Dorsal column sensory axons lack TrkC and are not rescued by local neurotrophin-3 infusions following spinal cord contusion in adult rats.

By reducing the progressive degeneration and disconnection of axons following spinal cord injury the functional outcome should improve. After direct transection of dorsal column sensory axons, neurotrophin-3 (NT-3) treatment can reduce degeneration and promote regeneration of the proximal stumps. Here, we tested in adult rats whether NT-3 infusion at the site of a moderate T9 spinal cord contusion would rescue sensory connections to the gracile nucleus in the medulla. Sensory projections were anterogradely traced bilaterally with injections of cholera toxin B (CTB) into the sciatic nerve 3 days before analysis. Seven days after the contusion plus intrathecal (subarachnoid) vehicle infusion as a control, the CTB-positive innervation of the gracile nucleus was reduced to approximately 25% of sham-operated rats. Intrathecal infusion of 10 microg/day of NT-3 did not affect this reduced innervation. To ensure good tissue penetration and high concentrations of NT-3 early after the injury, other rats received intraparenchymal infusions of vehicle or NT-3 near the injury site starting 2 days before until 7 days after the injury. This NT-3 treatment also did not affect the reduced innervation. This suggests that local NT-3 treatments cannot protect sensory axons from secondary degeneration after a contusive spinal cord injury. These results are likely because TrkC is not present in axons of the dorsal columns or gracile nucleus, or in other dorsal column cell types, even after the contusion. Together with published results, our data suggest that NT-3 is a peripherally--but not centrally--derived neurotrophic factor for sensory neurons.

Abnormal development of pacinian corpuscles in double trkB;trkC knockout mice.

Pacinian corpuscles depend on either Aalpha or Abeta nerve fibers of the large- and intermediate-sized sensory neurons for the development and maintenance of the structural integrity. These neurons express TrkB and TrkC, two members of the family of signal transducing neurotrophin receptors, and mice lacking TrkB and TrkC lost specific neurons and the sensory corpuscles connected to them. The impact of single or double targeted mutations in trkB and trkC genes in the development of Pacinian corpuscles was investigated in 25-day-old mice using immunohistochemistry and ultrastructural techniques. Single mutations on trkB or trkC genes were without effect on the structure and S100 protein expression, and caused a slight reduction in the number of corpuscles. In mice carrying a double mutation on trkB;trkC genes most of the corpuscles were normal with a reduction of 17% in trkB-/-;trkC+/- mice, and 8% in trkB +/-;trkC -/- mice. Furthermore, a subset of the remaining Pacinian corpuscles (23% in trkB-/-;trkC+/- mice; 3% in trkB+/-;trkC-/- mice) were hypoplasic or atrophic. Present results strongly suggest that the development of a subset of murine Pacinian corpuscles is regulated by the Trk-neurotrophin system, especially TrkB, acting both at neuronal and/or peripheral level. The precise function of each member of this complex in the corpuscular morphogenesis remains to be elucidated, though.

Haploinsufficiency in trkB and/or trkC neurotrophin receptors causes structural alterations in the aged hippocampus and amygdala.

Neurotrophins and their cognate trk receptors regulate key events, most notably survival and differentiation of specific neuron populations, during the development of the nervous system. Their functions in the adult and aged CNS are far less well understood. We have analysed mice aged 21-23 months with haploinsufficiencies of the trkB and/or trkC genes with regard to morphological alterations in the hippocampus and amygdala. Neuronal densities and total numbers of neurons in the dentate gyrus were significantly decreased in trkB+/-, trkC+/-, and trkB+/-/C+/- mutants. In the hippocampal area CA2/3, neuronal density and the total number of neurons were only reduced in double-heterozygous mice. Within the amygdala, neuronal densities were not altered. The lateral, basolateral and basomedial nuclei of the amygdala, as well as the dentate gyrus and area CA3, revealed significant increases in the densities of degenerated axonal fragments; the most pronounced changes were found in the double-heterozygous mice. Thus, partial impairment in trkB and/or trkC receptor expression caused region-specific neuron losses in the hippocampus and increased axonal fragmentation in both hippocampus and amygdala, which may result from degeneration of both intrinsic and extrinsic fibre systems. Together, these data indicate that endogenous ligands to the trkB and trkC receptors are essential to maintain neuronal integrity in the aged hippocampus and amygdala.

Reduction of NT-3 or TrkC results in fewer putative vagal mechanoreceptors in the mouse esophagus.

