Stimulation of trigeminal afferents improves motor recovery after facial nerve injury: functional, electrophysiological and morphological proofs.

Recovery of mimic function after facial nerve transection is poor: the successful regrowth of axotomized motoneurons to their targets is compromised by (1) poor axonal navigation and excessive collateral branching, (2) abnormal exchange of nerve impulses between adjacent regrowing axons, and (3) insufficient synaptic input to facial motoneurons. As a result, axotomized motoneurons get hyperexcitable and unable to discharge. Since improvement of growth cone navigation and reduction of the ephaptic cross talk between axons turn out be very difficult, we concentrated our efforts on the third detrimental component and proposed that an intensification of the trigeminal input to axotomized electrophysiologically silent facial motoneurons might improve specificity of reinnervation. To test our hypothesis we compared behavioral, electrophysiological, and morphological parameters after single reconstructive surgery on the facial nerve (or its buccal branch) with those obtained after identical facial nerve surgery but combined with direct or indirect stimulation of the ipsilateral infraorbital (ION) nerve. We found that in all cases, trigeminal stimulation was beneficial for the outcome by improving the quality of target reinnervation and recovery of vibrissa! motor performance.

Functional recovery of stepping in rats after a complete neonatal spinal cord transection is not due to regrowth across the lesion site.

Rats receiving a complete spinal cord transection (ST) at a neonatal stage spontaneously can recover significant stepping ability, whereas minimal recovery is attained in rats transected as adults. In addition, neonatally spinal cord transected rats trained to step more readily improve their locomotor ability. We hypothesized that recovery of stepping in rats receiving a complete spinal cord transection at postnatal day 5 (P5) is attributable to changes in the lumbosacral neural circuitry and not to regeneration of axons across the lesion. As expected, stepping performance measured by several kinematics parameters was significantly better in ST (at P5) trained (treadmill stepping for 8 weeks) than age-matched non-trained spinal rats. Anterograde tracing with biotinylated dextran amine showed an absence of labeling of corticospinal or rubrospinal tract axons below the transection. Retrograde tracing with Fast Blue from the spinal cord below the transection showed no labeled neurons in the somatosensory motor cortex of the hindlimb area, red nucleus, spinal vestibular nucleus, and medullary reticular nucleus. Retrograde labeling transsynaptically via injection of pseudorabies virus (Bartha) into the soleus and tibialis anterior muscles showed no labeling in the same brain nuclei. Furthermore, re-transection of the spinal cord at or rostral to the original transection did not affect stepping ability. Combined, these results clearly indicate that there was no regeneration across the lesion after a complete spinal cord transection in neonatal rats and suggest that this is an important model to understand the higher level of locomotor recovery in rats attributable to lumbosacral mechanisms after receiving a complete ST at a neonatal compared to an adult stage.

A high-throughput screen to identify novel compounds to promote neurite outgrowth.

Following spinal cord injury, a variety of inhibitory molecules hinder the success of axon regeneration. The motile tip of the axon, the growth cone, shares a similar cytoskeletal array as a migrating cell, and in general the cytoskeleton is regulated by a conserved set of signaling pathways that act downstream of guidance cue and growth factor receptors. We exploit these similarities by using migrating cells as a model system to screen for extracts that promote axon outgrowth. The screen is a high-throughput wound-healing assay performed by a 96-pin tool Biogrid robot where positive candidates are identified as extracts that stimulate complete wound healing. Testing of positive candidates on chick DRG explants has lead to the identification of extracts that promote neurite outgrowth on permissive and inhibitory substrates. Extracts can be fractionated to purity, identifying novel compounds that promote neurite outgrowth on inhibitory substrates.

Electro-acupuncture induced NGF, BDNF and NT-3 expression in spared L6 dorsal root ganglion in cats subjected to removal of adjacent ganglia.

