Axonal regeneration in lamprey spinal cord

Spinal cords of sea lamprey larvae were transected at one of two levels: (a) rostral, at the last gill, or (b) caudal, at the cloaca. Following various recovery times, regeneration of the posteriorly projecting giant reticulospinal axons (RAs) was demonstrated by intra-axonal injection of horseradish peroxidase (HRP). Regeneration of axons of anteriorly projecting dorsal cells (DCs) and giant interneurons (GIs) was demonstrated by intrasomatic HRP injection into cells located just below the transection scar. After 40 days of recovery, 55% of proximally transected RAs (rostral cut) regenerated at least as far as the center of the scar, whereas only 15% of distally transected RAs (caudal cut) did so. Maximum distance of regeneration was 5.3 mm beyond the scar for proximally transected RAs but only 38 u for distally transected RAs. Proximally transected RAs also branched more profusely than distally transected ones. These data (when combined with others in the literature) suggest that the regenerative capacity of RAs may decrease with distance of axotomy from the cell body. Distance of regeneration and degree of branching of proximally transected RAs peaked between 40 and 100 days. Thereafter, there appeared to be a tendency toward neurite retraction. Of axotomized GIs, 76% regenerated anteriorly at least as far as the center of a caudal transection scar (GIs are located only in the caudal part of the cord). The maximum distance of regeneration was 1.3 mm beyond the scar. Of DC axons, 56% regenerated anteriorly at least as far as the transection site. The maximum distance was 1.1 mm beyond the scar. DCs located just below a caudal transection regenerated at least as well as those located below a rostral transection. Axonal regeneration was also demonstrated for a few lateral cells, edge cells, and crossed caudally projecting interneurons.     

Axonal regeneration in the adult lamprey spinal cord

Larval sea lampreys recover from complete spinal transection by a process involving directionally specific axonal regeneration. In order to determine whether this is also true of adults, 14 adult lampreys were transected at the level of the 5th gill and allowed to recover for 10 weeks. Müller and Mauthner cells and their giant reticulospinal axons (GRAs) were impaled with microelectrodes and injected with horseradish peroxidase (HRP). The tissue was processed for HRP histochemistry and wholemounts of brain and spinal cord were prepared. All animals recovered coordinated swimming; 61 of 121 (50%) neurites emanating from 30 axons regenerated caudal to the scar into the distal stump. Of the neurites which had grown beyond the scar, 92% were correctly oriented, i.e., caudalward and ipsilateral to the parent axon. Retransection in two additional animals eliminated the recovered swimming. Thus, behavioral recovery in adult sea lampreys is accompanied by directionally specific axonal regeneration.     

Preferential regeneration of spinal axons through the scar in hemisected lamprey spinal cord.

Axons of lamprey spinal cord can regenerate across a complete spinal transection. Thus, unlike the scar of injured mammalian spinal cords, the scar in the lamprey is not an absolute impediment to regeneration. However, it is still not known whether the scar is a relative impediment or whether it provides a favorable environment for regeneration compared to the spinal cord parenchyma. In order to answer this question, the cords of 12 large larval sea lampreys (4-5 years old) were hemisected at the level of the third gill and the animals allowed to recover for 10 weeks. The large reticulospinal neurons (Müller and Mauthner cells) or their giant axons were injected intracellularly with HRP and their regenerating neurites visualized in central nervous system (CNS) wholemounts. Forty-five of seventy-one regenerating neurites (64%) grew beyond the level of the hemisection. Of these, 36 (82%) regenerated through the scar and remained on the same side of the cord as their parent axons, while only 8 (18%) crossed the midline and grew around the scar. Thus, regenerating neurites of giant reticulospinal axons tended to grow through the hemisection scar rather than around it. Once they passed the level of injury, they continued to elongate in their appropriate paths. It is possible that this tendency for axons to regenerate through the scar reflects the greater amount of empty spaces on the hemisected side. In order to rule this out, 13 animals received contralateral simultaneous hemisections at the level of the 3rd and 7th gills. This procedure created large numbers of degenerating axons and potential empty spaces both rostral and caudal to the scars within both hemicords; 92 of 158 neurites (58%) regenerated beyond the level of their respective hemisections. All of these grew through the scar and none crossed to the contralateral side. Distal to either hemisection, neurites remained on their correct side regardless of whether the contralateral cord contained normal CNS parenchyma or axonal debris and empty spaces produced by Wallerian degeneration. Moreover, in hemisected and double hemisected animals, as well as in completely transected control animals, neurites regenerating in their correct direction grew further than those that were misrouted. Because lamprey spinal axons grow preferentially through a scar rather than around it, the scar may play a positive role in supporting axonal regeneration.     

