The action potential of spinal axons in vitro

Despite the trauma of dissection and special metabolic requirements, the physiological properties of funiculi of the mammalian spinal cord can be studied in vitro. They are adequately oxygenated by diffusion at 0.88 atm. pO(2) and remain in a functionally normal state for over 12 hours. The internal consistency of several kinds of data presented in this and the foregoing papers (5, 38) serves to characterize certain properties of central myelinated axons whether excised or in situ. (1) Spinal tracts support a large spike potential in vitro whose form, duration, and velocity are comparable to those of alpha fibers in vitro and spinal tracts in vivo. (2) Properties consistent with a large L fraction are found in central axons whether excised or in situ. (3) Following conduction there has been identified post-spike supernormality with exponential time course (7.5 msecs. half-time) which is the result of activity intrinsic to parent fibers of dorsal columns. The supernormality is similar in form and magnitude both in excised and intact funiculi. (4) In excised funiculi the action potential of parent axons includes a large negative after-potential whose form and duration correspond satisfactorily with this supernormality. This potential appears not to result from activity arising in broken collaterals. (5) Central axons, excised or intact, fire spontaneously in the presence of citrate ion, and when synchronized by stimulation develop periodic oscillations at about 400 C.P.S. but show no such behavior in the presence of excess potassium ion. Certain characteristics peculiar to central axons indicate that they occupy an extreme position in the spectrum of properties encountered in conducting tissues. Dorsal column myelinated axons differ from their peripheral counterparts, even though they are parts of the same cell, in the following ways. The maintenance of the column spike potential is more critically dependent on CO(2) and the entire tissue mass has a higher oxygen consumption. The negative after-potential is much larger and the positive after-potential, non-existent following a single volley, is more difficult to develop by repetitive stimulation. Unlike peripheral nerve, central axons are not incited to spontaneous activity by manipulation of certain constituents normally present in their environment. However, when induced by the application of citrate the resulting rhythmic behavior has twice the frequency of that in peripheral nerve. In general, the recovery process in central axons is more invariant than that in peripheral axons when they are subjected to similar changes in their artificial environments.     

Abnormal impulse discharge in primary afferent axons injured in the peripheral versus the central nervous system

Chronic injury to sensory axons in the rat peripheral nerve induces pathophysiologic changes in the axolemma at the cut nerve end, which are reflected in spontaneous ectopic impulse discharge and hyperexcitability to a range of depolarizing stimuli. We asked whether sensory axons injured in the central nervous system (CNS) also respond in this way. Primary afferent axons were severed in the sciatic nerve and, alternatively, in the midcervical or upper lumbar dorsal column (DC). Measurements of abnormal discharge from myelinated afferents showed high levels of spontaneous activity generated at the nerve injury site, especially during the period 3-16 days postoperatively, but comparatively little activity generated at the DC lesion site at any postoperative time. There was a corresponding difference in ectopic hyperexcitability to mechanical and adrenergic stimulation, and to depolarization with topical K+. DC lesion sites were not made more excitable by concurrent transection of the sciatic nerve, or by placing an autologous graft of excised sciatic nerve tissue into the DC defect at the time of initial surgery. Transection sites on dorsal roots L4 and L5 yielded abnormal discharge similar to that of sciatic nerve neuromas, indicating that the relative silence of DC transection sites was related to the CNS environment and not to position with respect to the sensory cell body.     

Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation

The peripheral branch of primary sensory neurons regenerates after injury, but there is no regeneration when their central branch is severed by spinal cord injury. Here we show that microinjection of a membrane-permeable analog of cAMP in lumbar dorsal root ganglia markedly increases the regeneration of injured central sensory branches. The injured axons regrow into the spinal cord lesion, often traversing the injury site. This result mimics the effect of a conditioning peripheral nerve lesion. We also demonstrate that sensory neurons exposed to cAMP in vivo, when subsequently cultured in vitro, show enhanced growth of neurites and an ability to overcome inhibition by CNS myelin. Thus, stimulating cAMP signaling increases the intrinsic growth capacity of injured sensory axons. This approach may be useful in promoting regeneration after spinal cord injury.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

Forced expression of Phox2 homeodomain transcription factors induces a branchio-visceromotor axonal phenotype

