Peripheral specification of sensory connections in the spinal cord

During development, primary sensory neurons innervate peripheral targets and then form central connections appropriate to these targets. Sensory neurons induced to innervate novel peripheral targets by surgical manipulations in Rana catesbeiana tadpoles form central connections appropriate to their novel targets even when they must project into novel areas of the spinal cord to do so. The selectivity displayed by sensory neurons innervating different peripheral targets suggests that they may acquire distinctive biochemical properties from their targets that endow them with affinities for appropriate central neurons.     

Barium action potentials in regenerating axons of the lamprey spinal cord

Intracellular recordings were obtained from growing tips of regenerating giant axons in the lamprey spinal cord, the recording sites verified by Lucifer yellow injection. In the presence of extracellular Ba++ (3-6 mM), tetraethylammonium (10-15 mM), and 4-aminopyridine (4-6 mM), action potentials showed prolonged plateaus. The fast initial phase of the action potential, but not the plateau (Ba++-spike), was blocked by tetrodotoxin (10(-6) gm/ml). The Ba++ spike was associated with increased membrane conductance and could be terminated with hyperpolarizing current pulses. Normal axons did not generate similar Ba++ spikes. However, TTX-resistant, voltage-dependant conductance changes could be elicited in normal axons if much higher concentrations of Ba++ (18-30 mM) were used. Their rate of rise was slower than in regenerating axons (0.6 V/sec vs 3.2 V/sec; n = 5), and the response did not outlast the current pulse. The Ba++ responses in normal and regenerating axons were blocked by ions known to block voltage-gated Ca++ conductances (Co++, Ni++, or Cd++). Therefore, these spikes probably represent Ba++ entry through voltage-dependent Ca++ channels, suggesting the presence of a higher-than-average voltage-dependent Ca++ conductance in the growing axon. However, Ca++-dependent spikes could not be obtained under any conditions in either normal or regenerating axons. Simultaneous intracellular recordings from growth cones and axons indicated that the Ba++ spike was initiated, in most cases, at the growth cone. The Ba++ spikes were recorded in regenerating axons for as long as 50 days following cord transection and were not correlatable with the "dying-back" phenomenon in cut axons, which usually is over before day 6. The concept of a higher-than-average voltage-dependent Ca++ conductance in growing tips of regenerating axons is in agreement with the hypothesis that Ca++ is important in regeneration and that regeneration may be related to the process of chemical synaptic transmission.     

Heat production associated with synaptic transmission in the bullfrog spinal cord

By using heat detectors made with pyroelectric film, rapid heat production by the bullfrog spinal cord in response to dorsal root stimulation has been demonstrated. The heat production rises to its peak in about 100 ms after the arrival of afferent impulses and falls slowly with a time course comparable to that of the dorsal root potential. Stimulation of the ventral roots produces no detectable heat. The heat production was reversibly suppressed by immersion of the cord in a low Ca2+, high Mg2+ salt solution, indicating that the underlying exothermic process is associated with intraspinal synaptic transmission. The source of this 'synaptic heat' is located near the boundary between the dorsal column and the substantia gelatinosa in the vicinity of the stimulated dorsal roots.     

Regeneration of axons and synaptic connections by touch sensory neurons in the leech central nervous system

In studies of axonal regeneration, it has been difficult to determine (a) whether growth along the normal pathway is important for restoration of connections with previous targets and (b) whether the new synapses resemble the old in strength and location. To address these problems at the level of individual nerve cells, we have studied touch (T) sensory neurons in the leech after their axons have been severed and we have confirmed that their axons regenerate electrical connections with some of their usual synaptic targets in the central nervous system. Injections of horseradish peroxidase and Lucifer Yellow dye into separate T cells in unoperated animals showed that T cell axons typically run close to one another within single ganglia or from ganglion to ganglion. Knowledge of one T cell's arborizations thus revealed the groundplan of others in the same ganglia and the sites of apparent contact with its synaptic targets. For regenerating axons, those sprouts that encountered the normal pathway (as marked by homologous axons) grew preferentially along it. Despite the striking coincidence of old and new pathways, regenerated branching patterns within the ganglionic neuropils were usually incomplete and sometimes had atypical branches. Synaptic connections with normal targets (other T cells as well as S and C cells) were abnormally weak physiologically. The numbers of apparent contacts seen with the light microscope were also lower than normal. In addition, the strength of the synaptic potentials, normalized to the number of contacts (calculated as microvolts per contact), was generally smaller in the regenerated connections than in the controls, and smallest at earliest times, during the first 6 weeks following injury. It thus appears to be characteristic of T cell regeneration that axon regrowth is aided by the recognition of specific pathways and that successful regeneration, as assayed anatomically and physiologically, occurs frequently but usually incompletely.     

