Terminal processes of serotonin neurons in the caudal spinal cord of the molly,Poecilia latipinna,project to the leptomeninges and urophysis

Summary The caudal neurosecretory complex of poeciliids has previously been shown to be innervated by extranuclear and intrinsic serotonergic projections. In the present study, immunohistochemical techniques were used to characterize fibers originating from serotonin neurons intrinsic to the caudal spinal cord. Bipolar and multipolar neurons were oriented ventromedially, and contained numerous large granular vesicles. Three types of serotonergic fibers were distinguished based on their distribution and morphology. Intrinsic Type-A fibers branched into varicose segments near the ventrolateral surface of the spinal cord and contacted the basal lamina beneath the leptomeninges. Type-B fibers coursed longitudinally to enter the urophysis, where they diverged and terminated around fenestrated capillaries. Labelled vesicles in Type-A and Type-B terminals were the same size as those in labelled cells and in unlabelled neurosecretory terminals in the urophysis. Type-C small varicose fibers branched within the neuropil of the caudal neurosecretory complex. Serotonin may be secreted into the submeningeal cerebrospinal fluid, the urophysis, and the caudal vein by Type-A and Type-B fibers, whereas, Type-C fibers may be processes of serotonergic interneurons in the neuroendocrine nucleus. The possibility that urotensins I and II or arginine vasotocin were colocalized in the processes of the intrinsic serotonin neurons was investigated immunohistochemically. The negative results of these experiments suggest that serotonin-containing neurons may represent a neurochemically distinct subpopulation in the caudal neurosecretory complex.     

Termination areas of primary afferent fibers of the trigeminal nerve in the rat

The cobalt-labelling technique was used to investigate the termination areas of trigeminal primary afferent fibers. The familiar somatotopic arrangement of fibers and terminals of the three divisions of the trigeminal nerve was recognized both in the spinal tract and in the nuclear complex of the trigeminus. The spinal tract could be traced as far as the 3rd cervical segment of the spinal cord where fibers crossed to the contralateral side. The different divisions of the nuclear complex could be unambiguously defined on the basis of the pattern of fiber terminations. The nucleus principalis was characterized by the even distribution of terminals in the nucleus. The nucleus spinalis was characterized by small bundles of fibers of intranuclear origin, which broke up the even distribution pattern of terminals. The presence of mesencephalic trigeminal fibers in the nucleus oralis distinguished this nucleus from the nucleus interpolaris. The nucleus caudalis was recognized on the ground of its striated structure. Primary trigeminal afferent fibers were located in the following sites: in the solitary nucleus, in the lateral part of the reticular formation, in the dorsal-column nuclei and in the superior vestibular nuclei. Primary fiber terminations could not be observed in the cerebellum.     

Neonatal and adult myosin heavy chain isoforms in a nerve-muscle culture system

When adult mouse muscle fibers are co-cultured with embryonic mouse spinal cord, the muscle regenerates to form myotubes that develop cross-striations and contractions. We have investigated the myosin heavy chain (MHC) isoforms present in these cultures using polyclonal antibodies to the neonatal, adult fast, and slow MHC isoforms of rat (all of which were shown to react specifically with the analogous mouse isoforms) in an immunocytochemical assay. The adult fast MHC was absent in newly formed myotubes but was found at later times, although it was absent when the myotubes myotubes were cultured without spinal cord tissue. When nerve-induced muscle contractions were blocked by the continuous presence of alpha-bungarotoxin, there was no decrease in the proportion of fibers that contained adult fast MHC. Neonatal and slow MHC were found at all times in culture, even in the absence of the spinal cord, and so their expression was not thought to be nerve-dependent. Thus, in this culture system, the expression of adult fast MHC required the presence of the spinal cord, but was probably not dependent upon nerve-induced contractile activity in the muscle fibers.     

In vivo imaging of the mouse spinal cord using two-photon microscopy

In vivo imaging using two-photon microscopy in mice that have been genetically engineered to express fluorescent proteins in specific cell types has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task. We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.     

