Transient inability of neonatal rat motoneurons to reinnervate muscle

We studied the reinnervation of internal intercostal muscles of newborn rats. The distal halves were denervated by nerve section at various ages between birth and 6 weeks. Regardless of the age at denervation, neither evoked nor spontaneous nerve-muscle transmission reappeared until the animals were at least 3 weeks old. Older rats recovered a substantial degree of function within 7 days of nerve section. Normally the motor units in this muscle are narrowly distributed, so most axotomized motoneurons lost their entire synaptic periphery. Reinnervation was by axons which had been sectioned, and regenerated motor units were of normal size and number. There was no collateral sprouting from end plates left intact. Motoneurons axotomized at birth did regenerate axons the full length of the muscle within 7 days of operation. Their failure to reinnervate the muscle was due to delay in forming functional end plates. Nerve section in animals aged 1 month or older resulted in an abnormal pattern of reinnervation; reinnervated motor units were diffusely spread through large portions of the muscle, although they still did not overlap with the region left intact. This indicates that thoracic motoneurons respond to axotomy differently in neonatal rats than they do in adults.     

Critical periods in rat motoneuron development

Reinnervation of rat internal intercostal muscles was examined 2 weeks after intramuscular axotomy in the period embryonic Day 17 (E17) to postnatal Day 2 (PN2). The efficiency of reinnervation depended on the day of denervation. Responses to nerve stimulation were common only in muscles denervated in the interval E19–E21. Functional reinnervation was seldom seen in muscles denervated earlier, and was absent in muscles denervated shortly after birth. We suggest that there are “critical periods” in the sequence of differentiation of motoneurons. At E17 these neurons are very susceptible to cell death and any that lose contact with their muscle will die. Motoneurons axotomized later, while motor unit size is still increasing, can regenerate some functional nerve-muscle junctions. Motor unit size reaches its maximum by E21 and if motoneurons are exotomized after that time, in neonates, they cannot form more synaptic terminals although they can still extend axons. During this early postnatal period motoneurons normally reduce their number of nerve-muscle contacts. Finally, at 3 weeks of age, motoneurons reach their adult state when they are capable of regeneration relatively unhindered by restrictions on the number and location of their peripheral connections.     

Axonal degeneration within the tympanal nerve of Schistocerca gregaria

This study describes time course and ultrastructural changes during axonal degeneration of different neurones within the tympanal nerve of the locust Schistocerca gregaria. The tympanal nerve innervates the tergit and pleurit of the first abdominal segment and contains the axons of both sensory and motor neurones. The majority of axons (approx. 97%) belong to several types of sensory neurones: mechano- and chemosensitive hair sensilla, multipolar neurones, campaniform sensilla and sensory cells of a scolopidial organ, the auditory organ. Axons of campaniform sensilla, of auditory sensory cells and of motor neurones are wrapped by glial cell processes. In contrast, the very small and numerous axons (diameter <1 microm) of multipolar neurones and hair sensilla are not separated individually by glia sheets. Distal parts of sensory and motor axons show different reactions to axotomy: 1 week after separation from their somata, distal parts of motor axons are invaded by glial cell processes. This results in fascicles of small axon bundles. In contrast, distal parts of most sensory axons degenerate rapidly after being lesioned. The time to onset of degeneration depends on distance from the lesion site and on the type of sensory neurone. In axons of auditory sensory neurones, ultrastructural signs of degeneration can be found as soon as 2 days after lesion. After complete lysis of distal parts of axons, glial cell processes invade the space formerly occupied by sensory axons. The rapid degeneration of distal auditory axon parts allows it to be excluded that they provide a structure that leads regenerating axons to their targets. Proximal parts of severed axons do not degenerate.     