Ultrastructure and synaptic connections of cutaneous afferent fibres in the spinal cord

The combination of intracellular recording and staining techniques with electron microscopy offers a unique opportunity for correlating physiological and ultrastructural properties of single neurones. The spinal terminations of cutaneous primary afferent fibres have been studied by this method, and examination of the ultrastructural synaptology of their boutons sheds light on how sensory information may be processed at the first synapse in the spinal cord. The combination of intracellular staining techniques with other neuroanatomical tract-tracing methods can reveal some of the neuronal networks of the spinal cord and, particularly, the contributions made by primary afferent fibres to these networks.     

Neurotrophins promote regeneration of sensory axons in the adult rat spinal cord

We have investigated the effects of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) on the intraspinal regeneration of anterogradely labeled axotomized ascending primary sensory fibers in the adult rat. These fibers were allowed to grow across a predegenerated peripheral nerve graft and back into the thoracic spinal cord. In control animals that had been infused with vehicle for two weeks into the dorsal column, 3 mm rostral to the nerve graft, essentially no fibers had extended from the nerve graft back into the spinal cord. The number of sensory fibers in the rostral end of the nerve graft was not significantly different between control and neurotrophin-infused animals. With infusion of NGF, 37+/-2% of the fibers at the rostral end of the graft had grown up to 0.5 mm into the dorsal column white matter, 30+/-2% up to 1 mm, 19+/-3% up to 2 mm and 8+/-2% up to 3 mm, i.e., the infusion site. With infusion of NT-3, sensory fiber outgrowth was similar to that seen with NGF, but with BDNF fewer fibers reached farther distances into the cord. Infusion of a mixture of all three neurotrophins did not increase the number of regenerating sensory fibers above that seen after infusion of the individual neurotrophins. These findings suggest that injured ascending sensory axons are responsive to all three neurotrophins and confirm our previous findings that neurotrophic factors can promote regeneration in the adult central nervous system.     

Protein synthetic machinery and mRNA in regenerating tips of spinal cord axons in lamprey

Polyribosomes, mRNA, and other elements of translational machinery have been reported in peripheral nerves and in elongating injured axons of sensory neurons in vitro, primarily in growth cones. Evidence for involvement of local protein synthesis in regenerating central nervous system (CNS) axons is less extensive. We monitored regeneration of back‐labeled lamprey spinal axons after spinal cord transection and detected mRNA in axon tips by in situ hybridization and microaspiration of their axoplasm. Poly(A)+mRNA was present in the axon tips, and was more abundant in actively regenerating tips than in static or retracting ones. Target‐specific polymerase chain reaction (PCR) and in situ hybridization revealed plentiful mRNA for the low molecular neurofilament subunit and β‐tubulin, but very little for β‐actin, consistent with the morphology of their tips, which lack filopodia and lamellipodia. Electron microscopy showed ribosomes/polyribosomes in the distal parts of axon tips and in association with vesicle‐like membranes, primarily in the tip. In one instance, there were structures with the appearance of rough endoplasmic reticulum. Immunohistochemistry showed patches of ribosomal protein S6 positivity in a similar distribution. The results suggest that local protein synthesis might be involved in the mechanism of axon regeneration in the lamprey spinal cord. J. Comp. Neurol. 524:3614–3640, 2016. © 2016 Wiley Periodicals, Inc.     

Induced expression of polysialic acid in the spinal cord promotes regeneration of sensory axons

After spinal cord injury axonal regeneration is prevented by glial scar formation. In this study we examined whether induced expression of polysialic acid (PSA) in the lesion site would render the glial scar permissive to axonal regeneration after dorsal column transection. PSA was induced by lentiviral vector-mediated expression of polysialyltransferase (LV/PST). PSA expression increased astrocyte infiltration and permitted the penetration of regenerating axons across the caudal border of the lesion and into the lesion cavity. In LV/PST-injected animals with a peripheral nerve-conditioning lesion, 20 times more axons grew into the lesion cavity than those LV/GFP-injected plus conditioning lesion, and some axons grew across the cavity and extended to the rostral cord, while in LV/GFP group most ascending axons terminated at the caudal border of the lesion. Our result suggests that induced expression of PSA can provide a favorable environment for axonal regeneration.     