This study evaluated the effect of electro-acupuncture (EA) on the NGF, BDNF and NT-3 expression in spared L6 dorsal root ganglion (DRG) in cats subjected to bilateral removal of L1-L5 and L7-S2 DRG, using immunostaining, in situ hybridization and RT-PCR. The positive products of NGF, NT-3 protein and mRNA in the small and large neurons of spared L6 DRG in EA side increased greatly more than that of control side, while the increased BDNF was only noted in small and medium-sized neurons. RT-PCR demonstrated that the mRNA level for three factors was not influenced by EA in intact DRG, when a significant increase was seen in the spared L6 DRG of EA side. As it has been well known that DRG neurons project to the spinal cord wherein morphological plasticity has been present after DRG removal, the present results might have some bearing to the observed phenomenon.

Development of structural and functional connectivity in the thalamocortical somatosensory pathway in the wallaby.

Neuronal activity is implicated as a driving force in the development of sensory systems. In order for it to play a developmental role, however, the pathways involved must be capable of transmitting this activity. The relationship between afferent arrival, synapse formation and the onset of chemical neurotransmission has been examined using the advantageous model of a marsupial mammal, the wallaby (Macropus eugenii), to determine at what stage activity has the capacity to influence cortical development. It is known that thalamocortical afferents arrive in the somatosensory cortex on postnatal day (P)15 and that their growth cones reach to the base of the compact cell zone of the cortical plate. However, electronmicroscopy showed that thalamocortical synapses were absent at this stage. Glutamatergic responses were recorded in the cortex following stimulation of the thalamus in slices at this time but only in magnesium-free conditions. The responses were mediated entirely by N-methyl-d-aspartate (NMDA) receptors. From P28, responses could be recorded in normal magnesium and comprised a dominant NMDA-mediated component and a non-NMDA mediated component. At this time thalamocortical synapses were first identified and they were in the cortical plate. By P63 the non-NMDA-mediated component had increased relative to the NMDA-mediated component, and by P70 layer IV began to emerge and contained thalamocortical synapses. By P76 a fast non-NMDA-mediated peak dominated the response. This coincides with the appearance of cortical whisker-related patches and the onset in vivo of responses to peripheral stimulation of the whiskers.

Interplay between laminar specificity and activity-dependent mechanisms of thalamocortical axon branching.

Target and activity-dependent mechanisms of axonal branching were studied in the thalamocortical (TC) projection using organotypic cocultures of the thalamus and cortex. TC axons were labeled with enhanced yellow fluorescent protein (EYFP) by a single-cell electroporation method and observed over time by confocal microscopy. Changes in the firing activity of cocultures grown on multielectrode dishes were also monitored over time. EYFP-labeled TC axons exhibited more branch formation in and around layer 4 of the cortical explant during the second week in vitro, when spontaneous firing activity increased in both thalamic and cortical cells. Time-lapse imaging further demonstrated that branching patterns were generated dynamically by addition and elimination with a bias toward branch accumulation in the target layer. To examine the relationship between neural activity and TC branch formation, the dynamics of axonal branching was analyzed under various pharmacological treatments. Chronic blockade of firing or synaptic activity reduced the remodeling process, in particular, branch addition in the target layer. However, extension of branches was not affected by this treatment. Together, these findings suggest that neural activity can modify the molecular mechanisms that regulate lamina-specific TC axon branching.

Induction of axonal differentiation by silencing plasma membrane-associated sialidase Neu3 in neuroblastoma cells.