The alpha 1- and alpha 2-adrenoceptor and muscarinic responses of normal and axotomized bullfrog sympathetic ganglia

When neurones in bullfrog paravertebral sympathetic ganglia are studied by means of the sucrose-gap technique, muscarinic agonists produce a biphasic response (an initial hyperpolarization of ganglionic C cells followed by a depolarization of ganglionic B cells). Activation of ganglionic alpha 2-adrenoceptors promotes hyperpolarization. The present experiments with selective alpha 1- and alpha 2-adrenoceptor agonists and antagonists provided evidence for the existence of hitherto undescribed alpha 1-adrenoceptors, which are responsible for the production of depolarizing responses in these ganglia. Fifteen to twenty-five days after cutting postganglionic axons (axotomy), there was a nonselective depression of both alpha 1- and alpha 2-adrenoceptor mechanisms but little change in muscarinic responses. These results argue against the hypothesis that C cells assume all the properties of B cells after axotomy. Since the alpha-selective agonist phenylephrine failed to depolarize axotomized ganglia, it is unlikely that an alpha 1-adrenoceptor mechanism is prominent in axotomized neurones as it is in some immature adrenergic neurones. The data are consistent with the idea that axotomy selectively affects the properties of certain types of cation channels and raise questions as to the mechanisms involved in regulating the expression and maintenance of specific neurotransmitter responses on ganglionic neurones.     

Organization of motoneurones in the prothoracic ganglion of the cockroach Periplaneta americana (L.).

The location within the prothoracic ganglion of neurone somata with axons in identified peripheral nerves is examined by the cobalt iontophoresis technique. Axons are filled with cobalt by diffusion through their cut ends and the cobalt is then precipitated as the black sulphide inside the neurone. It is assumed that neurones with axons in peripheral nerves and somata in central ganglia are either motor or neuro-secretory. Fifteen nerves are examined and maps of the location of somata with axons in each nerve are presented. The axon distribution in peripheral nerves of three common inhibitory neurones is described. Dendritic morphology of one common inhibitory neurone and two coxal depressor motoneurones is illustrated. It is proposed that some individual neurones can be reliably identified from their soma dimensions and location within the ganglion. The number of motoneurones with somata in the prothoracic ganglion and their homology with cells in the other thoracic ganglia are discussed.     

Alterations in the morphology of ganglion cell dendrites in the adult rat retina after optic nerve transection and grafting of peripheral nerve segments

Summary Transected ganglion cell axons from the adult retina are capable of reinnervating their central targets by growing into transplanted peripheral nerve (PN) segments. Injury of the optic nerve causes various metabolic and morphological changes in the retinal ganglion cell (RGC) perikarya and in the dendrites. The present work examined the dendritic trees of those ganglion cells surviving axotomy and of those whose severed axons re-elongated in PN grafts to reach either the superior colliculus (SC), transplanted SC, or transplanted autologous thigh muscle. The elaboration of the dendritic trees was visualized by means of the strongly fluorescent carbocyanine dye DiI, which is taken up by axons and transported to the cell bodies and from there to the dendritic branches. Alternatively, retinofugal axons regrowing through PN grafts were anterogradely filled from the eye cup with rhodamine B-isothiocyanate. The transection of the optic nerve resulted in characteristic changes in the ganglion cell dendrites, particularly in the degeneration of most of the terminal and preterminal dendritic branches. This occurred within the first 1 to 2 weeks following axotomy. The different types of ganglion cells appear to vary in their sensitivity to axotomy, as reflected by a rapid degeneration of certain cell dendrites after severance of the optic nerve. The most vulnerable cells were those with small perikarya and small dendritic fields (type II), whereas larger cells with larger dendritic fields (type I and III) were slower to respond and less dramatically affected. Regrowth of the lesioned axons in peripheral nerve grafts and reconnection of the retina with various tissues did not result in a significant immediate recovery of ganglion cell dendrites, although it did prevent some axotomized cells from further progression toward posttraumatic cell death.     

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.     

Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats

To investigate the short-and long-term effects of axotomy on the survival of central nervous system (CNS) neurons in adult rats, retinal ganglion cells (RGCs) were labelled retrogradely with the persistent market diI and their axons interrupted in the optic nerve (ON) by intracranial crush 8 or 10 mm from the eye or in intraorbital cut 0.5 or 3 mm from the eye. Labelled RGCs were counted in flat-mounted retinas at intervals from 2 weeks to 20 months after axotomy. Two major patterns of RGC loss were observed: (1) an inital abrupt loss that was confined to the first 2 weeks after injury and was more severe when the ON was cut close to the eye; (2) a slower, persistent decline in RGC densities with one-half survival times that ranged from approximately 1 month after intraorbital ON cut to 6 months after intracranial ON crush. A small population of RGCs (approximately 5%) survived for as long as 20 months after intraorbital axotomy. The initial loss of axotomized RGCs presumably results from time-limited perturbations related to the position of the ON injury. A. persistent lack of terminal connectivity between RGCs and their targets in the brain may contribute to the subsequent, more protracted RGC loss, but the differences between intraorbital cut and intracranial crush suggest that additional mechanisms are involved. It is unclear whether the various injury-related processes set in motion in both the ON and the retina exert random effects on all RGCs or act preferentially on subpopulations of these neurons. © 1993 John Wiley & Sons, Inc.     

Upregulation of anti-apoptotic factors in upper motor neurons after spinal cord injury in adult zebrafish

Unlike mammals, fish motor function can recover within 6–8 weeks after spinal cord injury (SCI). The motor function of zebrafish is regulated by dual control; the upper motor neurons of the brainstem and motor neurons of the spinal cord. In this study, we aimed to investigate the framework behind the regeneration of upper motor neurons in adult zebrafish after SCI. In particular, we investigated the cell survival of axotomized upper motor neurons and its molecular machinery in zebrafish brain. As representative nuclei of upper motor neurons, we retrogradely labeled neurons in the nucleus of medial longitudinal fasciculus (NMLF) and the intermediate reticular formation (IMRF) using a tracer injected into the lesion site of the spinal cord. Four to eight neurons in each thin sections of the area of NMLF and IMRF were successfully traced at least 1–15 days after SCI. TUNEL staining and BrdU labeling assay revealed that there was no apoptosis or cell proliferation in the axotomized neurons of the brainstem at various time points after SCI. In contrast, axotomized neurons labeled with a neurotracer showed increased expression of anti-apoptotic factors, such as Bcl-2 and phospho-Akt (p-Akt), at 1–6 days after SCI. Such a rapid increase of Bcl-2 and p-Akt protein levels after SCI was quantitatively confirmed by western blot analysis. These data strongly indicate that upper motor neurons in the NMLF and IMRF can survive and regrow their axons into the spinal cord through the rapid activation of anti-apoptotic molecules after SCI. The regrowing axons from upper motor neurons reached the lesion site at 10–15 days and then crossed at 4–6 weeks after SCI. These long-distance descending axons from originally axotomized neurons have a major role in restoration of motor function after SCI.     

Regulation of immediate-early gene expression in rat retinal ganglion cells after axotomy and during regeneration through a peripheral nerve graft

