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.     

NADPH-Diaphorase in the central nervous system of the larval lamprey (Lampetra planeri)

The anatomy of the hippocampus, including the organization of its intrinsic neural circuits and afferents, is organized along a rostrocaudal axis. Dopamine D2 receptors are expressed in specific regions of the hippocampal complex (hippocampal subfields, entorhinal cortex, perirhinal cortex) and show differential expression along this axis. The dentate gyrus and CA3/CA4 subfields show higher numbers of D2 receptors in the rostral than in the caudal levels. In contrast, the subiculum shows the reverse gradient. We report here that Alzheimer's disease (AD) is associated with reduced expression of the dopamine D2 receptor, but the effects differ with respect to the rostrocaudal axis and area within the hippocampal complex. The number of D2 receptors is significantly reduced in the molecular layer of the dentate gyrus, CA3 subfield, and subiculum. For the dentate gyrus and subiculum, there were greater losses at more rostral levels. The CA3/CA4 subfields showed the greatest losses caudally. The entorhinal cortex, which shows only modest expression of D2 receptors in controls, does not exhibit reduced numbers in AD. The external laminae of the rostral perirhinal cortex showed more significant losses than more caudally in this cortical field. The regions showing loss of D2 receptors do not typically contain neuritic plaques, neurofibrillary tangles, or significant neuron loss. Thus other mechanisms must account for the unique gradient of D2 receptor loss in the hippocampus. The regions of reduced expression of dopamine D2 receptors do correlate well with the terminal zone of the dentate association pathway, the afferents from the anlygdala and perirhinal cortex, and the sources of those afferents within the amygdala and perirhinal cortex. The specific patterns of reduced D2 receptor expression in AD are likely to contribute significantly to the disrupted information flow into and out of the hippocampus and, thus, of functions subserved by this system. © 1994 Wiley-Liss, Inc.     

Regrowth of axons into the distal spinal cord through a Schwann-cell-seeded mini-channel implanted into hemisected adult rat spinal cord

Schwann cells (SCs) have been shown to be a key element in promoting axonal regeneration after being grafted into the central nervous system (CNS). In the present study, SC-supported axonal regrowth was tested in an adult rat spinal cord implantation model. This model is characterized by a right spinal cord hemisection at the eighth thoracic segment, implantation of a SC-containing mini-channel and restoration of cerebrospinal fluid circulation by suturing the dura. We demonstrate that a tissue cable containing grafted SCs formed an effective bridge between the two stumps of the hemicord 1 month after transplantation. Approximately 10 000 myelinated and unmyelinated axons (1 : 9) per cable were found at its midpoint. In addition to propriospinal axons and axons of peripheral nervous system (PNS) origin, axons from as many as 19 brainstem regions also grew into the graft without additional treatments. Most significantly, some regenerating axons in the SC grafts were able to penetrate through the distal graft-host interface to re-enter the host environment, as demonstrated by anterograde axonal labelling. These axons coursed toward, and then entered the grey matter where terminal bouton-like structures were observed. In channels containing no SCs, limited axonal growth was seen within the graft and no axons penetrated the distal interface. These findings further support the notion that SCs are strong promotors of axonal regeneration and that the mini-channel model may be appropriate for further investigation of axonal re-entry, synaptic reconnection and functional recovery following spinal cord injury.     

Localization and stoichiometry of electrogenic sodium bicarbonate cotransport in retinal glial cells.

An electrogenic Na+/HCO3- cotransport system was identified and characterized in freshly dissociated salamander Müller (glial) cells. Under voltage-clamp, these cells generated an outward current when external HCO3- concentration [( HCO3-]o) was raised. This current was Na(+)-dependent, Cl(-)-independent, and was blocked by the stilbenes 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate (DIDS) and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), and by harmaline, demonstrating that the current was generated by a Na+/HCO3- cotransport system. Substantially larger currents were evoked when [HCO3-]o was raised at the Müller cell endfoot as compared to other cell regions, indicating that cotransporter sites are localized preferentially to the endfoot. The reversal potential of the current, which varied as a function of HCO3- and Na+ transmembrane gradients, indicated that the cotransporter has a HCO3-:Na+ stoichiometry of 3:1.     

