Firing properties of axotomized central nervous system neurons recover after graft reinnervation

Axotomy produces changes in the electrical properties of neurons and in their synaptic inputs, leading to alterations in firing pattern. We have considered the possibility that these changes occur as a result of the target deprivation induced by the lesion. Thus, we have provided a novel target to axotomized central neurons by grafting embryonic tissue at the lesion site to study the target dependence of discharge characteristics. The extracellular single-unit electrical activity of abducens internuclear neurons was recorded in the alert behaving cat in control, after axotomy, and after axotomy plus the implantation of cerebellar primordium. As recently characterized (de la Cruz et al. [2000] J. Comp. Neurol. 427:391-404), firing alterations induced by axotomy included an overall decrease in firing rate and a loss of eye-related signals, i.e., eye position and velocity neuronal sensitivities, that do not resume to normality with time. The grafting of a novel target to the injured abducens internuclear neurons restored the normal firing and sensitivities as recorded in the majority of units. To study the reinnervation of the implant, we performed anterograde labeling with biocytin combined with electron microscopy visualization. Axons of abducens internuclear neurons grew into the transplant sprouting into granule cell and molecular layers, as characterized by the immunostaining for gamma-aminobutyric acid and calbindin D-28k. Ultrastructural examination of labeled axons and boutons revealed the establishment of synaptic contacts, mainly axodendritic, with different cell types of the grafted cerebellar cortex. Therefore, these data indicate that axotomized central neurons resume to normal firing after the reinnervation of a novel target.     

Dendritic amputation redistributes sprouting evoked by axotomy in lamprey central neurons

In the previous paper (Hall and Cohen, 1988), we showed that axotomy of anterior bulbar cells (ABCs) in the hindbrain of the larval lamprey results in the sprouting of axonlike neurites from either the end of the proximal axon stump, the dendritic tips, or both, depending on the site of axotomy. Here we show that, unlike axotomy, dendritic amputation (dendrotomy) does not by itself induce sprouting from ABCs. However, dendrotomy does induce sprouting from dendrites in the immediate vicinity of the dendritic lesion in cells that have been previously axotomized. We found that dendrotomy acts primarily to rearrange the distribution of sprouts induced by axotomy rather than serving as an additional stimulus to neurite outgrowth. We propose that (1) dendritic sprouting in ABCs occurs because the dendritic tips become attractive sites for sprout initiation when they are either directly injured (as with dendrotomy) or are situated relatively close to the site of injury (as with axotomy close to the soma), and (2) the axon stump, dendritic stumps, and uninjured dendritic tips of the cell compete to initiate a limited total amount of sprouting induced by axotomy. The probability that a given locus will support sprouting is determined both by its proximity to the nearest lesion site and by whether there are other attractive potential sprouting sites in the cell.     

Laminin supports neurite outgrowth from explants of axotomized adult rat retinal neurons

The influence of laminin on neurite outgrowth from explants of adult rat retina and its distribution in normal and lesioned rat optic nerves were examined. Neurite outgrowth required the presence of laminin in the substratum, and as with a goldfish retinal explant system, was markedly stimulated by prior axotomy. Except for blood vessels and the nerve sheath, normal rat optic nerve was devoid of laminin immunoreactivity. Unlike results seen in the goldfish optic nerve, injury to the rat optic nerve induced no observable increase in laminin content or change in its distribution. The differences in the in vivo regenerative capacities of these two species may in part be related to the differences in their abilities to provide a proper substratum for axon regrowth.     