To determine mechanisms of structural plasticity in adult CNS neurons, we investigated the expression of immediate early genes (IEGs) in the rat retina. Gene products of different IEG families (JUN and FOS proteins) and cAMP-responsive element binding protein (CREBP) were examined by immunohistochemistry under three different paradigms. Normal rats which were not axotomized were compared with axotomized animals, where retinal ganglion cells (RGCs) were axotomized by intraorbital optic nerve cut and retrogradely labeled with fluorogold (FG). Under these circumstances, RGCs show only transient sprouting, followed by continuous retrograde RGC degeneration. In the third group, after the optic nerve lesion, adult rats additionally received a sciatic nerve graft to the transected optic nerve stump. This allows some RGCs to regenerate an axon into the grafted nerve. In both groups, the time course of RGC survival and JUN, CREB, and FOS protein expression was monitored. In normal animals, JUN-Immunoreactivity (JUN-Ir) was not detectable in the retinal ganglion cell layer. JUN-Ir was induced in about 70% of all FG-positive RGCs 5 days after axotomy. The expression of JUN-Ir started to decline 8 days after axotomy. Only a few JUN-Ir-positive RGCs were found after 2 weeks. In transplanted animals, however, the numbers of JUN-Ir-positive RGCs were significantly higher 2 and 3 weeks after transplantation compared to animals that exclusively received axotomy. Furthermore, in grafted rats about 70% of the regenerating RGCs expressed JUN-Ir 2 weeks after grafting as compared to only 38% JUN-positive RGCs among the surviving but not regenerating RGCs. In normal animals CREBP-Ir was constitutively expressed in nearly all cells of the retinal ganglion cell layer. The decline in number of CREBP-Ir-positive cells paralleled the axotmy-induced RGC death. FOS-Ir-positive cells were not found in the ganglion cell layer at any time. These results demonstrate a selective and transient JUN expression of RGCs after axotomy which is sustained during axonal regeneration. This suggests that sciatic nerve grafts are able to regulate the expression of JUN proteins in axotomized RGCs of adult rats. 1994 John Wiley & Sons, Inc.     

High correlation between microvillous olfactory receptor cell abundance and sensitivity to pheromones in olfactory nerve-sectioned goldfish

1. To determine whether microvillous olfactory receptor cells mediate responses to pheromonal cues, the olfactory nerves of mature male goldfish were axotomized and both the olfactory and behavioral sensitivity of these animals to olfactory stimuli investigated after which the histological condition of their olfactory epithelia was determined. 2. Behavioral responsiveness to food odor returned within 2 weeks but responsiveness to sexually-active females (pheromones) took 4–10 weeks to return. 3. Electro-olfactogram recordings from the olfactory epithelium of axotomized fish found that olfactory responsiveness to amino acids and pheromones changed little during the first week subsequent to axotomy. However, olfactory sensitivity decreased rapidly during the second week. During the course of the third week, electro-olfactogram sensitivity to amino acids remained while exposure to pheromones evoked no recordable electro-olfactogram. During week 4, sensitivity to amino acids increased further, and weak sensitivity to some pheromones became evident. Further recovery of electro-olfactogram sensitivity to all odorants was slow and erratic over the next 6 months, particularly to the pheromones. 4. Histological examination of the olfactory epithelia of axotomized fish demonstrated that while ciliated receptor cells were present within 2 weeks, microvillous receptor cells took approximately 4 weeks to regenerate. 5. Together these data suggest that microvillous receptor cells mediate responsiveness to pheromones in this species. Accepted: 22 August 1996     

Axotomy of developing rat spinal motoneurons: Cell survival,soma size,muscle recovery,and the influence of testosterone

During the period of synapse elimination, motoneurons are impaired in their ability to generate or regenerate axonal branches: following partial denervation of their target muscle, young motoneurons do not sprout to nearby denervated fibers and after axonal injury, they fail to reinnervate the muscle. In the rat levator ani (LA) muscle, which is innervated by motoneurons in the spinal nucleus of the bulbocavernosus (SNB), synapse elemination ends relatively late in development and can be regulated by testosterone. We took advantage of this system to determine if the end of synapse elimination and the development of regenerative capabilities by motoneurons share a common mechanism, or, alternatively, if these two events can be dissociated in time. Axotomy on or before postnatal day 14 (P14) caused the death of SNB motoneurons. By P21, toward the end of synapse elimination in the LA muscle, SNB motoneurons had developed the ability to survive axonal injury. Altering testosterone levels by castration on P7 followed by 4 weeks of either testosterone propionate or control injections did not change the ability of SNB motoneurons to survive axonal injury during development, although these same treatments alter the time course of synapse elimination in the LA muscle. Thus, we dissociated the inability of SNB motoneurons to recover from axonal injury from their developmental elimination of synaptic terminals. We also measured the effect of early axotomy on motoneuronal soma size and on target muscle weight. Axotomy on P14 caused a long-lasting decrease in the soma size of surviving SNB motoneurons, whereas motoneurons axotomized on P28 recovered their normal soma size. Axotomy on or before P7 caused severe atrophy of the target muscles, matching the extensive loss of motoneurons. However, target muscle recovery after axotomy on P14 was as good as recovery after axotomy at later ages, despite greater motoneuronal death after axotomy on P14. This result may reflect an increase in motor unit size, a decrease in polyneuronal innervation by SNB motoneurons that survive axotomy on P14, or a combination of the two. © 1995 John Wiley & Sons, Inc.     