A reduction of 70% of the plasma membrane-associated sialidase Neu3 activity, due to a corresponding reduction of the enzyme expression by transducing cells with a short hairpin RNA encoding a sequence target (complementary messenger of mouse Neu3), caused neurite elongation in Neuro2a murine neuroblastoma cells. The differentiation process was accompanied in parallel by an increase of the acetylcholinesterase activity, a moderate increase of the c-Src expression and by the presence of the axonal marker tau protein on the neurites. The sphingolipid pattern and turnover in transduced and control cells were characterized by thin layer chromatography, mass spectrometry and metabolic radiolabeling after feeding cells with tritiated sphingosine. Control cells contained about 2 nmol of gangliosides/mg cell protein. GM2 was the main compound, followed by GD1a, GM3 and GM1. In Neu3 silenced cells, the total ganglioside content remained quite similar, but GM2 increased by 54%, GM3 remain constant, and GM1 and GD1a decreased by 66% and 50%, respectively. Within the organic phase sphingolipids, ceramide decreased by 50%, whereas the sphingomyelin content did not change in Neu3 silenced cells.

Axonal plasticity is associated with motor recovery following amphetamine treatment combined with rehabilitation after brain injury in the adult rat.

Clinical and laboratory studies have suggested that amphetamine treatment when paired with rehabilitation results in improved recovery of function after stroke or traumatic brain injury. In the present study, we investigated whether new anatomical pathways developed in association with improved motor function after brain damage and amphetamine treatment linked with rehabilitation. Following a unilateral sensorimotor cortex lesion in the adult rat, amphetamine (2 mg/kg) was administered in conjunction with physiotherapy sessions on postoperative days two and five. Physiotherapy was continued twice daily for the first 3 weeks after injury, and then once daily until week six. Performance on skilled forelimb reaching and ladder rung walking was used to assess motor improvement. Our results show that animals with sensorimotor cortical lesions receiving amphetamine treatment linked with rehabilitation had significant improvement in both tasks. Neuroanatomical tracing of efferent pathways from the opposite, non-damaged cortex resulted in the novel finding that amphetamine treatment linked with rehabilitation, significantly increased axonal growth in the deafferented basilar pontine nuclei. These results support the notion that pharmacological interventions paired with rehabilitation can enhance neuronal plasticity and thereby improve functional recovery after CNS injury.

Formation of the thalamocortical projection regulated differentially by BDNF- and NT-3-mediated signaling.

During development thalamocortical (TC) axons establish lamina-specific connections with cortical cells, and in later developmental stages TC projections are modified by activity-dependent processes. Recent studies have demonstrated that brain-derived neurotrophic factor and neurotrophin-3 are expressed in the cortex with distinct developmental time courses, and are involved not only in the formation of the TC projection but also in the subsequent refinement processes. Evidence further suggests that these actions of neurotrophins are achieved in cooperation with membrane-associated molecules expressed in cortical cells.

Pax6 guides a relay of pioneer longitudinal axons in the embryonic mouse forebrain.

We have characterized a system of early neurons that establish the first two major longitudinal tracts in the embryonic mouse forebrain. Axon tracers and antibody labels were used to map the axon projections in the thalamus from embryonic days 9.0-12, revealing several distinct neuron populations that contributed to the first tracts. Each of the early axon populations first grew independently, pioneering a short segment of new tract. However, each axon population soon merged with other axons to form one of only two shared longitudinal tracts, both descending: the tract of the postoptic commissure (TPOC), and, in parallel, the stria medullaris. Thus, the forebrain longitudinal tracts are pioneered by a relay of axons, with distinct axon populations pioneering successive segments of these pathways. The extensive merging of tracts suggests that axon-axon interactions are a major guidance mechanism for longitudinal axons. Several axon populations express tyrosine hydroxylase, identifying the TPOC as a major pathway for forebrain dopaminergic projections. To start a genetic analysis of pioneer axon guidance, we have identified the transcription factor Pax6 as critical for tract formation. In Pax6 mutants, both longitudinal tracts failed to form due to errors by every population of early longitudinal axons. Taken together, these results have identified potentially important interactions between series of pioneer axons and the Pax6 gene as a general regulator of longitudinal tract formation in the forebrain.

Differential growth of axons from sensory and motor neurons through a regenerative electrode: a stereological, retrograde tracer, and functional study in the rat.