Intraganglionic laminar endings (IGLEs) represent major vagal afferent structures throughout the gastrointestinal tract. Both morphological and functional data suggested a mechanosensory role. Elucidation of their functional significance in a particular organ would be facilitated by the availability of animal models with significantly altered numbers of IGLEs. The present study was aimed at searching for mouse strains fulfilling this criterion in the esophagus. Anterograde wheat germ agglutinin-horseradish peroxidase tracing (WGA-HRP) from nodose ganglion was used in order to label esophageal IGLEs in mice deficient for neurotrophin-3 (NT-3) or tyrosine kinase C-receptor (TrkC) and in control littermates. This approach was feasible only in heterozygous mutants which are viable. IGLEs were counted in tetramethylbenzidine (TMB) processed wholemounts using a standardised protocol. Quantification of myenteric neurons was done in cuprolinic blue-stained specimens. Nodose neuron counts were performed in cryostat sections stained with cresyl violet. Numbers of IGLEs in the esophagus were significantly reduced in both heterozygous NT-3 (NT-3+/-) and heterozygous TrkC (TrkC+/-) mutants (65% and 40% reduction, respectively). Numbers of nodose neurons were also significantly reduced in NT-3+/- mice (48% reduction), while their reduction in TrkC+/- mutants was insignificant (11% reduction). There was no reduction of myenteric neurons in the esophagus of either mutant strain. The numeric deficiency of IGLEs was unlikely to be secondary to reduction of myenteric neurons. Although only heterozygous mutants could be studied, these results suggest that esophageal IGLEs share neurotrophin dependence on NT-3/TrkC with spinal proprioceptors and some cutaneous mechanosensors. This concurs with their proposed function as vagal mechanosensors crucial for reflex peristalsis.

Neural crest stem cell and cardiac endothelium defects in the TrkC null mouse.

TrkC null mice have multiple cardiac malformations. Since neural crest cells participate in cardiac outflow tract septation, the aim of this study was to determine at the cellular level the putative neural crest defect. We have identified three types of progenitor cells: stem cells that undergo self-renewal and can generate many cell types, cells that are restricted in their developmental potentials, and cells that are committed to the smooth muscle cell lineage. In TrkC null mice, there is a greater than 50% decrease in stem cell numbers and an equivalent increase in fate-restricted cells. The outflow tract wall is thickened and the endothelial tube is disorganized. We conclude that deletion of the TrkC gene causes precocious fate restrictions of the neural crest stem cell and a defect of the outflow tract endothelium, both of which may contribute to the outflow tract malformations that occur in TrkC null mice.

Runx3 is essential for the target-specific axon pathfinding of trkc-expressing dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons project their axons to specific target layers in the gray matter of the spinal cord, according to their sensory modality (Neuron 30 (2001), 707; Cell 101 (2000), 485; Neuron 31 (2001), 59; J. Comp. Neurol. 380 (1997), 215; Sensory Neurons, Oxford Univ. Press, New York, 1992, p. 131). Expression of runt-related Runx/AML genes (Mech. Dev. 109 (2001), 413) on subtypes of DRG neurons suggests their involvement in lamina-specific afferent differentiation and maturation. Here we show that Runx3-/- mice display severe limb ataxia and abnormal posture and that most of them die shortly after birth. They show that proprioceptive afferent axons fail to reach the ventral horn and have a smaller dorsal funiculus in their spinal cords. Despite the strong resemblance of this phenotype to that of knockout mice deficient in neurotrophin-3 (NT-3) (Cell 77 (1994), 503; Nature 369 (1994), 658) and its receptor, trkC, (Nature 368 (1994), 249), which show proprioceptive afferent loss through selective neuronal cell death, Runx3-/- mice maintain normal number of TrkC/trkC positive DRG neurons throughout development. Our results suggest that Runx3 controls the target-specific axon pathfinding of trkC-expressing DRG neurons in the spinal cord.

Ruffini endings are absent from the periodontal ligament of trkB knockout mice.

To clarify the role of neurotrophin receptors in the development of Ruffini endings, periodontal ligaments and trigeminal ganglia of trkA, trkB, and trkC knockout mice were immunostained for protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), parvalbumin (PV), and calretinin (CR). Innervation patterns of PGP 9.5- and CGRP-immunoreactive fibers were examined in the periodontal ligament of the knockout mice. PGP 9.5-positive fibers in the incisal periodontal ligaments of trkA and trkC knockout mice form Ruffini endings distinguished by dendritic ramifications and branches. However, Ruffini endings were not present in the periodontal ligament of trkB knockout mice. Only free nerve endings were observed in tissue of trkB knockout mice. Compared with trkA and trkC knockouts, the proportion of CR-positive neurons in mandibular and maxillary regions of the trigeminal ganglion of trkB knockout mice is decreased. These findings indicate that the development of periodontal Ruffini endings is regulated by trkB-dependent and CR-coexpressing neurons.