Astrogliosis in different zones of the spinal cord gray matter after sciatic nerve axotomy in the newborn rat: a morphometric and immunohistochemical study

Astrocytic response following unilateral sciatic nerve axotomy was examined in the spinal gray matter of newborn rats. Using an antiserum to glial fibrillary acidic protein (GFAP), immunoreactive astrocytes were studied in the ventral, dorsal and transitional region between the dorsal and ventral gray matters (TDVG) at intervals of one day, one week, two weeks and one month postaxotomy. The axotomized side showed an obvious increase in the number of immunoreactive astrocytes at one week, two weeks and one month after surgery. The numerical density per area of the glial cells (N(a)) was determined in all groups on both the intact and axotomized sides, and it increased in all groups at the axotomized sides. The percentage of glial cell increase (Pgi) was also determined. At the ventral horn Pgi increased at day one and continued to increase in all groups, while the increase in TDVG and the dorsal horn occurred at later time points. The total motoneuron count in the ventral horn at the axotomized and intact sides was done at all time points, and the percentage of motoneuron reduction (Pmr) was calculated, the highest Pmr being noticed at one month (41%). A nonlinear regression for Pmr and Pgi showed that the rate of Pgi was approximately double that of Pmr. The rate of glial cell increase at each time point (one day, one week, two weeks and one month groups) was calculated, and the highest rate of glial cell increase in the ventral horn occurred one week after axotomy, while the highest rate in the dorsal horn and TDVG occurred at the second week. The conclusion of the study is that there may be an initial post-axotomic proliferative phase of the glial cells, which was followed by a differentiation phase. Also a gradient of an increase in the rate glial cell proliferation was noticed from the ventral horn toward the dorsal horn, maybe due to stimulation by a paracrine factor.     

Differential regulation of motor neuron survival and choline acetyltransferase expression following axotomy

Although it is well known that motor neuron survival following axotomy is enhanced with maturation, the ability of surviving neurons to express the cholinergic enzyme choline acetyltransferase (ChAT) following axotomy has not been closely examined. Moreover, the utility of the facial nucleus in studies of motoneuron response to injury and to trophic factors, coupled with the increasing importance of the mouse in gene targeting, compelled us to investigate the age dependence of neuronal survival and ChAT expression in the mouse facial nucleus following axotomy. We cut the facial nerve at postnatal day (P)4, 7, 14, 21, and 28 or in the adult and used Nissl staining and ChAT immunocytochemistry to quantitate survival and ChAT expression, respectively, following 1, 2, or 3 weeks' survival at each age. We confirm in this model that the rate and extent of motor neuron death following axotomy is reduced with increasing maturity. The surviving neurons maintain a high ChAT content through P21; however, axotomy from P28 through adulthood results in a striking reduction in ChAT immunoreactivity. That is, although axotomy at P21 results in 61% motor neuron survival, with virtually all of the surviving neurons being ChAT positive, axotomy in the adult results in 72% survival but only 9% of the neurons are ChAT positive. Thus, surviving motor neurons in the adult animals are only weakly cholinergic. These results indicate that a change in the regulation of ChAT expression occurs following P21 so that cell survival and enzyme levels are uncoupled. We suggest that the putative factor or factors that enhances motor neuron survival in maturity is not capable of maintaining ChAT expression. © 1995 John Wiley & Sons, Inc.     