The pattern of dendritic sprouting and retraction induced by axotomy of lamprey central neurons

We have investigated some of the factors controlling the distribution of axonal and dendritic sprouting following axotomy of a subset of Muller giant interneurons (anterior bulbar cells or ABCs) in the hindbrain of the larval sea lamprey (Petromyzon marinus). Sprouts originated from different sites in the cell depending on the distance of the axonal lesion from the soma. When the axon was cut close to the soma (within 500 microns), the dendritic tips sprouted profusely, whereas the proximal axon stump showed few sprouts and frequently disappeared entirely. Axotomy further from the soma (1000-1400 microns) resulted in less sprouting from the dendrites and more from the axon stump, with the total amount of dendritic plus axonal sprouting remaining constant. Axotomy at sites distant from the soma (1 cm or more) did not result in dendritic sprouting. No sprouts were ever observed emerging from the soma proper or from the axon stump except at the lesion site. Neuritic sprouts from dendrites and axon were similar in their gross morphology. Sprouts resembled axons rather than dendrites whatever their sites of origin; they followed linear, rostrocaudally oriented paths in the "basal plate" region of the hindbrain. Dendritic and axonal sprouts grew both rostrally and caudally within the brain. Either "close" or "distant" axotomy resulted in the retraction of the dendritic tree and of both dendritic and axonal sprouts by several months postaxotomy. Reaxotomy close to the soma 30 d after a distant axotomy accelerated the onset of this evoked dendritic retraction. Reaxotomy close to the soma also induced sprouting significantly sooner than did close axotomy alone. These results suggest that axotomy close to the soma causes axonal regeneration to be shunted into ectopic locations at the dendritic tips. The emerging sprouts then follow guidance cues appropriate for regenerating ABC axons.     

Newly born dentate granule neurons after pilocarpine-induced epilepsy have hilar basal dendrites with immature synapses

Neurogenesis in the subgranular zone of the dentate gyrus persists throughout the lifespan of mammals, and the resulting newly born neurons are incorporated into existing hippocampal circuitry. Seizures increase the rate of neurogenesis in the adult rodent brain and result in granule cells in the dentate gyrus with basal dendrites. Using doublecortin (DCX) immunocytochemistry to label newly generated neurons the current study focuses on the electron microscopic features of DCX-labeled cell bodies and dendritic processes in the dentate gyrus of rats with pilocarpine-induced epilepsy. At the base of the granule cell layer clusters of cells that include up to six DCX-labeled cell bodies were observed. The cell bodies in these clusters lacked a one-to-one association with an astrocyte cell body and its processes, a relationship that is typical for newly born granule cells in control rats. Also, DCX-labeled basal dendrites in the hilus had immature synapses while those in control rats lacked synapses. These results indicate that increased neurogenesis after seizures alters the one-to-one relationship between astrocytes and DCX-labeled newly generated neurons at the base of the granule cell layer. The data also suggest that the synapses on DCX-labeled hilar basal dendrites contribute to the persistence of hilar basal dendrites on neurons born after pilocarpine-induced seizures.     

Retinopetal neurons and fibers in the lamprey

The structure of the retinopetal system was studied in lampreys (Lampetra fluviatilis) using the HRP method. Preliminary chemical destruction of the retina or the optic nerve transsection ceased the anterograde axonal transport in the retinofugal fibres, and permitted observing the centrifugal visual projections without marking the retinofugal fibres. The cells of retinopetal system were found in the periventricular tegmental zone and in the reticular zone of mesencephalon. Retinopetal fibres followed the axial optic tract.     

Grafts of fetal central nervous system tissue rescue axotomized clarke's nucleus neurons in adult and neonatal operates

Many conditions are thought to contribute to neuron death after axotomy, including immaturity of the cell at the time of injury, inability to reestablish or maintain target contact, and dependence on trophic factors produced by targets. Exogenous application of neurotrophic factors and transplants of peripheral nerve and embryonic central nervous system (CNS) tissue temporarily rescue axotomized CNS neurons, but permanent rescue may require transplants that are normal targets of the injured neurons. We examined the requirements for survival of axotomized Clarke's nucleus (CN) neurons. Two months after hemisection of the spinal cord at the T8 segment, there was an ipsilateral 30% loss of neurons at the L1 segment in adult operates and a 40% loss in neonates. Transplants of embryonic spinal cord, cerebellum, and neocortex inserted into the T8 segment at the time of hemisection prevented virtually all of the cell death in both adults and neonates, but transplants of embryonic striatum were ineffective. None of the grafts prevented the somal atrophy of CN neurons caused by axotomy. Retrograde transport of fluoro-gold from the cerebellum demonstrated that 33% of all CN neurons at L1 project to the cerebellum, 50% of these died following a T8 hemisection, but all these projection neurons were rescued by a transplant of embryonic spinal cord. These results suggest that the rescue of axotomized CN neurons is relatively specific for the normal target areas of these neurons, but this specificity is not absolute and may depend on the distribution and synthesis of particular neurotrophic agents. © 1994 Wiley-Liss, Inc.     