Regeneration of monoamine-containing axons in the developing and adult spinal cord of the rat following intraspinal 6-OH-dopamine injections or transections

Summary Growth of descending noradrenaline (NA) and 5-hydroxytryptamine (5-HT) axons in the rat spinal cord during ontogenesis and following mechanical or chemical, 6-hydroxydopamine (6-OH-DA) induced, axotomy, was studied with the Falck-Hillarp histochemical fluorescence method for monoamines.The major NA and 5-HT axon bundles and terminal innervation areas are present already at birth and an essentially mature pattern of innervation is reached after two weeks.Complete degeneration of both 5-HT and NA nerves in the distal segment is obtained by a transection of the spinal cord. Sprouting of the cut monoamine fibers into the necrotic zone and scar tissue is vigorous in both immature and mature animals, but regeneration into the distal segment is very poor.Selective degeneration of the descending NA axons and terminals is obtained by a localized intraspinal 6-OH-DA injection. Thus, the 5-HT fiber systems as well as all other parts of the spinal cord are left intact. The method should therefore prove useful for evaluating the exact functional role of the NA and 5-HT neuron systems in the spinal cord.Reinnervation of the distal part of the spinal cord by new NA fibers following 6-OH-DA induced denervation is described. This process is faster in younger animals but takes place also in adult animals. The present evidence suggests that reinnervation mainly is the result of downgrowth of the axotomized fibers, but growth in the form of collateral sprouting from a few possibly surviving fibers in the distal region may also contribute. Reinnervation lead to a normal innervation pattern within 1–2 months in the various age groups.It is suggested that the poor regeneration of many spinal nerve tracts often reported in the literature following transection of the spinal cord is due to extraneuronal factors such as scar tissue and impaired circulation rather than to the nerves per se since reinnervation by NA nerves was very poor following mechanical transection but good following chemical, 6-OH-DA-induced axotomy.     

Thoracic Hemisection in Rats Results in Initial Recovery Followed by a Late Decrement in Locomotor Movements,with Changes in Coordination Correlated with Serotonergic Innervation of the Ventral Horn

Lateral thoracic hemisection of the rodent spinal cord is a popular model of spinal cord injury, in which the effects of various treatments, designed to encourage locomotor recovery, are tested. Nevertheless, there are still inconsistencies in the literature concerning the details of spontaneous locomotor recovery after such lesions, and there is a lack of data concerning the quality of locomotion over a long time span after the lesion. In this study, we aimed to address some of these issues. In our experiments, locomotor recovery was assessed using EMG and CatWalk recordings and analysis. Our results showed that after hemisection there was paralysis in both hindlimbs, followed by a substantial recovery of locomotor movements, but even at the peak of recovery, which occurred about 4 weeks after the lesion, some deficits of locomotion remained present. The parameters that were abnormal included abduction, interlimb coordination and speed of locomotion. Locomotor performance was stable for several weeks, but about 3–4 months after hemisection secondary locomotor impairment was observed with changes in parameters, such as speed of locomotion, interlimb coordination, base of hindlimb support, hindlimb abduction and relative foot print distance. Histological analysis of serotonergic innervation at the lumbar ventral horn below hemisection revealed a limited restoration of serotonergic fibers on the ipsilateral side of the spinal cord, while on the contralateral side of the spinal cord it returned to normal. In addition, the length of these fibers on both sides of the spinal cord correlated with inter- and intralimb coordination. In contrast to data reported in the literature, our results show there is not full locomotor recovery after spinal cord hemisection. Secondary deterioration of certain locomotor functions occurs with time in hemisected rats, and locomotor recovery appears partly associated with reinnervation of spinal circuitry by serotonergic fibers.     

Development of motor systems: a comparative approach.

Anurans offer a unique opportunity to study the development of neuronal connections. Transition from the aquatic limbless tadpole to the juvenile occurs over a protracted period of time during which the animal is accessible for experimental studies. Moreover, tract-tracing studies have demonstrated that their descending brain stem pathways show remarkable similarities in origin, course and site of termination to those of mammals. A developmental sequence in the formation of descending pathways to the spinal cord has been shown implying that reticulospinal and vestibulospinal fibers innervate spinal segments very early in development, whereas the red nucleus projects spinalwards definitely later in development. In anurans, this developmental sequence parallels the changes observed in locomotor pattern. The ingrowth of descending pathways into the spinal cord possibly occurs along so-called 'pre-existing' tracts. Several hypotheses on guidance cues in axonal pathfinding will be discussed.     