Acrylamide Neuropathy: Changes in the Composition of Proteins of Fast Axonal Transport Resemble Those Observed in Regenerating Axons

Proteins conveyed by fast axonal transport along sensory and motor axons of rat sciatic nerve were labelled with L-[35S]methionine and characterized by one- and two-dimensional electrophoresis on polyacrylamide gels, followed by fluorography. Nerves from normal or bis-acrylamide-treated animals were compared with nerves from acrylamide-treated animals and nerves regenerating after a crush axotomy. In both sensory and motor axons significant changes in the pattern of labelled bands on one-dimensional gels occurred after 10 days of acrylamide treatment (50 mg/kg daily, i.p.). These changes resembled those seen in regenerating axons, but were less pronounced. No changes were detectable after shorter periods of treatment, even though the onset of the neuropathy, assessed by a behavioral test, occurred on days 4-6 of treatment. Two-dimensional separations of the labelled proteins revealed increased labelling of growth-associated protein 43 in acrylamide-treated animals, but again this was less pronounced than in regenerating nerves. Acrylamide treatment induces changes in composition of fast-transported protein that are qualitatively similar to those seen after axotomy. Since these changes are not detectable until the neuropathy is advanced, it is unlikely that they are causative factors. Instead, they are most likely a result of the cell body reaction previously observed in acrylamide intoxication, a reaction that resembles that produced by axotomy.     

Axonal transport of class II and III beta-tubulin: evidence that the slow component wave represents the movement of only a small fraction of the tubulin in mature motor axons.

Pulse-labeling studies demonstrate that tubulin synthesized in the neuron cell body (soma) moves somatofugally within the axon (at a rate of several millimeters per day) as a well-defined wave corresponding to the slow component of axonal transport. A major goal of the present study was to determine what proportion of the tubulin in mature motor axons is transported in this wave. Lumbar motor neurons in 9-wk-old rats were labeled by injecting [35S]methionine into the spinal cord 2 wk after motor axons were injured (axotomized) by crushing the sciatic nerve. Immunoprecipitation with mAbs which recognize either class II or III beta-tubulin were used to analyze the distributions of radioactivity in these isotypes in intact and axotomized motor fibers 5 d after labeling. We found that both isotypes were associated with the slow component wave, and that the leading edge of this wave was enriched in the class III isotype. Axotomy resulted in significant increases in the labeling and transport rates of both isotypes. Immunohistochemical examination of peripheral nerve fibers demonstrated that nearly all of the class II and III beta-tubulin in nerve fibers is located within axons. Although the amounts of radioactivity per millimeter of nerve in class II and III beta-tubulin were significantly greater in axotomized than in control nerves (with increases of +160% and +58%, respectively), immunoassay revealed no differences in the amounts of these isotypes in axotomized and control motor fibers. We consider several explanations for this paradox; these include the possibility that the total tubulin content is relatively insensitive to changes in the amount of tubulin transported in the slow component wave because this wave represents the movement of only a small fraction of the tubulin in these motor fibers.     

Neurotrophic agents prevent motoneuron death following sciatic nerve section in the neonatal mouse

We have examined the ability of different neurotrophic and growth factors to prevent axotomy-induced motoneuron cell death in the developing mouse spinal cord. After postnatal unilateral section of the mouse sciatic nerve, most motoneuron (MN) loss occurs in the lateral motor column of the fourth lumbar segment (L4). Significant axotomy-induced cell death occurred after surgery performed on or before postnatal day (PN) 5. In contrast, no significant cell loss was found when axotomy was performed after PN10. Axotomy on PN2 or PN5 resulted in a 44% loss of L4 motoneurons by 7 days, and a 66% loss of motoneurons by 10 days postsurgery. Implantation of gelfoam presoaked in various neurotrophic factors at the lesion site rescued axotomized motoneurons. Nerve growth factor (NGF), nedurotrophin-4/5 (NT-4/5) and ciliary neurotrophic factor (CNTF) rescued 20%–30% of motoneurons, whereas brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and insulin-like growth factor 1 (IGF-1) rescued virtually all motoneurons from axotomy-induced death. By contrast, platelet-derived growth factor (PDGF)-AA, PDGF-AB, basic fibroblast growth factor (bFGF), and interleukin (IL-6) were ineffective on motoneuron survival following axotomy. NGF, BDNF, NT-3, IGF-1, and CNTF also prevented axotomy-induced atrophy of surviving motoneurons. These data show that mouse lumbar motoneurons continue to be vulnerable to axotomy up to about 1 week after birth and that a number of trophic agents, including the neurotrophins, CNTF, and IGF-1, can prevent the death of these neurons following axotomy. Our studies confirm and extend previous reports on the time course of axotomy-induced mouse motoneuron death and the survival promoting effects of neurotrophic factors. 1994 John Wiley & Sons, Inc.     