Determinants of directional specificity in the regeneration of lamprey spinal axons

The projection patterns of regenerating spinal axons in the larval sea lamprey (Petromyzon marinus) were determined by intracellular injection of HRP. Four hundred and eighty-six of 562 stained axons and axon-like neurites (87%) arising from Muller and Mauthner axons, giant interneurons, and dorsal cells terminated in an orientation similar to that of their counterpart control cells. Therefore, lamprey spinal axons regenerate selectively along their normal projection paths. During the first 4 weeks of recovery, i.e., before any had regenerated beyond the transection site, 91 of 114 axons and long neurites (80%) projected in the proper direction. Thus, the correctness of the final projection patterns did not result from selective retraction of randomly directed long neurites. When the cords were doubly transected 1 cm apart, orientation of regenerating neurites remained normal both within the 1 cm island and in the adjacent spinal cord. This suggests that the directional specificity of axonal regeneration was determined neither by the location of the scar nor by the availability of channels formed by the degenerating fibers. Finally, removing 1 cm of spinal cord eliminated potential synaptic targets for regenerating axons on either side of the lesion, but did not affect the direction of axonal growth. These findings are consistent with the hypothesis that the regeneration of lamprey spinal axons is guided by local chemical cues that persist long after the pathways are formed early in development.     

Experimental evidence for saltatory propagation of the Mauthner axon impulse in the tench spinal cord

External longitudinal current recording was applied in situ to the exposed spinal cord of the tench (Tinca tinca) for the study of impulse propagation in the Mauthner axon, a giant nerve fibre whose myelin sheath is not interrupted by nodes of Ranvier. Impulses in the antidromically excited Mauthner axon were recorded from the dorsal surface of the spinal cord; the time-lag between the main peaks of the bipolarly recorded current signal and unipolarly recorded reference signal was measured at regular intervals along the cord. Using a slot width of the bipolar electrodes,d= 0.65mm and electrode displacements= 0.5mm, the latency plotted as a function of distance showed small fluctuations, but no clear-cut steps over a 12.5 mm stretch of spinal cord. However, with improved spatial resolution (d= 0.24mm, s= 0.1mm) and electrical insulation of the spinal cord from the underlying tissues, it was possible to demonstrate steps in the latency plot occurring at 0.5–0.3 mm intervals and indicating a saltatory propagation of the Mauthner axon impulse. The distances between the latency steps and their distribution was comparable to the known distribution of the Mauthner axon collaterals suggesting that the myelin-free regions of the collaterals may be equivalents of Ranvier nodes.     

Barium action potentials in regenerating axons of the lamprey spinal cord

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

Regeneration of motoneuron axons into the adult frog spinal cord after ventral-to-dorsal-root anastomosis

Motoneuron axons routed into the adult frog spinal cord via a ventral-to-dorsal-root anastomosis regenerated into the white and the gray matters. The distribution, growth patterns, and arborizations of regenerated ventral root axons were compared to those of regenerated dorsal root axons within the same environment. Within the spinal white matter, regenerating ventral root axons behaved very similarly to regenerating dorsal root axons. Here, the regenerating ventral root axons grew longitudinally beneath the pia and radially toward the spinal gray matter, particularly within the dorsolateral fasciculus. The location of the regenerating axons and the patterns of their growth within the white matter suggest that glial endfeet and radial glial processes play a major role in the determination of these axonal growth patterns. When motor axons entered the gray matter, their arborizations were very similar to those of regenerated dorsal root axons, suggesting that these two very distinct populations of axons respond similarly to local cues within the spinal gray matter. One difference between the arborizations of these two populations of axons was the relative number of varicosities along axonal branches. Regenerated motoneuronal arborizations within the spinal gray matter had fewer en passant varicosities than regenerated dorsal root axonal arborizations. This difference may reflect the synaptogenetic response of the two types of axons to targets within the gray matter. The low number of en passant varicosities associated with the ventral root axonal aborizations suggests that these axons do not synapse with all available targets and that the rules governing synaptic specificity during development may apply during regeneration in the adult frog spinal cord.     