The activity-dependent plasticity of segmental and intersegmental synaptic connections in the lamprey spinal cord

Activity-dependent synaptic plasticity has been proposed as a contributory factor in the patterning of rhythmic network activity. However, its role has not been examined in detail. Here, paired or triple intracellular recordings have been made from identified neurons in the lamprey locomotor network to examine the potential relevance of activity-dependent synaptic plasticity in segmental and intersegmental spinal networks. Segmental inputs from glutamatergic excitatory interneurons (EIN) to ipsilateral glycinergic crossed caudal (CC) interneurons were facilitated or depressed during spike trains at 5-20 Hz. Connections between EINs were depressed. Glycinergic inputs from small ipsilateral inhibitory interneurons were depressed in motor neurons, but were facilitated in CC interneurons. Excitatory inputs from small crossing interneurons to motor neurons were depressed, whereas inhibitory inputs were unaffected. With the exception of connections between EINs, significant effects occurred with stimulation that mimicked interneuron spiking during network activity. Intersegmental EIN synaptic properties were also investigated. EIN inputs did not differ significantly when examined from zero to four segments rostral to motor neurons or CC interneurons. However, caudally located EINs evoked greater activity-dependent facilitation than did rostral EINs. Whilst the amplitude or plasticity of EIN inputs in the rostral or mid-trunk regions of the spinal cord did not differ, EINs in the caudal trunk region evoked greater facilitation. Synaptic transmission between locomotor network neurons thus exhibits activity-dependent plasticity in response to physiologically relevant stimulation. Activity-dependent plasticity could thus contribute to the patterning of the rhythmic network output.     

Reinnervation of a denervated skeletal muscle by spinal axons regenerating through a collagen channel directly implanted into the rat spinal cord

In the present study, the continuity between the central nervous system (CNS) and the peripheral nervous system (PNS) was restored by mean of a collagen channel in order to reinnervate a skeletal muscle. Three groups of animals were considered. In the first group, one end of the collagen channel was implanted in the cervical spinal cord of adult rats. The other end was connected to a 30-mm autologous peripheral nerve graft (PNG) implanted into the denervated biceps brachii muscle. The gap between the spinal cord and the proximal nerve stump varied from 3 to 7 mm. In the second group of animals, the distal end of the PNG graft was ligatured in order to compare the survival of the growing axons in the presence and in the absence of a muscular target. In the third group of animals, the extraspinal stump of the collagen channel was ligatured. Our study demonstrates that spinal neurons and dorsal root ganglion (DRG) neurons can grow long axons through the collagen channel over a 7-mm gap and reinnervate a denervated skeletal muscle. The results also indicate that the presence of a PNG at the extraspinal stump of the collagen channel is essential for axonal regrowth and that the muscle target contributes to the long-term maintenance of the regenerating axons. These data might be interesting for clinical application when the continuity between the CNS and PNS is interrupted such as in root avulsion.     

Functional connections are established in the deafferented rat spinal cord by peripherally transplanted human embryonic sensory neurons

Functionally useful repair of the mature spinal cord following injury requires axon growth and the re-establishment of specific synaptic connections. We have shown previously that axons from peripherally grafted human embryonic dorsal root ganglion cells grow for long distances in adult host rat dorsal roots, traverse the interface between the peripheral and central nervous system, and enter the spinal cord to arborize in the dorsal horn. Here we show that these transplants mediate synaptic activity in the host spinal cord. Dorsal root ganglia from human embryonic donors were transplanted in place of native adult rat ganglia. Two to three months after transplantation the recipient rats were examined anatomically and physiologically. Human fibres labelled with a human-specific axon marker were distributed in superficial as well as deep laminae of the recipient rat spinal cord. About 36% of the grafted neurons were double labelled following injections of the fluorescent tracers MiniRuby into the sciatic and Fluoro-Gold into the lower lumbar spinal cord, indicating that some of the grafted neurons had grown processes into the spinal cord as well as towards the denervated peripheral targets. Electrophysiological recordings demonstrated that the transplanted human dorsal roots conducted impulses that evoked postsynaptic activity in dorsal horn neurons and polysynaptic reflexes in ipsilateral ventral roots. The time course of the synaptic activation indicated that the human fibres were non-myelinated or thinly myelinated. Our findings show that growing human sensory nerve fibres which enter the adult deafferentated rat spinal cord become anatomically and physiologically integrated into functional spinal circuits.     