Characterization and localization of two forms of gonadotropin-releasing hormone (GnRH) in the spinal cord of the frog Rana ridibunda

Two molecular variants of gonadotropin-releasing hormone (GnRH) have been previously characterized in the brain of amphibians, i.e., mammalian GnRH (mGnRH) and chicken GnRH-II (cGnRH-II). The aim of the present study was to identify the molecular forms of gonadotropin-releasing hormone and to localize gonadotropin-releasing hormone-containing elements in the spinal cord of the frog Rana ridibunda using highly specific antisera against mGnRH and cGnRH-II. High-performance liquid chromatography (HPLC) analysis combined with radioimmunoassay (RIA) detection revealed that frog spinal cord extracts contained both mGnRH and cGnRH-II. Immunohistochemical labeling revealed that the frog spinal cord was devoid of GnRH-containing cell bodies. In contrast, numerous GnRH-immunoreactive fibers were observed throughout the entire length of the cord. mGnRH immunoreactivity was only detected in the rostral region of the cord and consisted of varicose processes located in the vicinity of the central canal. cGnRH-II-positive fibers were found throughout the spinal cord, the density of immunoreactive processes decreasing gradually toward the caudal region. Two main cGnRH-II-positive fiber tracts with a rostrocaudal orientation were observed: a relatively dense fiber bundle surrounding the central canal, and a more diffuse plexus in the white matter. In addition, short, varicose cGnRH-II-positive processes with a radial orientation were present throughout the gray matter. These fibers were particularly abundant ventromedially and formed a diffuse network that ramified laterally to end in the vicinity of motoneurons. Taken together, these data indicate that the frog spinal cord, like the frog brain, contains two forms of GnRH. The presence of numerous cGnRH-II-immunoreactive fibers in the ventral horn suggests that cGnRH-II may influence the activity of a subpopulation of motoneurons.     

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.     

Repair of spinal cord transection and its effects on muscle mass and myosin heavy chain isoform phenotype.

A number of significant advances have been developed for treating spinal cord injury during the past two decades. The combination of peripheral nerve grafts and acidic fibroblast growth factor (hereafter referred to as PNG) has been shown to partially restore hindlimb function. However, very little is known about the effects of such treatments in restoring normal muscle phenotype. The primary goal of the current study was to test the hypothesis that PNG would completely or partially restore 1) muscle mass and muscle fiber cross-sectional area and 2) the slow myosin heavy chain phenotype of the soleus muscle. To test this hypothesis, we assigned female Sprague-Dawley rats to three groups: 1) sham control, 2) spinal cord transection (Tx), and 3) spinal cord transection plus PNG (Tx+PNG). Six months following spinal cord transection, the open-field test was performed to assess locomotor function, and then the soleus muscles were harvested and analyzed. SDS-PAGE for single muscle fiber was used to evaluate the myosin heavy chain (MHC) isoform expression pattern following the injury and treatment. Immunohistochemistry was used to identify serotonin (5-HT) fibers in the spinal cord. Compared with the Tx group, the Tx+PNG group showed 1) significantly improved Basso, Beattie, and Bresnahan scores (hindlimb locomotion test), 2) less muscle atrophy, 3) a higher percentage of slow type I fibers, and 4) 5-HT fibers distal to the lesion site. We conclude that the combined treatment of PNG is partially effective in restoring the muscle mass and slow phenotype of the soleus muscle in a T-8 spinal cord-transected rat model.     

Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury

Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.     

Projections of the optic tectum and the mesencephalic nucleus of the trigeminal nerve in the tegu lizard (Tupinambis nigropunctatus)

Summary Fibers undergoing Wallerian degeneration following tectal lesions were demonstrated with the Nauta and Fink-Heimer methods and traced to their termination. Four of the five distinct fiber paths originating in the optic tectum appear related to vision, while one is related to the mesencephalic nucleus of the trigeminus. The latter component of the tectal efferents distributes fibers to 1) the main sensory nucleus of the trigeminus, 2) the motor nucleus of the trigeminus, 3) the nucleus of tractus solitarius, and 4) the intermediate gray of the cervical spinal cord.The principal ascending bundle projects to the nucleus rotundus, three components of the ventral geniculate nucleus and the nucleus ventromedialis anterior ipsilaterally, before it crosses in the supraoptic commissure and terminates in the contralateral nucleus rotundus, ventral geniculate nucleus and a hitherto unnamed region dorsal to the nucleus of the posterior accessory optic tract.Fibers leaving the tectum dorso-medially terminate in the posterodorsal nucleus ipsilaterally and the stratum griseum periventriculare of the contralateral tectum. The descending fiber paths terminate in medial reticular cell groups and the rostral spinal cord contralaterally and in the torus and the lateral reticular regions ipsilaterally. The ipsilateral fascicle also issues fibers to the magnocellular nucleus isthmi.     