Ultrastructural characteristics of the dendrites of spinal neurons

Some kinds of dendritic protrusions were found in the different parts of the spinal cord grey matter connected with the long descending fibre systems. The small simple dendritic protrusions were localized in the medial part of the ventral horn. As a rule they were invaginated in the axon terminal which made the simultaneous synaptic contacts with them and with the basic trunk of dendrite. In the lateral parts of the V-VII laminae of the grey matter the dendritic branches were getting longer and more complex, the breaking forms and "crest" synapses appeared. There were some inclusions in the matrix which were probably the precursor of the spine apparatus and were called spine-like protrusions. Statistically authentic difference was determined between the size of the dendritic protrusions in the medial and lateral parts of the ventral horn grey matter.     

Neurofilament sidearm proteolysis is a prominent early effect of axotomy in lamprey giant central neurons

In the accompanying paper, it was shown that axotomy of lamprey spinal axons induces the rapid formation of condensed neurofilamentous masses in the proximal axon stump near the lesion. In this study, we used immunocytochemical and Western blot analysis to characterize these masses further and to determine the time course of their formation and dispersal. We show that monoclonal antibodies specific to the “rod” domain of lamprey neurofilament protein strongly stain such masses in tissue sections without staining other axonal neurofilaments. Antibodies specific for the neurofilament “sidearm” domain fail to recognize neurofilamentous masses but stain other axonal neurofilaments. Western blots of spinal cord segments from the lesion site were compared to unlesioned cord and to samples of cord distant from the lesion. We found that a neurofilament rodrspecific antibody identified breakdown products of the same size as the rod domain in samples from the lesion site, but not elsewhere. Other lesion-specific neurofilament breakdown products were recognized by a sidearm-specific antibody. This lesion-specific pattern of neurofilament proteolysis was visible at 1 day postlesion and was still present 3 weeks later. Immunocytochemistry showed masses of rod-staining neurofilaments in axon stumps by 12 hours postlesion that remained for 1–2 weeks postaxotomy; these dispersed with the onset of regeneration. Such neurofilament rod staining was also prominent in distal axon stumps undergoing Wallerian degeneration. We conclude that axotomy induces neurofilament sidearm proteolysis near the lesion, permitting antibody access to the rod domain. We suggest that sidearm loss causes the high packing density of neurofilaments within neurofilamentous masses near the lesion site and that neurofilament sidearm proteolysis can be used to distinguish degenerative from regenerative changes in lesioned lamprey axons. © 1995 Wiley-Liss, Inc.     

Mechanosensitive neurons in the spinal cord of the lamprey

‘Fictive locomotion’ in the lamprey in vitro spinal cord-notochord preparation can be entrained by side to side movements of the spinal cord-notochord which mimic swimming movements even after transection of dorsal and ventral roots. This study provides direct evidence of mechonosensitive neurons intrinsic to the spinal cord. Neurons with axons located in the lateral aspect of the spinal cord discharge in response to moderate bending movements of the spinal cord. Movements of this amplitude will inevitably occur during normal swimming.     

Inhibitors of mitogen-activated protein kinases protect axotomized developing neurons

Axotomy kills developing neurons by mechanisms dependent on protein synthesis and influenced by the redox status. Amongst the redox-regulated transduction systems regulating gene expression are the mitogen-activated protein kinases (MAPKs). In the chick embryo, inhibitors of two different MAPK pathways, including notably the p38 kinase pathway, reduce the number of dying axotomized retinal ganglion cells. The regulation of the genetic events associated to axotomy-induced death thus seems to involve MAPKs.     