What causes motor neurons to project into the periphery is not well understood. We here show that forced expression of the homeodomain protein Phox2b, shown previously to be necessary and sufficient for branchio-visceromotor neuron development, and of its paralogue Phox2a imposes a branchiomotor-like axonal phenotype in the spinal cord. Many Phox2-transfected neurons, whose axons would normally stay within the confines of the neural tube, now project into the periphery. Once outside the neural tube, a fraction of the ectopic axons join the spinal accessory nerve, a branchiomotor nerve which, as shown here, does not develop in the absence of Phox2b. Explant studies show that the axons of Phox2-transfected neurons need attractive cues to leave the neural tube and that their outgrowth is promoted by tissues, to which branchio-visceromotor fibers normally grow. Hence, Phox2 expression is a key step in determining the peripheral axonal phenotype and thus the decision to stay within the neural tube or to project out of it.     

Dynamics of the transganglionic movement of horseradish peroxidase in primary sensory neurons

Summary The dynamics of horseradish peroxidase (HRP) transport in primary sensory neurons were studied in rats by demonstration of the reaction product in spinal nerves, spinal ganglia, dorsal roots and in the spinal cord at different survival times after application of the enzyme to the transected sciatic nerve and to the spinal cord. Using tetramethylbenzidine as the chromogen according to Mesulam (1978), transganglionic transport of HRP was shown in both the disto-proximal direction after peripheral application, and proximo-distal direction after central application. Significant differences in staining intensity between the central and peripheral processes of primary sensory neurons were found after all survival times used in this study. After peripheral application the number of labeled axons and the staining intensity were higher in spinal nerves than in dorsal roots; an inverse situation occurred after central application. These differences as well as the time sequences in staining of different parts of primary sensory neurons suggest that HRP applied to a peripheral nerve and to the spinal cord, respectively, enters the perikarya of spinal ganglion cells in any case before continuing its movement in a cellulifugal direction. Lysosomal degradation of the major portion of the applied HRP is supposed. However, in the post-perikaryal portion of a considerable number of neurons HRP-transport still occurs to a varying extent, thus resulting in labeling of nerve endings. In some neurons a post-perikaryal transport could not be detected light microscopically. The transport rates differ: the calculated transport rate of disto-proximal, cellulipetal movement in the fastest transporting neurons was 7.5 mm/h, that of the disto-proximal cellulifugal movement 2.5 to 3 mm/h.This work was partly supported by the Hartmann Müller-Stiftung I want to thank Miss Regula Eichholzer for the technical assistance     

Effects of GABA, THIP, and potassium on excitability of myelinated axons of isolated amphibian spinal roots

The effects of direct applications of GABA (gamma-aminobutyric acid) and the GABAA agonist, THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) on the excitability of myelinated axons of individual dorsal and ventral spinal roots (lumbar VI and (or) VII) of the isolated bullfrog peripheral nerve are reported. Increases evoked by the GABA agonists (0.01-10 mM) in the amplitude of half-maximal A-fiber compound action potentials indicate the presence of depolarizing responses with apparently greater localization to the dorsal roots, and a sensitivity to GABA twofold greater than that for THIP. The changes evoked by GABA and THIP, as well as potassium have components that closely resemble those of sensory and motor fibers in the more distal, desheathed nerve bundle but are smaller and delayed, differences attributable to a closely attached root sheath that acts as a diffusion barrier. These results confirm the likely existence of GABAA receptors on both dorsal and ventral spinal roots.     

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Injured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.     

Amyloid precursor protein at node of Ranvier modulates nodal formation

Amyloid precursor protein (APP), commonly associated with Alzheimer disease, is upregulated and distributes evenly along the injured axons, and therefore, also known as a marker of demyelinating axonal injury and axonal degeneration. However, the physiological distribution and function of APP along myelinated axons was unknown. We report that APP aggregates at nodes of Ranvier (NOR) in the myelinated central nervous system (CNS) axons but not in the peripheral nervous system (PNS). At CNS NORs, APP expression co-localizes with tenascin-R and is flanked by juxtaparanodal potassium channel expression demonstrating that APP localized to NOR. In APP-knockout (KO) mice, nodal length is significantly increased, while sodium channels are still clustered at NORs. Moreover, APP KO and APP-overexpressing transgenic (APP TG) mice exhibited a decreased and an increased thickness of myelin in spinal cords, respectively, although the changes are limited in comparison to their littermate WT mice. The thickness of myelin in APP KO sciatic nerve also increased in comparison to that in WT mice. Our observations indicate that APP acts as a novel component at CNS NORs, modulating nodal formation and has minor effects in promoting myelination.     