Evidence for functional regeneration in the adult lamprey spinal cord following transection

We report here that following partial spinal transections in adult lampreys, the fibers of the spinal cords can regenerate and restore some intersegmental coordination to the central pattern generator for locomotion, as tested in the isolated cord preparation. However, the regeneration by this test is not successful in all animals.     

Dorsal root axonal regeneration in the adult frog spinal cord

The frog dorsal root provides a useful model for the study of axonal regeneration in an adult vertebrate CNS. We have used the model to compare the regeneration of two very different types of axons within the same CNS environment and have found that regenerating dorsal root, as well as rerouted motoneuron axons, display similar growth patterns in the spinal cord. Both sensory and motor axons grow preferentially in some regions and not in others. They both regenerate effectively longitudinally as well as radially within the dorsolateral fasciculus (DLF). By contrast, fewer sensory and motor axons regenerate longitudinally or radially in the dorsal funiculus (DF). This similar preferential growth of two very different populations of axons suggests that the growth patterns reflect regional differences in the cellular environment of the cord. The DLF has fascicles of unmyelinated axons separated by radial glial processes and, after dorsal root injury, is mildly gliotic. By contrast, DF has very large myelinated axons, which widely separate the radial glial processes that traverse the region. After dorsal root injury, this region is markedly gliotic and contains myelin, debris and oligodendroglia, and microglial macrophages. Our data suggest that unmyelinated axons and radial glial processes are more preferred substrates for axonal growth than myelin debris, oligodendroglia and macrophages. It is not surprising, then, that regions of the adult mammalian CNS that are characterized by large myelinated axons fail to support axonal growth. Moreover, there is some evidence that regions of the adult mammalian CNS that are characterized by unmyelinated axons support axonal growth.     

The collagenous lesion scar--an obstacle for axonal regeneration in brain and spinal cord injury

After CNS trauma a sheet-like, collagen type IV (Coll IV) immunopositive basement membrane (BM) develops in the lesion zone as well as at newly formed blood vessels. The basic scaffold of this BM is composed of Coll IV, laminin and nidogen but numerous other proteins some of which are discussed to be inhibitory for axonal regeneration, i.e. chondroitin- and heparansufate-proteoglycans, are associated with BM. This review will focus on the collagenous wound healing scar, discuss its composition and summarize the experimental results that demonstrate its role in the failure of axonal regeneration in the injured mammalian CNS.     

Spontaneous axonal regeneration in rodent spinal cord after ischemic injury

Here we present evidence for spontaneous and long-lasting regeneration of CNS axons after spinal cord lesions in adult rats. The length of 200 kD neurofilament (NF)-immunolabeled axons was estimated after photochemically induced ischemic spinal cord lesions using a stereological tool. The total length of all NF-immunolabeled axons within the lesion cavities was increased 6- to 10-fold at 5, 10, and 15 wk post-lesion compared with 1 wk post-surgery. In ultrastructural studies we found the putatively regenerating axons within the lesion to be associated either with oligodendrocytes or Schwann cells, while other fibers were unmyelinated. Immunohistochemistry demonstrated that some of the regenerated fibers were tyrosine hydroxylase- or serotonin-immunoreactive, indicating a central origin. These findings suggest that there is a considerable amount of spontaneous regeneration after spinal cord lesions in rodents and that the fibers remain several months after injury. The findings of tyrosine hydroxylase- and serotonin-immunoreactivity in the axons suggest that descending central fibers contribute to this endogenous repair of ischemic spinal cord injury.     