Discharge characteristics of axotomized abducens internuclear neurons in the adult cat

The aim of the present work was to characterize the axotomy-induced changes in the discharge properties of central nervous system neurons recorded in the alert behaving animal. The abducens internuclear neurons of the adult cat were the chosen model. The axons of these neurons course through the contralateral medial longitudinal fascicle and contact the medial rectus motoneurons of the oculomotor nucleus. Axotomy was carried out by the unilateral transection of this fascicle (right side) and produced immediate oculomotor deficits, mainly the incapacity of the right eye to adduct across the midline. Extracellular single-unit recording of abducens neurons was carried out simultaneously with eye movements. The main alteration observed in the firing of these axotomized neurons was the overall decrease in firing rate. During eye fixations, the tonic signal was reduced, and, on occasion, a progressive decay in firing rate was observed. On-directed saccades were not accompanied by the high-frequency spike burst typical of controls; instead, there was a moderate increase in firing. Similarly, during the vestibular nystagmus, neurons hardly modulated during both the slow and the fast phases. Linear regression analysis between firing rate and eye movement parameters showed a significant reduction in eye position and velocity sensitivities with respect to controls, during both spontaneous and vestibularly induced eye movements. These firing alterations were observed during the 3 month period of study after lesion, with no sign of recovery. Conversely, abducens motoneurons showed no significant alteration in their firing pattern. Therefore, axotomy produced long-lasting changes in the discharge characteristics of abducens internuclear neurons that presumably reflected the loss of afferent oculomotor signals. These alterations might be due to the absence of trophic influences derived from the target.     

Quantitative cytochemistry of RNA in axotomized feline rubral neurons

One-sided lateral funiculotomy at the C-2 segment induced axon reaction in the contralateral red nucleus of adult cats. Two to 60 days postoperatively the animals were sacrificed and the mesencephalon was fixed in ethanol-acetic acid, 3:1. Ten μm paraffin sections including both red nuclei were stained for RNA with azure B after incubation in DNAse. Cytophotometric measurements of RNA content of neurons from the caudal 600–1000 μm of each red nucleus were made with a Zeiss Cytoscan system using an automatic scanning stage. In contrast to the heightened RNA synthesis that has been reported for axotomized peripheral (extrinsic) neurons, the axotomized central (intrinsic) neurons of the red nucleus showed no evidence of accumulation of cytoplasmic or nucleolar RNA. Rather depletion of cellular RNA occurred. Further indication of the regressive nature of rubral axon reaction derived from morphometric measurements that showed cytoplasmic, nuclear and nucleolar atrophy of the neurons of the red nucleus contralateral to operation with the exception of a possible transient cytoplasmic enlargement 9 days postoperatively. From the cytophotometric and morphometric data here reported we are led to suggest that the frequenyly observed failure of axonal repair in mammalian CNS results from the innately regressive nature of the axon reaction of many mammalian central neurons.     

Effects of axotomy on lamprey spinal neurons

The central axonal processes of dorsal cells of larval sea lampreys were cut by a high spinal transection. Subsequent histologic changes included a loss of cytoplasmic basophilia, nuclear eccentricity, the development of a perinuclear basophilic shell, and a slight decrease in cell diameter and increase in nuclear diameter resulting in a 26% increase in the ratio of nucleus to cell diameter. These changes were maximal at 3 weeks and were related to the proximity of the cell to the transection site. Electrophysiologic changes included increased input resistance, increased voltage and current thresholds for spike initiation, decreased maximal rate of rise of action potentials, increased spike width, and increased spike overshoot. There was also an increase in axonal conduction velocity and a reduction in resting membrane potential. The electrophysiologic changes tended to lag behind the histologic changes by 1 to 2 weeks and were not clearly related to the proximity of the cell to the transection site. The cellular electrophysiologic changes could result from a decrease in the densities of sodium and potassium channels of the membrane, but there is no evidence yet to support this or any other specific hypothesis.     