Polyimide regenerative electrodes (RE) constitute a promising neural interface to selectively stimulate regenerating fibers in injured nerves. The characteristics of the regeneration through an implanted RE, however, are only beginning to be established. It was recently shown that the number of myelinated fibers distal to the implant reached control values 7 months postimplant; however, the functional recovery remained substantially below normal [J Biomed Mater Res 60 (2002) 517]. In this study we sought to determine the magnitude, and possible selectivity, of axonal regeneration through the RE by counting sensory and motor neurons that were retrogradely labeled from double tracer deposits in the sciatic nerve. Adult rats had their right sciatic nerves transected, and the stumps were placed in silicone tubes; some simply were filled with saline (Tube group), and others held a RE in its center (RE group). Simultaneously, the proximal stump was exposed to Diamidino Yellow. Two months later the nerves were bilaterally excised distal to the implant, and exposed to Fast Blue. Electrophysiological recordings, and skin nociceptive responses confirmed previous findings of partial functional recovery. In controls, an average of 20,000 and 3080 neurons were labeled in L4-L5 dorsal root ganglia (with minor contributions from L3 and/or L6), and in the ventral horn of the lumbar spinal cord, respectively. In the regenerating side, 35% of the DRG neurons were double-labeled, without differences between groups. In contrast, only 7.5% of motoneurons were double-labeled in the RE group, vs. 21% in the Tube group. Moreover, smaller ganglion cells regenerated better than large neurons by a significant 13.8%. These results indicate that the RE is not an obstacle for the re-growth of sensory fibers, but partially hinders fiber regeneration from motoneurons. They also suggest that fine fibers may be at an advantage over large ones to regenerate through the RE.

Development of the amygdalohypothalamic projection in the mouse embryonic forebrain.

The amygdalohypothalamic projection, a major component of the stria terminalis, is involved in the conduction of emotional and olfactory information integrated in the amygdala to the hypothalamus to elicit emotional reactions. Despite the extensive studies on functional aspects of the amygdaloid complex, developmental mechanisms of the amygdala and related structures are still poorly understood. To investigate the development of the amygdalohypothalamic projection in the mouse embryonic brain, carbocyanine dye was applied to the amygdala to label the growing axons anterogradely and to the hypothalamus to label the amygdaloid neurons retrogradely. The initial outgrowth of the stria terminalis was found to be as early as E11.5. The pathway crossed in a saddle over the internal capsule, another prominent connection in the developing forebrain of the mammalian embryo. Bipolar immature neurons were distributed along the stria terminalis at the telencephalo-diencephalic boundary, and the internal capsule was also surrounded by these cells. These cells expressed immunoreactivities to calretinin and the lot-1 antigen which has been shown to be involved in guidance of the developing lateral olfactory tract. Ultrastructural analysis revealed an adherens-like junction between the stria terminalis and the apposed cells, implying contact-mediated guidance. These results suggest that, in the development of the stria terminalis, the axonal outgrowth is guided by a mechanism similar to that of the developing lateral olfactory tract, a major amygdalopetal connection.

Tracing developing pathways in the brain: a comparison of carbocyanine dyes and cholera toxin b subunit.