Mechanical properties of the lamprey spinal cord: Uniaxial loading and physiological strain

During spinal cord injury, nerves suffer a strain beyond their physiological limits which damages and disrupts their structure. Research has been done to measure the modulus of the spinal cord and surrounding tissue; however the relationship between strain and spinal cord fibers is still unclear. In this work, our objective is to measure the stress–strain response of the spinal cord in vivo and in vitro and model this response as a function of the number of fibers. We used the larvae lamprey (Petromyzon Marinus), a model for spinal cord regeneration and animal locomotion. We found that physiologically the spinal cord is pre-stressed to a longitudinal strain of 10% and this strain increases to 15% during swimming. Tensile measurements show that uniaxial, longitudinal loading is independent of the meninges. Stress values for uniaxial strains below 18%, are homogeneous through the length of the body. However, for higher uniaxial strains the Head section shows more resistance to longitudinal loading than the Tail. These data, together with the number of fibers obtained from histological sections were used in a composite-material model to obtain the properties of the spinal cord fibers (2.4 MPa) and matrix (0.017 MPa) to uniaxial longitudinal loading. This model allowed us to approximate the percentage of fibers in the spinal cord, establishing a relationship between uniaxial longitudinal strains and spinal cord composition. We showed that there is a proportional relationship between the number of fibers and the properties of the spinal cord at large uniaxial strains.     

Protein composition and synthesis in the adult mouse spinal cord

Propepties of spinal cord proteins were studied in adult mice subjected to unilateral crush or electrical stimulation of sciatic nerve. The protein composition of spinal tissue was determined using SDS-polyacrylamide gel electrophoresis coupled with subcellular fractionation. Comparisons of mouse spinal cord and brain revealed similarities in the types but differences in the concentrations of myelin associated proteins, nuclear histones and other proteins. Comparisons with sciatic nerve proteins demonstrated differences in types of proteins but similarities in the concentration of myelin proteins and nuclear histones. The short term (<2 hrs.) incorporation of radioactive amino acids into spinal cord proteins revealed heterogeneous rates of incorporation. Neither nerve crush six days prior to testing nor sciatic nerve stimulation had a significant effect on the protein composition or amino acid incorporation rates of spinal cord tissue. These observations suggest that known differences in spinal cord function following alterations in nerve input may be dependent upon different mechanisms than have been found in the brain.     

Immunocytochemical localization of carbonic anhydrase in the spinal cords of normal and mutant (shiverer) adult mice with comparisons among fixation methods

The peroxidase-antiperoxidase technique was used for immunocytochemical localization of carbonic anhydrase in the mouse spinal cord to detect whether this antigen was normally present in myelinated fibers, in oligodendrocytes in both white and gray matter, and in astrocytes, and to determine where the carbonic anhydrase might be localized in the spinal cords of dysmyelinating mutant (shiverer) mice. The most favorable methods for treating tissue were: 1) immersion in formalin-ethanol-acetic acid followed by paraffin embedding, or 2) light fixation with paraformaldehyde and preparation of vibratome sections. Carnoy's solution, followed by paraffin embedding, extracted myelin from the tissue, while aqueous aldehydes, when used before paraffin embedding, reduced staining everywhere except at sites of compact myelin. The latter conclusion was based, in part, on the almost complete loss of this antigen from the shiverer cord, where compact myelin is known to be virtually absent but where membrane-bound carbonic anhydrase was demonstrated enzymatically. When the optimal methods were used with normal mouse cords, carbonic anhydrase was found throughout the white matter columns and in the oligodendrocytes in gray and white matter. The staining of the white matter was attributed to myelinated fibers because of the similarity in distribution to both a histological myelin stain and the immunocytochemical staining for myelin basic protein. In the mutant mice the oligodendrocyte cell bodies and processes, which were stained in all areas of the spinal cord, were particularly numerous at the periphery of the sections. In contrast to the oligodendrocytes, the fibrous astrocytes appeared to lack carbonic anhydrase, or to have lower than detectable levels, since the astrocyte marker, glial fibrillary acidic protein, had a very different distribution from that of carbonic anhydrase. Even finer localization was obtained in vibratome sections, where the antibody against carbonic anhydrase permitted visualization of the processes connecting oligodendrocytes to myelinated fibers in the normal adult spinal cord.     