Amphibian sympathetic ganglia as a model system for investigating regeneration in the vertebrate peripheral nervous system

1. The paravertebral sympathetic ganglion of the bullfrog serves as an excellent experimental system in which to study the response of vertebrate neurones to axotomy and the mechanisms associated with regeneration. 2. Various types of lesions to the axons (axotomy) of these neurones promote distinct and reproducible changes in the electrophysiological properties of the cell bodies which are not a consequence of changes in cell body morphology. 3. The axotomy-induced increase in spike width and decrease in the amplitude of the action potential after-hyperpolarization may allow an increase in Ca2+ influx and thereby promote regrowth. 4. The axotomy-induced decrease in after-hyperpolarization duration may reflect the disconnection of the neurone with its target and the loss of available nerve growth factor (NGF) from the target. 5. Experiments with NGF antibodies provide evidence that an NGF-like substances serves to maintain the normal electrophysiological characteristics of amphibian sympathetic neurones.     

Changes in synthesis of specific proteins following axotomy: Detection with two-dimensional gel electrophoresis

Changes in protein synthesis during development and following axotomy were analyzed by two-dimensional gel electrophoresis. The two major postganglionic nerves emerging from the superior cervical sympathetic ganglia (SCSG) of adult rats were either cut or crushed unilaterally. At intervals ranging from 1 to 112 days after surgery both SCSG were removed and incubated for 1 hr in the presence of ¹⁴C-leucine. Proteins were extracted and subjected to two-dimensional electrophoretic separation and autoradiography. With this technique, proteins are separated on the basis of isoelectric point and molecular weight. Also, intact SCSG from 1, 2, 7, and 14 day old rats were labeled and analyzed. It was found that a minority of the separated proteins exhibited some detectable change in relative rate of synthesis following axotomy. Actin exhibited a slight (< 20%) increase in relative synthesis rate while tubulin did not change significantly. There were small but significant differences in the protein patterns following nerve crush, as opposed to nerve cut. Comparison of protein synthesis patterns from developing rat SCSG with those from intact and from axotomized adult SCSG failed to demonstrate any marked similarity between the developmental and the axotomy patterns.     

In vivo application of mitochondrial pore inhibitors blocks the induction of apoptosis in axotomized neonatal facial motoneurons

Axotomy induces apoptosis in motoneurons of neonatal rodents. To identify the key players in motoneuron apoptosis, we assessed the progression of apoptosis at 4 h intervals following facial motoneuron axotomy. The mitochondrial release of cytochrome c, caspase-3 activation and nuclear condensation were first observed in the motoneuron cell bodies 16 h postaxotomy. In vivo application of inhibitors of the mitochondrial permeability transition pore, Bongkrekic acid and cyclosporin A prevented cytochrome c release as well as caspase-3 activation and attenuated motoneuron apoptosis. Similarly, in vivo application of RU360, an inhibitor of the mitochondrial calcium uniporter, also protected axotomized motoneurons from apoptosis. Taken together, our results show that cytochrome c release and subsequent caspase-3 activation are critical events that precipitate the apoptotic death of axotomized neonatal motoneurons in vivo. In addition, these results provide evidence that application of mitochondrial pore inhibitors in vivo can block the induction of apoptosis following motoneuron axotomy.