The present study examined the efficiency of fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylinodocarbocyanine perchlorate and cholera toxin B subunit in tracing the crossed tectal projection to the nucleus rotundus of the thalamus (tectorotundal pathways) of paraformaldehyde-fixed and living chick embryos. The tracers were injected into the optic tectum under three experimental conditions (carbocyanine postfix, carbocyanine in vivo, and cholera toxin B subunit in vivo) and the anterograde transport of the nucleus rotundus was monitored and compared. In the carbocyanine postfix method, small crystals of carbocyanine dye were inserted into the tectum of paraformaldehyde-fixed embryos. A 6-month post-insertion period was required to label the crossed tectorotundal pathway. Results showed that tectal neurons did not begin to innervate the ipsilateral nucleus rotundus until embryonic day 9 and the contralateral nucleus rotundus until embryonic day 17. This slow progression of labeling through the crossed tectal projection resulted in significant contrast of the labeling between the ipsilateral and contralateral nuclei rotundus. In the carbocyanine in vivo method, a small volume of carbocyanine dye solution was injected into the tectum of living embryos. A 8- to 12-h survival period was sufficient enough to label the tectorotundal pathway. By embryonic day 8, the labeled axons terminated in the ipsilateral nucleus rotundus and the crossed tectorotundal projection was first detected by embryonic day 10. Similarly, in the cholera toxin B subunit in vivo method, a small volume of cholera toxin B subunit solution was injected into the tectum of living embryos. After a 6- to 10-h survival period, heavily labeled axons were found to innervate bilaterally the nucleus rotundus by embryonic day 8. This appeared to be the earliest schedule for detecting the crossed tectorotundal projection, compared with that of both the postfix and in vivo methods of carbocyanine dye. Based on the differences in the detectability of the crossed tectorotundal projection between the postfix and in vivo methods, the present data suggest that the former method is of limited purpose for labeling tectal collaterals during embryogenesis. Moreover, given the rapid transport rate and absence of photobleaching, which is often seen when using carbocyanine dye, the cholera toxin B subunit in vivo method appears to be the tracer of choice for investigating embryonic pathways.

Neurotrophin-3 is required for appropriate establishment of thalamocortical connections.

In the vertebrate brain, the thalamus serves as a relay and integration station for diverse neuronal information en route from the periphery to the cortex. Formation of the thalamocortical tract occurs during pre- and postnatal development, with distinct thalamic nuclei projecting to specific cortical regions. The molecular forces that underlie the invasion by axons into specific cortical layers followed by activity-dependent maturation of synapses are poorly understood. We show that genetic ablation of neurotrophin-3 (NT-3) in the mouse neocortex results in reduction of a set of anatomically distinct axonal bundles projecting from thalamus through cortical white matter. These bundles include thalamocortical axons that normally establish connections with retrosplenial and visual cortex, sites of early postnatal NT-3 expression. These results implicate neurotrophins in the critical stage of precise thalamocortical connections.

Layer-specific thalamocortical innervation in organotypic cultures is prevented by substances that alter neural activity.

Cortical layer IV is the major target of thalamocortical axons and many previous studies have shown that the development of this layer-specific innervation can be modelled in vitro by organotypic cocultures of thalamus and cortex. The mechanisms causing thalamic axons to terminate in layer IV are unknown. We used these in vitro models to test the possibility that neural activity plays a part in this termination process by adding substances that raise or lower levels of neural activity to the cocultures. We found that addition of tetrodotoxin or 2-amino-5-phosphonovalerate, to block activity, or potassium, to raise it, all interfered with termination in layer IV. These findings suggest that termination in layer IV requires neural activity at an appropriate level in the thalamocortical system. They also add support to recent findings that show that the importance of neural activity in development may extend to an earlier period than thought previously, to include the correct targeting of axons as well as the later refinement of connections.

Drosophila Nedd4, a ubiquitin ligase, is recruited by Commissureless to control cell surface levels of the roundabout receptor.

Crossing the midline produces changes in axons such that they are no longer attracted to the midline. In Drosophila, Roundabout reaches high levels on axons once they have crossed the midline, and this prohibits recrossing. Roundabout protein levels are regulated by Commissureless. We show that Commissureless binds to and is regulated by the ubiquitin ligase DNedd4. We further show that the ability of Commissureless to regulate Roundabout protein levels requires an intact DNedd4 binding site and ubiquitin acceptor sites within the Commissureless protein. The ability of Commissureless to regulate Robo in the embryo also requires a Commissureless/DNedd4 interaction. Our results show that changes in axonal sensitivity to external cues during pathfinding across the midline makes use of ubiquitin-dependent mechanisms to regulate transmembrane protein levels.