Entrainment of the spinal pattern generators for swimming by mechano-sensitive elements in the lamprey spinal cord in vitro

Imposed sinusoidal bending of a mobile region of the curarized spinal cord/notochotd preparation of the lamprey results in phase-locking (i.e. ‘entrainment’) of the ‘fictive swimming’ motor pattern (recorded in ventral roots) to the bending movements. This entrainment phenomenon occurs both with intact ventral and dorsal roots and with all roots cut (i.e. a completely isolated spinal cord). It is proposed that mechano-sensitive elements within the spinal cord contribute in part to the entrainment.     

Colocalization of dopamine and GABA in spinal cord neurones in the sea lamprey

In this study, double immunofluorescence methods were used to investigate possible colocalization of the neurotransmitters dopamine [DA] and GABA in rostral spinal cord neurones in the upstream migrating adult sea lamprey (Petromyzon marinus). Double immunofluorescence revealed that all the DA-immunoreactive (ir) cerebrospinal fluid-contacting (CSF-c) cells, approximately 30% of the medioventral DA-ir cells, and most of the DA-ir cells located in the grey lateral to the central canal were also GABA-ir. The results also revealed some DA-ir cells located dorsally to the central canal, which increases the number of dopaminergic cell types known in lamprey. Double-labelled fibres were mainly distributed in the ventral column, and double-labelled boutons contacted some dorsal GABA-ir CSF-c cells, as well as some non-CSF-c GABA-ir cells and ventromedial dendrites of motoneurones. The findings reveal colocalization of dopamine and GABA in some cells and fibres, which suggests co-release of these substances in some synaptic terminals. Although dopaminergic/GABAergic CSF-c cells have been reported in some other vertebrates, the other double-labelled spinal populations appear exclusive to lampreys.     

Differential expression of class 3 and 4 semaphorins and netrin in the lamprey spinal cord during regeneration

To explore the role of axon guidance molecules during regeneration in the lamprey spinal cord, we examined the expression of mRNAs for semaphorin 3 (Sema3), semaphorin 4 (Sema4), and netrin during regeneration by in situ hybridization. Control lampreys contained netrin-expressing neurons along the length of the spinal cord. After spinal transection, netrin expression was downregulated in neurons close (500 mum to 10 mm) to the transection at 2 and 4 weeks. A high level of Sema4 expression was found in the neurons of the gray matter and occasionally in the dorsal and the edge cells. Fourteen days after spinal cord transection Sema4 mRNA expression was absent from dorsal and edge cells but was still present in neurons of the gray matter. At 30 days the expression had declined to some extent in neurons and was absent in dorsal and edge cells. In control animals, Sema3 was expressed in neurons of the gray matter and in dorsal and edge cells. Two weeks after transection, Sema3 expression was upregulated near the lesion, but absent in dorsal cells. By 4 weeks a few neurons expressed Sema3 at 20 mm caudal to the transection but no expression was detected 1 mm from the transection. Isolectin I-B(4) labeling for microglia/macrophages showed that the number of Sema3-expressing microglia/macrophages increased dramatically at the injury site over time. The downregulation of netrin and upregulation of Sema3 near the transection suggests a possible role of netrin and semaphorins in restricting axonal regeneration in the injured spinal cord.     

Development of GABA-immunoreactive cells in the spinal cord of the sea lamprey, P. marinus