Morphology of physiologically identified rubrospinal axons in the spinal cord of the cat

Intra-axonal injection of HRP into physiologically identified rubrospinal neurons has shown that axon collaterals are given off at different cervical segments from stem axons. These collaterals spread in a delta-like fashion in laminae V–VII (occasionally in IX) and extend very widely in a rostrocaudal direction (1.0–5.1 mm).     

Metamorphosis and the regenerative capacity of spinal cord axons in Xenopus laevis

Throughout the vertebrate subphylum, the regenerative potential of central nervous system axons is greatest in embryonic stages and declines as development progresses. For example, Xenopus laevis can functionally recover from complete transection of the spinal cord as a tadpole but is unable to do so after metamorphosing into a frog. Neurons of the reticular formation and raphe nucleus are among those that regenerate axons most reliably in tadpole and that lose this ability after metamorphosis. To identify molecular factors associated with the success and failure of spinal cord axon regeneration, we pharmacologically manipulated thyroid hormone (TH) levels using methimazole or triiodothyronine, to either keep tadpoles in a permanently larval state or induce precocious metamorphosis, respectively. Following complete spinal cord transection, serotonergic axons crossed the lesion site and tadpole swimming ability was restored when metamorphosis was inhibited, but these events failed to occur when metamorphosis was prematurely induced. Thus, the metamorphic events controlled by TH led directly to the loss of regenerative potential. Microarray analysis identified changes in hindbrain gene expression that accompanied regeneration-permissive and -inhibitory conditions, including many genes in the permissive condition that have been previously associated with axon outgrowth and neuroprotection. These data demonstrate that changes in gene expression occur within regenerating neurons in response to axotomy under regeneration-permissive conditions in which normal development has been suspended, and they identify candidate genes for future studies of how central nervous system axons can successfully regenerate in some vertebrates.     

A new population of neurons with crossed axons in the lamprey spinal cord

Neurons with contralateral, rostrally and caudally projecting axons were studied in whole mounts of lamprey spinal cord using retrograde labelling techniques with fluorescent dextran-amines, cobalt-lysine or horseradish peroxidase. A previously unknown large population (180-300 cells per hemisegment) of small (less than 25 microns) cells with contralateral projecting axons is described. Their axons extend over less than 5 segments rostrally or caudally. The number of these cells per segment was relatively constant in the rostral half of the spinal cord, but increased significantly in the caudal half. In comparison, medium-sized cells with contralateral axons corresponding to previously identified premotor interneurons were far less numerous (14-21 per hemisegment) and their axons extended more than 5 segments. Contralaterally projecting edge cells (intraspinal stretch receptor neurons) with principal rostral or caudal axons plus short collaterals in the other direction were distributed throughout the length of the spinal cord, whereas large and giant cells with a varied morphology were found in the caudal half.     

Excitatory amino acid receptors in primary cultures of glial cells from the retina.

Binding of 3H-L-aspartate to membranes from retinal glial cells in primary culture was characterized. Binding kinetics showed a saturable, reversible binding to three populations of sites with KB = 40, 200, and 1,300 nM. The first two were present at 1 day in vitro (DIV), whereas the latter two were observed at 12 DIV. The possibility of the 40 nM site being neuronal cannot be discarded, since some neurons are present at 1 DIV. In 12 DIV cultures, the presence or absence of sodium determined two different pharmacological patterns, comparable to those described for electrogenic glutamate transport in Müller cells, and QA metabotropic receptors in astrocytes, respectively. Results suggest that, as has been shown for some receptors in nerve tissue, the properties of glial cell receptors undergo age-dependent changes. In turn, this could be related to changes in the function of neurotransmitter substances during development.     

Spontaneous regeneration of intrinsic spinal cord axons in a novel spinal cord slice culture model