Amyloid precursor protein at node of Ranvier modulates nodal formation

Amyloid precursor protein (APP), commonly associated with Alzheimer disease, is upregulated and distributes evenly along the injured axons, and therefore, also known as a marker of demyelinating axonal injury and axonal degeneration. However, the physiological distribution and function of APP along myelinated axons was unknown. We report that APP aggregates at nodes of Ranvier (NOR) in the myelinated central nervous system (CNS) axons but not in the peripheral nervous system (PNS). At CNS NORs, APP expression co-localizes with tenascin-R and is flanked by juxtaparanodal potassium channel expression demonstrating that APP localized to NOR. In APP-knockout (KO) mice, nodal length is significantly increased, while sodium channels are still clustered at NORs. Moreover, APP KO and APP-overexpressing transgenic (APP TG) mice exhibited a decreased and an increased thickness of myelin in spinal cords, respectively, although the changes are limited in comparison to their littermate WT mice. The thickness of myelin in APP KO sciatic nerve also increased in comparison to that in WT mice. Our observations indicate that APP acts as a novel component at CNS NORs, modulating nodal formation and has minor effects in promoting myelination.     

Peripherally-derived BDNF promotes regeneration of ascending sensory neurons after spinal cord injury

Background

The blood brain barrier (BBB) and truncated trkB receptor on astrocytes prevent the penetration of brain derived neurotrophic factor (BDNF) applied into the peripheral (PNS) and central nervous system (CNS) thus restrict its application in the treatment of nervous diseases. As BDNF is anterogradely transported by axons, we propose that peripherally derived and/or applied BDNF may act on the regeneration of central axons of ascending sensory neurons.

Methodology/Principal Findings

The present study aimed to test the hypothesis by using conditioning lesion of the sciatic nerve as a model to increase the expression of endogenous BDNF in sensory neurons and by injecting exogenous BDNF into the peripheral nerve or tissues. Here we showed that most of regenerating sensory neurons expressed BDNF and p-CREB but not p75NTR. Conditioning-lesion induced regeneration of ascending sensory neuron and the increase in the number of p-Erk positive and GAP-43 positive neurons was blocked by the injection of the BDNF antiserum in the periphery. Enhanced neurite outgrowth of dorsal root ganglia (DRG) neurons in vitro by conditioning lesion was also inhibited by the neutralization with the BDNF antiserum. The delivery of exogenous BDNF into the sciatic nerve or the footpad significantly increased the number of regenerating DRG neurons and regenerating sensory axons in the injured spinal cord. In a contusion injury model, an injection of BDNF into the footpad promoted recovery of motor functions.

Conclusions/Significance

Our data suggest that endogenous BDNF in DRG and spinal cord is required for the enhanced regeneration of ascending sensory neurons after conditioning lesion of sciatic nerve and peripherally applied BDNF may have therapeutic effects on the spinal cord injury.     

Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury.

Regeneration is abortive following adult mammalian CNS injury. We have investigated whether increasing the intrinsic growth state of primary sensory neurons by a conditioning peripheral nerve lesion increases regrowth of their central axons. After dorsal column lesions, all fibers stop at the injury site. Animals with a peripheral axotomy concomitant with the central lesion show axonal growth into the lesion but not into the spinal cord above the lesion. A preconditioning lesion 1 or 2 weeks prior to the dorsal column injury results in growth into the spinal cord above the lesion. In vitro, the growth capacity of DRG neurite is also increased following preconditioning lesions. The intrinsic growth state of injured neurons is, therefore, a key determinant for central regeneration.     