The temporal sequence of morphological and molecular changes in axotomized feline motoneurons leading to the formation of axons from the ends of dendrites

At 8-12 weeks post axotomy, unusual distal processes (UDPs) with axon-like structural (uniform diameter, tortuous) and molecular (growth-associated protein [GAP]43, absence of microtubule-associated protein [MAP]2a/b immunoreactivity) features emerge from distal motoneuron dendrites (Rose et al. [2001] Eur J Neurosci 13:1166-1176). In this study, we determine the time course of molecular and morphological changes associated with the formation of axons from dendrites. Motoneurons innervating neck muscles in the adult cat were permanently axotomized for 2, 4, 20, or 35 weeks and intracellularly stained with Neurobiotin. Computer-assisted reconstructions were used to map the location of MAP2a/b and GAP-43 immunoreactivity. At 2 and 4 weeks post axotomy, all UDPs had short appendages, giving them an arboreal appearance. They were immunoreactive for GAP-43 and lacked immunostaining for MAP2a/b. Axon-like UDPs were not seen until 8-12 weeks post axotomy. By 20 and 35 weeks post axotomy, some axon-like UDPs acquired morphological features of axons with synaptic connections (right-angled branching, bouton-like specializations). GAP-43 immunoreactivity was not detected in any axotomized motoneurons by 20 weeks post axotomy, whereas all UDPs remained devoid of MAP2a/b immunoreactivity even at 35 weeks post axotomy. These molecular changes accompanied structural modifications to proximal regions of "dendrites" giving rise to UDPs. The distance from the ends of the UDPs to the soma did not change. Thus, all UDPs begin as simple, arboreal structures with molecular features of growing axons, but over a period of 35 weeks, some UDPs slowly acquire morphological and molecular features of motoneuron axons with synaptic connections. These results suggest a new modus operandi for axonal growth and the establishment of new synaptic connections after injury.     

Cell proliferation in the lamprey central nervous system

After spinal cord transection, axons regenerate both in larval and adult lampreys. It is not known to what degree cells proliferate, even in the uninjured animal. Therefore, we have determined the prevalence of mitosis in the lamprey central nervous system (CNS). Bromodeoxyuridine (BrdU) was injected and incorporated for 4 hours into 2- to 5-year-old larvae, animals undergoing metamorphosis, and young adults. Labeled cells were counted in the rhombencephalon (where most supraspinal projecting neurons are located) and spinal cord. A mitotic index (MI) was calculated as the percentage of nuclei that were labeled. There was a seasonal variation in mitotic activity, with higher MIs occurring in summer. Within the summer, there was an additional transient spike in mitosis, especially in the rhombencephalon. There was no correlation between age and MI within the range of developmental stages examined. Baseline MIs in the rhombencephalon and spinal cord were approximately 0.15% and 0.20%, respectively. In most animals, the highest mitotic rates in both the rhombencephalon and spinal cord were seen in the ependyma, but many labeled cells were found in nonependymal regions as well. During the summer spike, almost all of the additional mitosis in the rhombencephalon was in the ependyma, but this finding was not true in the spinal cord. Many BrdU-labeled cells in the spinal cord and rhombencephalon were also stained by monoclonal antibodies specific for lamprey glial keratin but were never labeled by anti-neurofilament antibodies. These results suggest that (1) neurogenesis is uncommon in the lamprey CNS; (2) during most of the year, baseline gliogenesis occurs mainly in the ependyma with substantial contribution by nonependymal areas. During the summer, a spike of mitotic activity occurs in the ependyma of the rhombencephalon and throughout the spinal cord.     