A substantial problem in research concerned with axonal repair is the use of a wide variety of lesion models and the complexity associated with the respective in vivo lesion paradigms. Organotypic slice cultures are a potential in vitro alternative because the cytoarchitectonic tissue organization is well preserved and the slices can be maintained in culture for several weeks. Until now no spinal cord slice culture model for the study of axonal growth has been available. Here we present a spinal cord slice culture model that is well suited for the study of axonal growth. The spinal cord slices were cut not in the transverse but in the sagittal longitudinal plane such that several spinal cord segments were included in the slice culture. In these cultures the typical ventro-dorsal polarity of the spinal cord was maintained and intrinsic spinal cord axons formed a strong fibre tract extending along the longitudinal axis of the slice. The axons became myelinated during the culture period and synaptic contacts were present in these cultures. After mechanical lesions the intrinsic spinal cord axons had a substantial potential for axonal growth and regeneration. The number of regenerating axons crossing the lesion site decreased with increasing maturation of the culture, but even in mature cultures a small number of crossing fibres were present. This slice culture model could provide an important tool for several aspects of spinal cord research in the fields of axonal growth and regeneration, synapse formation, formation of intrinsic spinal cord circuits and myelination.     

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.     

Oligodendroglia in the avian retina: immunocytochemical demonstration in the adult bird.

Immunohistochemical techniques were used in conjunction with an avian-specific probe for oligodendrocyte (OLG) marker, the antibody for transferrin binding protein (TfBP), to study the characteristics and distribution of OLGs in the retina of chickens and quails. For comparison, other antibodies such as myelin basic protein, Rip, and those for labeling Müller cells and microglia were used. A large population of OLGs was found to be distributed throughout the retina, with the distinct pattern of a central-to-peripheral gradient. It was possible to detect a spectrum of OLG morphology that bore a resemblance to the subtype of the mammalian central nervous system. In addition to these mature OLGs, limited numbers of TfBP-positive (TfBP(+)) cells with the morphology of immature OLGs were found in the immediate vicinity of the optic head. The majority of OLGs appeared in the ganglion cell layer throughout the retina, whereas OLGs in the nerve fiber layer were seen mainly in the central zone of the retina, near the optic nerve head. Double-labeling experiments showed that OLGs were associated with myelin only in the central region, where the majority of retinal OLGs occurred, but not toward the periphery of the retina. The present study is the first comprehensive analysis of the morphological features and spatial distribution of OLGs in the adult avian retina and provides in vivo evidence for the existence of a substantial population of both mature and immature OLGs in the retina of adult birds. The putative functions of TfBP(+) OLGs including myelination and the tropic role of the ganglion cells are discussed in conjunction with the physical properties of TfBP and structural characteristics of the avascular retina of birds.     

The need for cellular elements during axonal regeneration in the sea lamprey spinal cord.

Spinal axons in the larval sea lamprey regenerate following a complete spinal transection. It is not known whether regenerating growth cones require contact with cellular elements or whether the basement membrane and collagenous meninx primitiva which surround the spinal cord are sufficient for neurite out-growth. To determine this, a freeze lesion was made which severed axons, destroyed neuronal perikarya, and greatly reduced the number of glial cells. After at least 10 weeks of recovery, 50 neurites from 31 Müller and Mauthner axons were labeled by intracellular injection of HRP. Eighty-six percent of these neurites did not regenerate into the lesion site. No neurites grew through the lesion. No animals recovered coordinated swimming. These results suggest that glial and/or neuronal surfaces are required for axonal regeneration. Moreover, a monolayer of glial cells appears to be suboptimal and a three-dimensional matrix of cells may be necessary to promote regeneration in the lamprey spinal cord.     

Characteristics of excitatory amino acid uptake in cultures from neurons and glia from the retina

3H-D-Aspartate uptake was biochemically characterized in cultures from chick retina enriched in glial (Müller) cells or neurons during progressive days in vitro (DIV). In the neuronal cultures a high-affinity, Na(+)-dependent system was found with Km = 8-13 microM and pharmacological characteristics in agreement with those of reuptake systems in other regions of the CNS. The uptake system in glial cells showed a lower affinity, with Km = 100-135 microM. In both cases, uptake was temperature and energy dependent. A sharp increase in the Vmax of uptake was observed in both neuronal and glial cultures at 5 DIV, at which time morphologically mature synapses have been shown to be present in retinal cultures. A parallel increase in the pharmacological specificity of the uptake system in neuronal cultures was observed, with a rise in the efficiency of D-Asp, L-Asp, L-Glu, and DL-asp- beta-hydroxamate for inhibiting 3H-D-Aspartate uptake. Results suggest the possibility of reuptake participating in the regulation of extracellular glutamate concentration during development.     

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

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