Direct synaptic contacts of catecholamine axons on the preganglionic sympathetic neurons in the rat thoracic spinal cord

Preganglionic sympathetic neurons were labelled by retrograde transport of horseradish peroxidase, while catecholamine axon varicosities were marked by the uptake of 5-hydroxydopamine in the intermediolateral nucleus of the rat. The direct synaptic contacts from the catecholamine axons to the preganglionic sympathetic neurons were demonstrated. Catecholamine axons formed symmetric synapses.     

Relative numbers of several types of synaptic connections in the substantia gelatinosa of the cat spinal cord

The relative numbers of axo-dendritic, axo-axonic, dendroaxonic and dendro-dendritic synapses were determined by classifying and recording all such specialized contacts in sample areas of the substantia gelatinosa. The samples were taken from segments L1-L5 of the cat spinal cord. In the glomerular complexes 97% of all synapses were recorded as axon-dendritic. In substantia gelatinosa deprived of glomerular complexes by dorsal root section, 96.5% were axo-dendritic. The remainders were about equally divided between axo-amonic, dendro-dendritic and dendro-axonic synapses.     

Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons

Most current studies quantify axon regeneration by immunostaining regeneration-associated proteins,representing indirect measurement of axon lengths from both sensory neurons in the dorsal root ganglia and motor neurons in the spinal cord.Our recently developed method of in vivo electroporation of plasmid DNA encoding for enhanced green fluorescent protein into adult sensory neurons in the dorsal root ganglia provides a way to directly and specifically measure regenerating sensory axon lengths in whole-mount nerves.A mouse model of sciatic nerve compression was established by squeezing the sciatic nerve with tweezers.Plasmid DNA carrying enhanced green fluorescent protein was transfected by ipsilateral dorsal root ganglion electroporation 2 or 3 days before injury.Fluorescence distribution of dorsal root or sciatic nerve was observed by confocal microscopy.At 12 and 18 hours,and 1,2,3,4,5,and 6 days of injury,lengths of regenerated axons after sciatic nerve compression were measured using green fluorescence images.Apoptosis-related protein caspase-3 expression in dorsal root ganglia was determined by western blot assay.We found that in vivo electroporation did not affect caspase-3 expression in dorsal root ganglia.Dorsal root ganglia and sciatic nerves were successfully removed and subjected to a rapid tissue clearing technique.Neuronal soma in dorsal root ganglia expressing enhanced green fluorescent protein or fluorescent dye-labeled microRNAs were imaged after tissue clearing.The results facilitate direct time course analysis of peripheral nerve axon regeneration.This study was approved by the Institutional Animal Care and Use Committee of Guilin Medical University,China(approval No.GLMC201503010)on March 7,2014.     

Glial scar size, inhibitor concentration, and growth of regenerating axons after spinal cord transection

A mathematical model has been formulated in accordance with cell chemotaxis and relevant experimental data. A three-dimensional lattice Boltzmann method was used for numerical simulation. The present study observed the effects of glial scar size and inhibitor concentration on regenerative axonal growth following spinal cord transection. The simulation test comprised two parts: (1) when release rates of growth inhibitor and promoter were constant, the effects of glial scar size on axonal growth rate were analyzed, and concentrations of inhibitor and promoters located at the moving growth cones were recorded. (2) When the glial scar size was constant, the effects of inhibitor and promoter release rates on axonal growth rate were analyzed, and inhibitor and promoter concentrations at the moving growth cones were recorded. Results demonstrated that (1) a larger glial scar and a higher release rate of inhibitor resulted in a reduced axonal growth rate. (2) The axonal growth rate depended on the ratio of inhibitor to promoter concentrations at the growth cones. When the average ratio was < 1.5, regenerating axons were able to grow and successfully contact target cells.     

The origin, distribution and synaptic relationships of substance P axons in rat spinal cord.