The Nodes of Ranvier: Molecular Assembly and Maintenance

Action potential (AP) propagation in myelinated nerves requires clustered voltage gated sodium and potassium channels. These channels must be specifically localized to nodes of Ranvier where the AP is regenerated. Several mechanisms have evolved to facilitate and ensure the correct assembly and stabilization of these essential axonal domains. This review highlights the current understanding of the axon intrinsic and glial extrinsic mechanisms that control the formation and maintenance of the nodes of Ranvier in both the peripheral nervous system (PNS) and central nervous system (CNS).Axons conduct electrical signals, called action potentials (APs), among neurons in a circuit in response to sensory input, and between motor neurons and muscles. In mammals and other vertebrates, many axons are myelinated. Myelin, made by Schwann cells and oligodendrocytes in the peripheral nervous system (PNS) and central nervous system (CNS), respectively, is a multilamellar sheet of glial membrane that wraps around axons to increase transmembrane resistance and decrease membrane capacitance. Although myelin is traditionally viewed as a passive contributor to nervous system function, it is now recognized that myelinating glia also play many active roles including regulation of axon diameter, axonal energy metabolism, and the clustering of ion channels at gaps in the myelin sheath called nodes of Ranvier. Together, the active and passive properties conferred on axons by myelin, result in axons with high AP conduction velocities, low metabolic demands, and reduced space requirements as compared with unmyelinated axons. Thus, myelin and the clustering of ion channels in axons permitted the evolution of the complex nervous systems found in vertebrates. This review highlights the current understanding of the axonal intrinsic and glial extrinsic mechanisms that control the formation and maintenance of the nodes of Ranvier in both the PNS and CNS.     

Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons

We have examined the distribution of microtubule-associated protein 2 (MAP2) in the lumbar segment of spinal cord, ventral and dorsal roots, and dorsal root ganglia of control and beta,beta'-iminodipropionitrile- treated rats. The peroxidase-antiperoxidase technique was used for light and electron microscopic immunohistochemical studies with two monoclonal antibodies directed against different epitopes of Chinese hamster brain MAP2, designated AP9 and AP13. MAP2 immunoreactivity was present in axons of spinal motor neurons, but was not detected in axons of white matter tracts of spinal cord and in the majority of axons of the dorsal root. A gradient of staining intensity among dendrites, cell bodies, and axons of spinal motor neurons was present, with dendrites staining most intensely and axons the least. While dendrites and cell bodies of all neurons in the spinal cord were intensely positive, neurons of the dorsal root ganglia were variably stained. The axons of labeled dorsal root ganglion cells were intensely labeled up to their bifurcation; beyond this point, while only occasional central processes in dorsal roots were weakly stained, the majority of peripheral processes in spinal nerves were positive. beta,beta'- Iminodipropionitrile produced segregation of microtubules and membranous organelles from neurofilaments in the peripheral nervous system portion and accumulation of neurofilaments in the central nervous system portion of spinal motor axons. While both anti-MAP2 hybridoma antibodies co-localized with microtubules in the central nervous system portion, only one co-localized with microtubules in the peripheral nervous system portion of spinal motor axons, while the other antibody co-localized with neurofilaments and did not stain the central region of the axon which contained microtubules. These findings suggest that (a) MAP2 is present in axons of spinal motor neurons, albeit in a lower concentration or in a different form than is present in dendrites, and (b) the MAP2 in axons interacts with both microtubules and neurofilaments.     

Presence of an alpha-melanocyte-stimulating hormone-like epitope in the 150,000 dalton neurofilament subunit from diverse regions of the central nervous system: an immunohistochemical and immunoblot study in guinea pig

Monoclonal antibodies specific for the two higher molecular weight neurofilament (NF) subunits (NF200 and NF150), and antiserum to alpha-melanocyte-stimulating hormone (alpha-MSH) were used to probe the distribution of an alpha-MSH-like epitope in NF proteins of the guinea pig central nervous system using immunoblot and immunohistochemical methods. The anti-alpha-MSH antiserum recognized the same protein band as an anti-NF150 monoclonal antibody in immunoblots of proteins extracted from guinea pig cerebellum, spinal cord, retina, optic nerve, and neurohypophysis; it also stained axons and dendrites in sections of cerebellum, retina, and optic nerve. Although all cells of the pars intermedia and some in the pars distalis exhibited immunoreactivity with this antiserum, it did not stain axons in the neurohypophysis. Our immunoblot data demonstrate an alpha-MSH-like epitope in NF150 extracted from each of the regions studied. The lack of in situ recognition of this alpha-MSH-like epitope in neurophypophyseal axons, using the same immunohistochemical methods that demonstrate this epitope in axons of the cerebellum, retina, and optic nerve, suggests that NF150 is immunochemically heterogeneous in different regions of the guinea pig central nervous system.     