Arginase I and polyamines act downstream from cyclic AMP in overcoming inhibition of axonal growth MAG and myelin in vitro.

Elevation of cAMP can overcome myelin inhibitors to encourage regeneration of the CNS. We show that a consequence of elevated cAMP is the synthesis of polyamines, resulting from an up-regulation of Arginase I, a key enzyme in their synthesis. Inhibiting polyamine synthesis blocks the cAMP effect on regeneration. Either over-expression of Arginase I or exogenous polyamines can overcome inhibition by MAG and by myelin in general. While MAG/myelin support the growth of young DRG neurons, they become inhibitory as DRGs mature. Endogenous Arginase I levels are high in young DRGs but drop spontaneously at an age that coincides with the switch from promotion to inhibition by MAG/myelin. Over-expressing Arginase I in maturing DRGs blocks that switch. Arginase I and polyamines are more specific targets than cAMP for intervention to encourage regeneration after CNS injury.

Cortical and thalamic axon pathfinding defects in Tbr1, Gbx2, and Pax6 mutant mice: evidence that cortical and thalamic axons interact and guide each other.

During development, cortical areas establish precise reciprocal projections with corresponding thalamic nuclei. Pioneer axons from the cortex and thalamus first meet in the intermediate zone of the subcortical telencephalon (subpallium). Their close interactions in the subpallium suggest that they may use each other for guidance. To test this hypothesis, the development of corticothalamic and thalamocortical connections was studied in mice with mutations of transcription factor genes expressed specifically in the cortex (Tbr1), the dorsal thalamus (Gbx2), or both (Pax6). In Tbr1 mutants, cortical pioneer axons entered the subpallium at the appropriate time, but most stopped growing without entering the diencephalon. Surprisingly, thalamic axons (which do not express Tbr1) deviated into the external capsule and amygdala regions, without entering the cortex. Conversely, in most Gbx2 mutants, thalamic axons were reduced in number and grew no farther than the subpallium. Cortical axons (which do not express Gbx2) grew into the subpallium but did not enter the diencephalon. In one Gbx2- /- case, sparse thalamocortical and corticothalamic projections both developed, but in no case did one projection reach its target and not the other. In Pax6 mutants, neither corticothalamic nor thalamocortical axons reached their targets. These results suggest that thalamocortical and corticothalamic projections may not form independently. After reaching the subpallium, each projection may require a molecularly intact reciprocal projection for further guidance. This type of mechanism ensures that thalamocortical and corticothalamic axons project reciprocally. However, the exact nature of the interaction between cortical and thalamic pioneer axons remains to be elucidated.

Postnatal development of connectional specificity of corticospinal terminals in the cat.

The purpose of this study was to examine postnatal development of connectional specificity of corticospinal terminals. We labeled a small population of primary motor cortex neurons with the anterograde tracer biotinylated dextran amine. We reconstructed individual corticospinal segmental axon terminals in the spinal gray matter in cats of varying postnatal ages and adults. We found that at days 25 and 35 the segmental termination field of reconstructed axons was large, estimated to cover more than half of the contralateral gray matter. Branches and varicosities were sparse and had a relatively uniform distribution. When we examined the terminal fields of multiple axons, reconstructed over the same set of spinal sections (120-200 microm), we found that there was extensive overlap. By day 55, the morphology and termination fields had changed remarkably. There were many short branches, organized into discrete clusters, and varicosities were preferentially located within these clusters. The termination field of individual axons was substantially reduced compared with that of younger animals, and there was minimal overlap between the terminals of neighboring corticospinal neurons. In adults, a further reduction was seen in the spatial extent of terminals, branching, and varicosity density. Termination overlap was not substantially different from that in PD 55 animals. Development of spatially restricted clusters of short terminal branches and dense axonal varicosities occurred just prior to development of the motor map in primary motor cortex and may be necessary for ensuring that the corticospinal system can exert a dominant influence on skilled limb movement control in maturity.