Testis developmental phenotypes in neurotropin receptor trkA and trkC null mutations: role in formation of seminiferous cords and germ cell survival.

The objective of the present study was to determine if the neurotropin receptors trkC and trkA are involved in embryonic testis development. These receptors bind neurotropin 3 and nerve growth factor, respectively. The hypothesis tested was that the absence of trkC or trkA receptors will have detrimental effects on testis development and morphology. The trkA and trkC homozygote knockout (KO) mice generally die either at or shortly after birth. Therefore, heterozygote mice were mated to obtain homozygote gene KO mice at Embryonic Day (E) 13, E14, E17, and E19 of gestation, with E0 being the plug date. Gonads from approximately 80 embryos were collected and fixed, and each embryo was genotyped. To determine gonadal characteristics for each genotype, the number of germ cells, number of seminiferous cords, seminiferous cord area, and interstitial area were calculated at each developmental age. Germ cell numbers varied in trkA gene KO mice from those of wild-type mice at each age evaluated. In trkC gene KO mice, differences were detected in germ cell numbers when compared to wild-type mice at E17 and E19. At E19, germ cell numbers were reduced in both trkA and trkC gene KO mice when compared to wild-type animals. Apoptosis was evaluated in testes of wild-type, trkC gene KO, and trkA gene KO mice to determine if the alteration in germ cell numbers at each developmental age was influenced by different patterns of germ cell survival or apoptosis. No differences were found in germ cell apoptosis during embryonic testis development. Interestingly, trkA gene KO mice that survived to Postnatal Day 19 had a 10-fold increase in germ cell apoptosis when compared to germ cells in wild-type mice. Evaluation of other morphological testis parameters demonstrated that trkC KO testes had reduced interstitial area at E13, reduced number of seminiferous cords at E14, and reduced seminiferous cord area at E19. The trkA gene KO testes had a reduction in the number of seminiferous cords at E14. Histology of both trkA and trkC gene KO testes demonstrated that these gonads appear to be developmentally delayed when compared to their wild-type testis counterparts at E13 during testis development. The current study demonstrates that both trkA and trkC neurotropin receptors influence germ cell numbers during testis development and events such as seminiferous cord formation.

Target-derived neurotrophic factors regulate the death of developing forebrain neurons after a change in their trophic requirements.

Many neurons die as the normal brain develops. How this is regulated and whether the mechanism involves neurotrophic molecules from target cells are unknown. We found that cultured neurons from a key forebrain structure, the dorsal thalamus, develop a need for survival factors including brain-derived neurotrophic factor (BDNF) from their major target, the cerebral cortex, at the age at which they innervate it. Experiments in vivo have shown that rates of dorsal thalamic cell death are reduced by increasing cortical levels of BDNF and are increased in mutant mice lacking functional BDNF receptors or thalamocortical projections; these experiments have also shown that an increase in the rates of dorsal thalamic cell death can be achieved by blocking BDNF in the cortex. We suggest that the onset of a requirement for cortex-derived neurotrophic factors initiates a competitive mechanism regulating programmed cell death among dorsal thalamic neurons.

Defects in sensory axon growth precede neuronal death in Brn3a-deficient mice.

Brn3a/Brn-3.0 is a POU-domain transcription factor expressed in primary sensory neurons of the cranial and dorsal root ganglia and in specific neurons in the caudal CNS. Mice lacking Brn3a undergo extensive sensory neural death late in gestation and die at birth. To further examine Brn3a expression and the abnormalities that accompany its absence, we constructed a transgene containing 11 kb of Brn3a upstream regulatory sequence linked to a LacZ reporter. Here we show that these regulatory sequences direct transgene expression specifically to Brn3a peripheral sensory neurons of the cranial and dorsal root ganglia. Furthermore, expression of the 11 kb/LacZ reporter in the sensory neurons of the mesencephalic trigeminal, but not other Brn3a midbrain neurons, demonstrates that cell-specific transgene expression is targeted to a functional class of neurons rather than to an anatomical region. We then interbred the 11 kb/LacZ reporter strain with mice carrying a null mutant allele of Brn3a to generate 11 kb/LacZ, Brn3a knock-out mice. beta-Galactosidase expression in these mice reveals significant axonal growth defects, including excessive and premature branching of the major divisions of the trigeminal nerve and a failure to correctly innervate whisker follicles, all of which precede sensory neural death in these mice. These defects in Brn3a(-/-) mice resemble strongly those seen in mice lacking the mediators of sensory pathfinding semaphorin 3A and neuropilin-1. Here we show, however, that sensory neurons are able to express neuropilin-1 in the absence of Brn3a.