Immunofluorescence studies of neurofilaments in the rat and human peripheral and central nervous system

Localization of antisera to neurofilament antigens derived from rat peripheral nerve was carried out in tissues of rat and human peripheral and central nervous systems by indirect immunofluorescence. Unfixed and chloroform-methanol-fixed frozen sections of tissues were incubated in purified IgG of the experimental rabbit antisera and subsequently exposed to goat anti-rabbit IgG conjugated with fluorescein isothiocyanate. Control studies were conducted on identical tissue preparations incubated in the same concentrations of nonspecific rabbit IgG or in experimental rabbit IgG absorbed with extracts of rat peripheral nerve containing neurofilament antigen. Extensive immunofluorescence was observed in rat and human peripheral and central nervous systems. The distribution and configuration of immunofluorescence corresponded to neurofilament-rich structural components of these tissues. Prominent immunofluorescence was also noted in neuronal cell bodies of spinal sensory ganglia, especially in perikarya of the large neuronal type. Immunofluorescence of the central nervous system was located predominantly in myelinated axons of the white matter in cerebrum, cerebellum, brain stem, and spinal cord. Less intense immunofluorescence was also seen in neuronal perikarya and in short thin linear processes of grey matter.     

Special dendritic and axonal endings formed by the cerebrospinal fluid contacting neurons of the spinal cord

Summary The cerebrospinal fluid (CSF) contacting neurons have a dendritic process which protrudes into the central canal, and is provided with one long kinocilium and many shorter stereocilia (about 80 in the turtle) as revealed by scanning electron microscopy. The shape, number and arrangement of the cilia are similar to those of known receptor endings.The silver impregnated axons of these cells converge to a paired centrosuperficial tract forming terminal enlargements at the ventrolateral surface of the spinal cord. Lying among glial endfeet these terminals are ultrastructurally similar to those present in known neurosecretory areas. The nerve endings are attached to the basal lamina, and they comprise many synaptic vesicles (200 to 400 Å in diameter), as well as granular vesicles of different sizes (diameter 600 to 1800 Å). The axons may lie within finger-like protrusions on the surface of the spinal cord, or they may terminate around vessels.Morphological evidence suggests that these nerve terminals and the corresponding CSF contacting perikarya represent a spinal neurosecretory system possibly influenced by information taken up by its special dendrites protruding into the inner CSF space.     

Strategies to repair lost sensory connections to the spinal cord

Failure of injured axons to regenerate in the central nervous system (CNS) is the main obstacle for repair of stroke and traumatic injuries to the spinal cord and sensory roots. This regeneration failure is high-lighted at the dorsal root transitional zone (DRTZ), the boundary between the peripheral (PNS) and central nervous system where sensory axons enter the spinal cord. Injured sensory axons regenerate in the PNS compartment of the dorsal root but are halted as soon as they reach the DRTZ. The failure of regenerating dorsal root axons to re-enter the mature spinal cord is a reflection of the generally nonpermissive nature of the CNS environment, in contrast to the regeneration supportive properties of the PNS. The dorsal root injury paradigm is therefore an attractive model for studying mechanisms underlying CNS regeneration failure in general and how to overcome the hostile CNS environment. Here we review the main lines that have been pursued to achieve growth of injured dorsal root axons into the spinal cord: (i) modifying the inhibitory nature of the DRTZ by breaking down or blocking the effect of growth repelling molecules, (ii) stimulate elongation of injured dorsal root axons by a prior conditioning lesion or administration of specific growth factors, (iii) implantation of olfactory ensheathing cells to provide a growth supportive cellular terrain at the DRTZ, and (iv) replacing the regeneration deficient adult dorsal root ganglion neurons with embryonic neurons or neural stem cells.     

Effect of nitric oxide on hyperpolarization-activated current in substantia gelatinosa neurons of rats

Central sensitization is the hyperexcitability of spinal processing after peripheral nerve injury or inflammation. This phenomenon may be associated with nitric oxide (NO) signal pathway in synapse. Here, we have investigated the effect of NO on hyperpolarization-activated inward current (I(h)) in substantia gelatinosa (SG) neurons, using the whole-cell patch clamp technique. I(h) was increased by the application of sodium nitro prusside (SNP, a NO donor) or 8Br-cGMP. The stimulatory effects of NO were abolished by guanylyl cyclase inhibitor, ODQ, suggesting that the effect of NO was mediated by cGMP. However, this effect of NO was not prevented by the pretreatment with KT5823, PKG inhibitor. Taken together, the activation of I(h) in SG neurons could be mediated by NO-cGMP dependent pathway. These results reveal an involvement of NO in excitability of SG neuron via the activation of I(h) may be associated with central sensitization.