The lamprey spinal cord increases in length and size during all its life cycle; thus, it is expected that new cells will be generated. This expectation suggests that the locomotor circuits must be continuously remodeled. Key elements in the cellular network controlling locomotor behavior are inhibitory cells. Here, we studied the gamma-aminobutyric acid-immunoreactive (GABA-ir) cells in the lamprey spinal cord during postembryonic development. Three major populations of GABA-ir cells were identified according to their distribution: those located in the gray matter, those contacting the cerebrospinal liquid (LC cells), and those located in the white matter. The results show (1). the number of GABA-ir cells per segment increase from prolarvae (<10 mm) to adulthood; (2). the lower number of GABA-ir cells in 100 microm of spinal cord is 66 +/- 7, found in premetamorphic larvae, and the highest is 107 +/- 6, found in postmetamorphic animals; (3). the gray matter and LC GABA-ir cells show different variations in number depending on the developmental period. Thus, in the 10-mm larvae, the gray matter GABA-ir cells are more abundant than LC cells, whereas in the young postmetamorphic specimens, the contrary occurs. Most of the GABA-ir cells located in the white matter were classified as edge cells. They increase in number from the beginning of the prolarval period, where there are not white matter-positive cells, to the middle larval period, where there are 9 +/- 4 GABA-ir edge cells per segment. This value was unaltered in later periods, where GABA-ir edge cells represent 20-30% of the total number of edge cells per segment. The increase in number of GABA-ir cells in these populations during a specific point of the lamprey life cycle may indicate different inhibitory requirements of the locomotor circuit at different developmental periods.     

Alteration of neuronal synaptic complement during regeneration and axonal sprouting of rat spinal cord

Spinal cord hemisection results in limited regeneration of nerve fibers and the sprouting of intact nerve fibers proximal to the site of lesion. Electron microscopically the nerve fibers form new synaptic complexes. The following study was undertaken in the rat to determine the synaptic profile of neurons during the regenerative process. The spinal cords of 40 rats were hemisected at vertebral segment T-2 and 5 animals per group (in addition to 5 normals) were utilized 10, 20, 30, 45, 60, and 90 days posthemisection. Tissue was prepared by the Rasmussen technique for the light microscopic demonstration of boutons. In addition, five animals had the cord hemisected and the spinal cord tissue was prepared for electron microscopy 40 days postoperatively. Bouton counts were made on perikaryon and primary dendrite (of the same neuron) of neurons in lamina IV, lamina VII, and on motoneurons. Statistical analysis of counts from coded material was carried out utilizing a polynomial regression, analysis of variance, and a Neuman-Keuls a posteriori analysis. The number of boutons on the perikaryon of neurons on the operated side demonstrated a significant decrease in boutons at 10–20 days postoperative followed by a significant increase in boutons at 30 days to levels below normal innervation. This reinnervation was followed by a significant secondary loss of boutons from 30 to 60 days. This trend of bouton alteration was repeated (with minor variations) on the perikaryon of neurons on the unoperated side of the spinal cord. This general trend was repeated on the primary dendrite on both operated and unoperated side of the spinal cord. However, the number of boutons on primary dendrite usually returned to normal. The regenerated boutons (30 days) frequently contained granular vesicles (predominantly 900 Å). The boutons that degenerated 30–60 days had all combinations of synaptic vesicles. This is an indication that former and new synaptic complexes were degenerating. The alteration of boutons on perikaryon and primary dendrite following hemisection suggests: that there is a critical period of initial denervation (0–20 days); that reinnervation occurs within the first 30 days; and that a secondary denervation occurs after 30–60 days. This recombination of neuronal circuits may represent an accelerated view of a dynamic continuum which imparts information to denervated neurons about altered neuronal circuitry adding a further dimension to the plasticity of the adult nervous system.     

Motor axonal regeneration after partial and complete spinal cord transection

We subjected rats to either partial midcervical or complete upper thoracic spinal cord transections and examined whether combinatorial treatments support motor axonal regeneration into and beyond the lesion. Subjects received cAMP injections into brainstem reticular motor neurons to stimulate their endogenous growth state, bone marrow stromal cell grafts in lesion sites to provide permissive matrices for axonal growth, and brain-derived neurotrophic factor gradients beyond the lesion to stimulate distal growth of motor axons. Findings were compared with several control groups. Combinatorial treatment generated motor axon regeneration beyond both C5 hemisection and T3 complete transection sites. Yet despite formation of synapses with neurons below the lesion, motor outcomes worsened after partial cervical lesions and spasticity worsened after complete transection. These findings highlight the complexity of spinal cord repair and the need for additional control and shaping of axonal regeneration.     