Trk C receptor signaling regulates cardiac myocyte proliferation during early heart development in vivo.

Neurotrophin-3 (NT-3) is a member of the neurotrophin family of growth factors, best characterized by its survival- and differentiation-inducing effects on developing neurons bearing the trk C receptor tyrosine kinase. Through analysis of NT-3 and trk C gene-targeted mice we have identified NT-3 as critically regulating cardiac septation, valvulogenesis, and conotruncal formation. Although these defects could reflect cardiac neural crest dysfunction, the expression of NT-3 and trk C by cardiac myocytes prior to neural crest migration prompted analysis of cell-autonomous actions of NT-3 on cardiac myocytes. Retroviral-mediated overexpression of truncated trk C receptor lacking kinase activity was used to inhibit activation of trk C by endogenous NT-3, during early heart development in ovo. During the first week of chicken development, expression of truncated trk C reduced myocyte clone size by more than 60% of control clones. Direct mitogenic actions of NT-3 on embryonic cardiac myocytes were demonstrated by analysis of BrdU incorporation or PCNA immunoreactivity in control and truncated trk C-expressing clones. Inhibition of trk C signaling reduced cardiac myocyte proliferation during the first week of development, but had no effect at later times. These studies demonstrate that endogenous NT-3:trk C signaling regulates cardiac myocyte proliferation during cardiac looping and the establishment of ventricular trabeculation but that myocyte proliferation becomes NT-3 independent during the second week of embryogenesis.

Proprioceptive afferents survive in the masseter muscle of trkC knockout mice.

Peripheral innervation patterns of proprioceptive afferents from dorsal root ganglia and the mesencephalic trigeminal nucleus were assessed in trkC-deficient mice using immunohistochemistry for protein gene product 9.5 and parvalbumin. In trkC knockout mice, spinal proprioceptive afferents were completely absent in the limb skeletal muscles, M. biceps femoris and M. gastrocnemius, as previously reported. In these same animals, however, proprioceptive afferents from mesencephalic trigeminal nucleus innervated masseter muscles and formed primary endings of muscle spindles. Three wild-type mice averaged 35.7 spindle profiles (range: 31-41), six heterozygotes averaged 32.3 spindles (range: 27-41), and four homozygotes averaged 32.8 spindles (range: 26-42). Parvalbumin and Nissl staining of the brain stem showed approximately 50% surviving mesencephalic trigeminal sensory neurons in trkC-deficient mice. TrkC-/- mice (n = 5) had 309.4 +/- 15.9 mesencephalic trigeminal sensory cells versus 616.5 +/- 26.3 the sensory cells in trkC+/+ mice (n = 4). These data indicate that while mesencephalic trigeminal sensory neurons are significantly reduced in number by trkC deletion, they are not completely absent. Furthermore, unlike their spinal counterparts, trigeminal proprioceptive afferents survive and give rise to stretch receptor complexes in masseter muscles of trkC knockout mice. This indicates that spinal and mesencephalic trigeminal proprioceptive afferents have different neurotrophin-supporting system during survival and differentiation. It is likely that one or more other neurotrophin receptors expressed in mesencephalic trigeminal proprioceptive neurons of trkC knockout mice compensate for the lack of normal neurotrophin-3 signaling through trkC.

Effects of neurotrophin and neurotrophin receptor disruption on the afferent inner ear innervation.

Two neurotrophins and their two receptors appear to regulate the survival of vestibular and cochlear neurons in the developing ear. Mice lacking either brain derived neurotrophic factor (BDNF) or its associated receptor, Trk B, show a severe reduction in the number of vestibular neurons and a loss of all innervation to the semicircular canals. Mice lacking NT-3 or its receptor, Trk C, show a severe reduction of spiral neurons in the basal turn of the cochlea. Mice lacking both BDNF and NT-3 or Trk B and Trk C, reportedly lose all innervation to the inner ear. These two neurotrophins and their associated receptors are necessary for the normal afferent innervation of the inner ear.