lineage rather than to the oligodendrocyte lineage, as previously thought. To examine this possibility, sections of postnatal and adult cerebellum were doublelabelled with markers for rat microglia [the B₄ isolectin derived from Griffonia simplicifolia (GSI-B₄), the ED1 monoclonal antibody (mAb), and the OX-42 mAb] and anti-G^D3 mAbs (the mAbs R24 and LB₁). These immunolabellings showed that ramified microglia as well as amoeboid microglia are strongly G_D3⁺ in vivo. Moreover, most, if not all, cells that express high levels of G_D3 in sections of developing cerebellum appear to belong to the microglial lineage. These observations contradict previous suggestions that the strongly G_D3 ⁺ cells in the putative white matter regions of the developing brain are oligodendrocyte-type-2 astrocyte (0–2A) progenitor cells; the cells that give rise to oligodendrocytes in the CNS. The present study did, however, confirm that some 0–2A progenitor cells in sections of postnatal cerebellum are weakly G_D3⁺ in vivo. Amoeboid microglia are present in areas of the developing cerebellum where newly generated oligodendrocytes are found, suggesting that these cells play a role in the phagocytosis of the large numbers of oligodendrocytes that die as part of CNS development. © 1995 Wiley-Liss, Inc.     

Neurogenesis of subpopulations of rat lumbar dorsal root ganglion neurons including neurons projecting to the dorsal column nuclei

The time of birth of subpopulations of dorsal root ganglion (DRG) neurons was studied with immunohistochemistry for 5-bromodeoxyuridine (BrdU). Pregnant rats were injected with BrdU i.p. to label the neurons on one of the embryonic days (E) E11-E16. When they were adults, the rats were given injections of Fluoro-Gold (FG) into the gracile nucleus to identify DRG neurons projecting to this structure. Following a 5 day survival period, the animals were perfused with aldehyde fixative. Sections from the L3-L5 DRGs were processed for BrdU immunohistochemistry followed by either immunostaining for the antineurofilament antibody RT97, as marker of the light neuronal subpopulation, or histochemical staining for the B4 isolectin from Griffonia simplicifolia I, as marker of the small dark subpopulation. The results indicated that the DRG neurons were generated between E12 and E16. The RT97⁺ neurons were generated on E12–E15, with a peak at E13. FG⁺ neurons, the majority of which were RT97⁺, were also generated on E12–E15. The B4⁺ neurons were generated on E13–E16, with a peak around E14. The overall pattern of neurogenesis of the DRG neurons showed that the RT97⁺ neurons were produced prior to the B4⁺ neurons. These findings are in agreement with earlier observations that the large DRG neurons are generated earlier than the small dark neurons. Our findings also suggest the existence of a third neuronal subpopulation that might be produced at the latest period of DRG neurogenesis at E15–E16. © 1996 Wiley-Liss, Inc.     

Characterization of neurotrophin receptors by affinity crosslinking

Neurotrophic factors regulate the developmental survival and differentiation of specific neuronal populations. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of the nerve growth factor (NGF) protein family, also known as the neurotrophins. Insights into the different roles of neurotrophins can be gained by studying the expression of their functional receptors. Here we report the development of procedures for their radiolabeling and efficient crosslinking to specific cell-surface receptors. BDNF and NT-3 receptors in cell lines and tissue preparations expressing receptors for the 2 neurotrophins have been identified using this affinity crosslinking procedure. Like NGF, BDNF and NT-3 crosslinking to the low affinity NGF receptor (p75^NGFR) on PC12 cells. BDNF and NT-3 also crosslinked to cells expressing p145^trkB protein, producing an approximately 160 kD neurotrophin-receptor complex. Crosslinking of the 2 neurotrophins in vivo to specific trk family members in many areas of the central nervous system also produced a 160 kD receptor complex. However, in all brain regions a complex of approx. 100 kD could also be identified, all or most of which represents crosslinking to a truncated from of trkB. The broad distribution of BDNF and NT-3 receptors throughout the CNS suggests that neurotrophins may have yet unrecognized functions on specific neuronal populations. BDNF and NT-3 receptors were also found in brain areas in which the neurotrophins themselves are also synthesized, suggesting that beyond long-range trophic effects, these proteins may also act as autocrine or short-range paracrine regulators. © 1993 Wiley-Liss, Inc.