Synaptic interactions of identified nerve cells in the spinal cord of the sea lamprey

As part of a continuing study on the organization of the lamprey nervous system, two additional groups of interneurons have been identified by physiological and morphological criteria in the isolated spinal cord of Petromyzon marinus. These and other identified nerve cells were tested for synaptic interactions using separate intracellular microelectrodes for stimulation and recording.

• 1 Edge cells were identified by their unusual location in the lateral fiber tracts of the spinal cord. Their axons extended rostrally either on the ipsilateral or contralateral side of the cord.
• 2 Some edge cells showed polysynaptic EPSP's and IPSP's after stimulation of sensory dorsal cells, but interactions with other identified neurons were rare. A single cell was excited by a giant interneuron. One edge cell produced IPSP's in a contralateral edge cell, and another produced IPSP's in lateral cells on the opposite side of the spinal cord. Thus, some edge cells have an inhibitory function.
• 3 Lateral cells were distinguished from giant interneurons and edge cells by their cell bodies in the lateral grey of the spinal cord in the gill and trunk regions, their ipsilateral dendrites, and their long ipsilateral axons extending as far as the tail.
• 4 Lateral cells were excited and inhibited polysynaptically by sensory dorsal cells and, in turn, produced weak IPSP's in unidentified neurons. Stimulation of lateral cells produced neither visible movements peripherally nor synaptic potentials in other lateral cells, in giant interneurons, or in edge cells.
• 5 Giant interneurons were previously identified on the basis of their cell bodies in the caudal half of the spinal cord, their bilateral dendrites, and their long contralateral axons extending towards the brain. Giant interneurons exhibited unitary composite EPSP's when more caudal giant interneurons were stimulated. The two components of the EPSP were due to electrical and chemical transmission. Under the electron microscope a contact between a dendrite of one giant interneuron and the probable axon of another had separate junctions resembling chemical and electrical synapses.
• 6 Intracellular stimulation of sensory dorsal cells produced both monosynaptic and polysynaptic EPSP's in giant interneurons. Some dorsal cells produced unitary composite EPSP's Giant interneurons are part of a convergent, multispecific sensory system extending towards the brain.

     

The inulin space of the lamprey spinal cord

The distribution of [¹⁴C]inulin was measured in isolated spinal cords of larval and feeding stage adult forms of sea lamprey (Petromyzon marinus) and expressed in per cent of total cord wet weight. In larval cord the apparent inulin space reached a plateau value of 32–33% within 2.5 min. This correlates well with electrophysiological experiments in which 10⁻⁷ M tetrodotoxin added to the perfusion fluid blocked the responses of giant interneurons to both intracellular and rostral cord stimulation in 1 to 2 min. Thus the plateau level of inulin space probably represents the extracellular space.[¹⁴C]Mannitol did not reach a steady distribution space even after 30 min of incubation. Therefore, mannitol is not an accurate extracellular space indicator in the isolated lamprey spinal cord.The inulin space increased with increasing temperature of incubation. Average inulin spaces for larval spinal cords incubated at 5, 10 and 22 °C were approximately 26%, 33% and 42% respectively. The inulin space of isolated adult lamprey spinal cords was about 18–19%. Since in larvae the inulin space did not vary consistently with the sizes (and therefore presumably the ages) of the animals, it is likely that the reduction in inulin space during maturation does not occur gradually during the larval phase, but probably occurs during transformation. The difference between the inulin spaces of isolated larval and adult spinal cords is reflected qualitatively in the electron microscopic appearance of the extracellular space.We conclude that the inulin space in the lamprey spinal cord behaves similarly to the picture of the mammalian brain extracellular space which has emerged in recent years. Because of the rapidity of inulin diffusion in the lamprey cord and the unambiguous time-dependent behavior of the inulin space of the isolated lamprey cord, the latter would seem to be a useful model for the extracellular space of the vertebrate central nervous system.     