Tissue engineered, guided nerve tube consisting of aligned neural stem cells and astrocytes

Injury of the nervous system, particularly in the spinal cord, impairs the quality of life of the patient by resulting in permanent loss of neurologic function. The main limitation in spinal cord regeneration is the lack of extracellular matrix to guide nerves for functional recovery of the transected nerve tissue. In the present study, a tissue engineered nerve tube was prepared by wrapping neural stem cells (NSCs) on aligned fibers using a micropatterned film with astrocytes aligned along the microgrooves to support the NSCs. Initially the cell behavior on micropatterns and parallel fibers was investigated with cytoskeletal and nuclear staining, immunocytochemistry, and proliferation assay using the fiber and the film system separately. The results showed that both cells, NSCs in undifferentiated and astrocytes in differentiated form, were oriented in the direction of the guiding and support elements, the microgrooves, and the microfibers. They were able to grow and increase in number on these cell carriers. This trend was also maintained after the components were brought together in a nerve tube form and testing in coculture. The cells were able to survive and maintained their orientation in the 3D tissue engineered construct. The guided nerve tissue engineering approach tested in the present study with parallel NSCs and support cells in the tubular construct is expected to provide an appropriate environment for nerve regeneration in vivo.     

Mechanism of formation of intramedullary cavities and their role in the regeneration of the spinal cord

The spinal cord preparations of 38 dogs and 20 rabbits have been studied with the aim to investigate the influence of the cerebrospinal fluid on the spinal cord nervous tissue. The spinal cord preparations of 8 patients having trauma of the vertebral column with interruption of the spinal cord have also been studied. As demonstrate histological investigations, the cerebral tissue of the pieces, put into the flask with liquor, in the subarachnoidal space of the canine spinal cord, in diastasis between the ends of the cut spinal cord during 6 h up to 7 days, swells, becomes edematous. Cavities occupying about 30% of the area in the slices studied appear in it. At hemisection of the rabbit spinal cord without closure of the defect in the meninx vasculosa with the glue MK-6, the area of the cavity formation varies from 24 up to 35%, comparing the whole area of the preparation, while in rabbits with hemisection and successive gluing of the defect in the meninx vasculosa the area of the nervous tissue destruction makes 13-18%. It has been proved that the scar forming in the traumatized segment of the spinal cord does not present a continuous formation, but contains a large amount of cavities that prevent regeneration of nerve fibers. The experimental data concerning lysing effect of the cerebrospinal fluid on the traumatized nervous tissue are confirmed by the results obtained at investigating the preparations of the spinal cord of the patients died as the cause of the spinal cord trauma.     

Effects on neurite outgrowth and cell survival of a secreted fibroblast growth factor binding protein upregulated during spinal cord injury

The fibroblast growth factor binding protein (FGF-BP; GenBank accession no. NP_005121) is a secreted protein that mobilizes FGFs from the extracellular matrix, protects them from degradation, and enhances their biological activity. Several previous studies reported that FGF-BP is an early response gene upregulated during tissue repair processes including wound healing and atherogenesis. In this study we analyzed whether FGF-BP expression was impacted by spinal cord injury and could have an effect on neuronal cell viability. Immunohistochemical and in situ hybridization studies revealed a dramatic upregulation of FGF-BP protein and mRNA levels following unilateral hemisection and contusion injury of adult rat spinal cord. In spinal cord sections of laminectomized rats, increased FGF-BP expression was observed in the fibers and cell bodies ipsilateral to the lesion site but was absent in the uninjured spinal cord tissue contralateral to the lesion. Increased expression of FGF-BP was observed at all postinjury time points, examined with peak levels occurring at day 4, a time when injury-induced increased levels of FGF2 have also been reported to be maximal. Moreover, using PC12 cells as a neuronal model, we observed that exogenous FGF-BP increased the capacity of FGF2 to stimulate neurite outgrowth and to increase cell survival. At the molecular level, FGF-BP enhanced FGF2-induced protein tyrosine phosphorylation and AKT/PKB activation. Collectively, these results suggest that FGF-BP is an early response gene after spinal cord injury and that its upregulation in regenerating spinal cord tissue may provide a molecular mechanism for enhancing the initial FGF2-mediated neurotrophic effects occurring after such tissue damage.     

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1''-dioctadecyl-3,3,3'',3''-tetramethylindocarbocyanine perchlorate (DiI)¹. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching² (Fig. 1).The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)³. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself⁴ (Fig. 2), however, other options have been discussed as well⁵.This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.     

Surgical transplantation of mouse neural stem cells into the spinal cords of mice infected with neurotropic mouse hepatitis virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.