Morphological response of axotomized septal neurons to nerve growth factor

Septal efferent fibers from the neurons in the medial septal nucleus are destroyed by fimbria-fornix aspirative lesion. In the present study we used quantitative morphometric techniques to evaluate the response of these axotomized septal neurons to a constant infusion of nerve growth factor (NGF). By 2 weeks following the lesion, approximately 75% of the cholinergic neurons had degenerated in the untreated rats. The remaining cholinergic neurons showed few signs of the effect of the lesion when stained for a polyclonal antibody to ChAT and examined in 40-micron-thick sections. In 1-micron-thick sections the remaining ChAT-immunoreactive (IR) neurons also appeared no different from the intact ChAT neurons. However, non-ChAT-IR neurons had a shrunken nucleus, while all other morphometric parameters appeared normal. NGF infusion protected most of the ChAT-IR neurons from degenerating. The saved neurons had the same parameters as the undamaged ChAT-IR neurons when examined in either 40-micron- or 1-micron-thick sections. In addition, the shrunken appearance of the non-ChAT-IR neurons' nuclei was avoided by the NGF infusions. Enlarged ChAT-IR processes were evident in the dorsolateral quadrant of the septum following damage to the fimbria-fornix. NGF-infusions prevented the formation of these processes. Instead, in the treated animals the dorsal lateral quadrant contained a dense plexus of fine ChAT-IR varicosities. Taken together these results demonstrate that NGF not only can protect the cholinergic neurons from axotomy-induced degeneration but can also cause the saved neurons to maintain the same morphometric appearance as intact ChAT-IR neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiologic evidence of regeneration of lamprey spinal neurons

Morphologic evidence has shown that the anteriorly projecting axons of giant interneurons (GIs) can regenerate after spinal transection in larval sea lampreys (19). In the present study, we showed that the regenerating neurites of GIs were electrically excitable. We also showed evidence for regeneration of descending afferent connections to GIs. Spinal cords were transected at the level of the cloaca. After at least 70 days recovery, GIs located 1.5 to 17.0 mm below the scar were impaled with microelectrodes. Stimulating electrodes were placed at various distances above the scar. Six of 13 GIs located 4 to 17 mm below the scar could be activated antidromically. For 1 GI, the rostralmost point of stimulation which elicited these responses was 13.5 mm above the scar. For the others, the range was 0.5 to 4.5 mm. Estimated average conduction velocity in regenerated neurites was 0.50 m/s compared with 1.94 m/s for the parent axon. Twelve GIs could be orthodromically activated by fixed-latency EPSPs. The most rostral point of stimulation that could elicit such responses was 0.5 to 8.5 mm above the scar. There was an inverse relationship between the farthest distance of stimulation and the distance of the GI from the scar. These findings are consistent with the hypothesis that regeneration of axons across a spinal transection is limited to neurons whose cell bodies are situated within 1 to 2 cm from the transection, and that regenerating neurites grow only a few millimeters beyond the scar.     

Emergence of axons from distal dendrites of adult mammalian neurons following a permanent axotomy

The distinctive features of axons and dendrites divide most neurons into two compartments. This polarity is fundamental to the ability of most neurons to integrate synaptic signals and transmit action potentials. It is not known, however, if the polarity of neurons in the adult mammalian nervous system is fixed or plastic. Following axotomy, some distal dendrites of neck motoneurons in the adult cat give rise to unusual processes that, at a light microscopic level, resemble axons (Rose, P.K. & Odlozinski, M., J. Comp. Neurol., 1998, 390, 392). The goal of the present experiments was to characterize these unusual processes using well-established ultrastructural and molecular criteria that differentiate dendrites and axons. These processes were immunoreactive for growth-associated protein-43 (GAP-43), a protein that is normally confined to axons. In contrast, immunoreactivity for a protein that is widely used as a marker for dendrites, microtubule-associated protein (MAP)-2a/b, could not be detected in the unusual distal arborizations. At the electron microscopic level, unusual distal processes contained dense collections of neurofilaments and were frequently myelinated. These molecular and structural characteristics are typical of axons and suggest that the polarity of adult neurons in the mammalian nervous system can be disrupted by axotomy. If this transformation in neuronal polarity is common to other types of neurons, axon-like processes emerging from distal dendrites may represent a mechanism for replacing connections lost due to injury. Alternatively, the connections formed by these axons may be aberrant and therefore maladaptive.