Histopathological reactions and axonal regeneration in the transected spinal cord of hibernating squirrels

The failure of axonal regeneration in the transected spinal cord of mammals has been attributed to many factors, including an intrinsic lack of regenerative capacity of mature CNS neurons, mechanical obstruction of axonal elongation by glial-connective tissue scars, necrosis of spinal tissue resulting in cavitation, lack of trophic influences sufficient to sustain outgrowth, and contact inhibition resulting from the formation of aberrant synapses. Assessment of the relative importance of each of these factors requires animal models in which one or more of these pathological processes can be eliminated. We therefore examined the effects of spinal transection in the hibernating animal because, during hibernation, collagen formation is depressed while nerve regeneration and slow axonal transport are maintained. Midthoracic spinal transections were performed in hibernating ground squirrels and the spinal cords were examined histologically 1–6 months later. The lesion site was composed primarily of a loose accumulation of macrophages and showed minimal glial and collagenous scarring, or cavitation. There was extensive regeneration of intrinsic spinal cord and dorsal root fibers. These axons grew to the margin of the lesion where they turned abruptly and continued growing along the interface between the lesion and the spinal cord. We conclude (1) that mammalian spinal-cord neurons have considerable regenerative potential; (2) that such mechanical impediments as collagenous and glial scarring, cyst formation, and cavitation cannot provide the sole explanation of why regeneration in the mammalian CNS is abortive; and (3) that specific physical and chemical properties of the cells in the environment of the growth cone regulate the extent and orientation of regenerative axonal outgrowth.     

Subcellular distribution of serotonin in the lamprey spinal cord.

The subcellular distribution of serotonin (5-hydroxytryptamine; 5-HT) in the lamprey (Ichtyomyzon unicuspis, Lampetra fluviatilis) spinal cord was investigated by using ultracentrifugation on continuous density gradients combined with an electron microscopic analysis of the gradients and of immunostained tissue. Endogenous 5-HT was analyzed by high-performance liquid chromatography with electrochemical detection. After differential centrifugation, the highest levels of 5-HT were found in the particulate fractions. After ultracentrifugation of lysed synaptosomal fractions on continuous sucrose gradients and the subsequent sedimentation of the individual fractions, 5-HT showed a biphasic distribution in the gradient. The two peaks corresponded to 0.30-0.40 M and 0.85-1.05 M sucrose. Electron microscopy of intact tissue showed that some of the boutons were strongly immunoreactive to 5-HT with dense precipitates over large granular vesicles. The area around these large vesicles, however, also showed reaction product. Large granular vesicles could be clearly distinguished in the immunostained axonal varicosities. In tissue not processed for 5-HT immunoreactivity it was seen that the varicosities contained not only large dense-cored vesicles, but also small agranular vesicles. An electron microscopical analysis of the subcellular fractions revealed that the fraction corresponding to the "light" 5-HT peak contained numerous vesicular structures, which in most cases were electron lucent. In the "heavy" fractions, nerve ending particles containing vesicles of various sizes were observed. The results suggest that 5-HT in the lamprey spinal cord may be distributed in more than one subcellular compartment which, apart from the cytosol, possibly corresponds to small and large synaptic vesicles.     

Ontogeny of gamma-aminobutyric acid-immunoreactive neurons in the rhombencephalon and spinal cord of the sea lamprey

The development of neurons expressing gamma-aminobutyric acid (GABA) in the rhombencephalon and spinal cord of the sea lamprey (Petromyzon marinus) was studied for the first time with an anti-GABA antibody. The earliest GABA-immunoreactive (GABAir) neurons appear in late embryos in the basal plate of the isthmus, caudal rhombencephalon, and rostral spinal cord. In prolarvae, the GABAir neurons of the rhombencephalon appear to be distributed in spatially restricted cellular domains that, at the end of the prolarval period, form four longitudinal GABAir bands (alar dorsal, alar ventral, dorsal basal, and ventral basal). In the spinal cord, we observed only three GABAir longitudinal bands (dorsal, intermediate, and ventral). The larval pattern of GABAir neuronal populations was established by the 30-mm stage, and the same populations were observed in premetamorphic and adult lampreys. The ontogeny of GABAergic populations in the lamprey rhombencephalon and spinal cord is, in general, similar to that previously described in mouse and Xenopus.