Substance P positive (SP⁺) immunoperoxidase reaction product has been localized in light and electron microscopic preparations of rat lumbar spinal cord using an immunocytochemical method. SP⁺ reaction product was found to be highly concentrated in dorsal horn laminae I, II, portions of III, Lissauer's tract, s small nucleus in the dorsal part of the lateral funiculus, and in a longitudinal bundle of fibers just ventral to the central canal. Moderate accumulations of SP⁺ reaction product were observed in portions of laminae III-V, lamina X and in a narrow zone bordering fasciculus gracilis which expanded in its ventral aspect to include nucleus cornucommissuralis dorsalis and nucleus dorsalis. Remaining portions of the spinal gray matter exhibited extremely sparse staining. Light microscopic observations indicated that SP⁺ product was concentrated in fine axons and collaterals, which displayed densely-stained varicosities and terminal puncta. These varicosities and puncta were closely associated with neurons and blood vessels. Dorsal rhizotomy produced a marked reduction in the number of SP⁺ fibers in Lissauer's tract, dorsal horn laminae I-III and the nucleus of the dorsolateral funiculus. However, dorsal root fibers were not the only source of SP⁺ structures in these regions since some SP⁺ fibers remained following dorsal root resections. In addition, this residual population of SP⁺ fibers did not appear to be altered by a combination of dorsal rhizotomy and ipsilateral transverse hemisection of the spinal cord. In conjunction with the results of other investigators, this finding suggests that the SP⁺ fibers which remain after dorsal rhizotomy are derived from local circuit interneurons of the spinal cord. Electron microscopic observations revealed that certain axons and axon terminals contained concentrations of SP⁺ product which were associated with large, granular vesicles, while lesser amounts were in the terminal cytoplasm and associated with the exterior surfaces of small, agranular synaptic vesicles. SP⁺ terminals formed axodendritic, axosomatic and axoaxonal synapses, but the deposition of SP⁺ product often was not concentrated precisely at the “active sites” of these synaptic junctions. Some SP⁺ axons also made rudimentary contacts with astrocytic processes including those surrounding blood vessels. Since many of the SP⁺ terminals are similar in several respects to neuroendocrine terminals, it is possible that substance P may be released at several different types of non-synaptic sites as well as at conventional synaptic junctions. Thus our findings suggest that substance P could participate in a variety of neural functions ranging from those which are limited to synaptic junctions to those which are more generally distributed via an involvement of axonal plexuses as well as the glial, vascular and ventricular systems.     

Regeneration of sensory-motor synapses in the spinal cord of the bullfrog

Sensory fibers innervating muscles in the arm of the bullfrog form specific patterns of monosynaptic connections with motoneurons in the spinal cord. We show here that these normal patterns are re-established after interruption of the second dorsal root (DR2) in tadpoles and postmetamorphic frogs. DR2 was either cut or crushed, and 2 to 8 months later the extent and specificity of regeneration were assessed anatomically and physiologically. Horseradish peroxidase labeling of DR2 showed that sensory afferents had regenerated back into the spinal cord to form local arborizations, as in the normal adult. However, few long-tract fibers in the dorsal columns regenerated. Intracellular recording from different classes of motoneurons at the level of DR2 revealed that triceps muscle sensory afferents had regenerated to form functionally appropriate synapses. As in the normal adult, stimulation of the triceps nerves elicited larger monosynaptic EPSPs in triceps motoneurons than in non-triceps motoneurons. Thus, in the central nervous system of the bullfrog, specific monosynaptic connections are re-formed within the region of local regeneration.     

Specificity of neuromuscular connections during early development and following regeneration of motor axons in the bullfrog

The specificity of neuromuscular connectivity was examined in unoperated bullfrog (Rana catesbeiana) tadpoles and in tadpoles that had undergone transection of the three ventral roots that normally innervate the hindlimb. The specificity of motoneuron projections was assessed by applying small amounts of horseradish peroxidase to circumcribed hindlimb regions and mapping the locations of retrogradely labeled motoneurons within the lumbar lateral motor column (LMC). In unoperated tadpoles, the locations of retrogradely labeled motoneurons in the LMC were as circumscribed at early stages of development as in tadpoles examined after motoneuron number in the LMC had stabilized. Six to eight weeks after ventral root transection in young tadpoles, localization of retrogradely labeled motoneurons was almost as circumscribed as found in unoperated tadpoles. However, localization following regeneration became less precise in more advanced tadpoles. If the ventral roots of adult frogs were crushed rather than transected, motor axon regeneration was considerably more precise, confirming previous reports (Westerfield and Powell, 1983). The implications of these results for hypotheses of neuromuscular specificity are discussed.