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PubMed2006

Cerebellar granule cell: ascending axon and parallel fiber.

Morphometric data have indicated that most (> 95%) of the granule cell axonal synapses are located along the parallel fibers. The ascending axons of granule cells, however, exert powerful excitatory effects on the activities of Purkinje cells. To resolve this apparent conflict, we propose that some of the parallel fiber synapses overlying a functionally homogeneous granule cell patch can discharge in close synchrony with the ascending axon synapses from that same granule cell patch, thereby augmenting the excitatory action from ascending axons. Moreover, we also propose that age may be an important factor in the ascending axon synapses. We examined the synaptic varicosities along the ascending axons and parallel fibers in the anterior lobe of the male NIA C57BL/6j mouse. Between 2.5 and 10 months, the fraction of synaptic varicosities anatomically associated with the ascending axons increased from 2.6% to 6.6% whereas the fraction of synaptic varicosities that can discharge in synchrony with ascending axon synapses (including some parallel fiber synapses) increased from 17.3% to 39% (assuming a granule cell patch of 300 x 300 microm). The results of the present study may be relevant to understanding the role of the ascending axon vs. the parallel fibers. Differential age-related synaptic changes in these two portions of the granule cell axons may constitute an important age-dependent synaptic remodeling in the granule cell-Purkinje cell synaptic system.

Aging↗
PubMed2006

Axonal retraction and regeneration induced by N,N-diethyldithiocarbamate (DEDTC) in the central nervous system.

Dithiocarbamates (DTCs), such as disulfiram, have been used in aversion therapy for alcoholism even though an inherent toxicity is induced, which is related mainly to peripheral neuropathy and is associated with behavioural and neurological complications. At anatomical and histopathological levels, DTCs affect structural elements in nervous tissue, such as axonal degeneration and alterations in the cytoskeletal proteins of astrocytes. Therefore, given the axonal effects of DTCs and to gain further insight into axonal growth and axonal pathfinding in the central nervous system (CNS), here we established an in vivo experimental model of mouse development. Daily intraperitoneal injections of N,N-diethyldithiocarbamate (DEDTC), the first metabolite of disulfiram, were given from postnatal day 2 (P2) until P15. From P16 until P30, animals were not treated. Treatment induced considerable physiological alterations, such as growth delay, throughout postnatal development. Moreover, by immunohistochemistry techniques, we observed important alterations in the cytoskeletal glial protein at early stages of postnatal development. At later stages (P15), the immunoreactivity pattern detected by an antibody against axonal neurofilaments (anti-NF-H) showed alteration in the axonal distribution pattern followed by drastic axonal loss at P22, data that were corroborated using an anti-MBP (myelin basic protein) antibody. Using an antibody against the beta amyloid precursor protein (APP), we detected axonal injury. Furthermore, given that we observed axonal re-growth in adulthood in the in vivo model presented, we propose that this model would be a good system in which to identify new strategies for inducing regenerative growth in neural diseases in which axonal regeneration is blocked.

Aging↗
PubMed2006

Evidence for direct axonal toxicity in vincristine neuropathy.

There is a long-standing debate concerning the localization of the primary insult that results in distal axonal degeneration, or 'dying back' neuropathy. To address this question, we created an in vitro model of vincristine neuropathy in rat dorsal root ganglia (DRG). DRGs were grown in compartmentalized chambers, allowing for isolated exposure of the cell body or the axon to vincristine. Initial dose-finding studies identified a dose of vincristine that showed differential effects on cell death when delivered to either the cell body or the axonal compartment. At this dose of 0.05 microM, exposure of the cell bodies had no effect on the growth of axons, whereas addition of vincristine to the axonal compartment caused axonal shortening without affecting the growth of unexposed 'sister' axons. Toxicity was seen only with exposure of the growing axonal tips. These data support localized axonal toxicity as a cause of distal axonal degeneration due to vincristine.

Animals↗
PubMed1978

Propagating calcium spikes in an axon of Aplysia.

1. Intracellular recordings were obtained from the axon of the giant neurone R2 of Aplysia in order to study the ionic dependency of action potentials. 2. The overshoot potential of the axon spike increases with Na concentration in the manner predicted for a Na electrode. The maximum rate of rise (Vm) is linearly related to Na concentration. The overshoot potential is insensitive to Ca concentration at Na concentrations as low as 250 mM. 3. Tetrodotoxin (TTX) or replacement of Na with Tris abolishes action potentials in the axon but not soma of R2. Addition of 4-aminopyridine to a Na-free solution permits axon spikes to be generated. These action potentials are blocked by 30 mM-Co2+, but not by TTX. The overshoot potential and Vm of these action potentials increase monotonically with Ca concentration. 4. Axonal action potentials can be generated when an equimolar concentration of Sr is substituted for all of the Ca and Mg in Na-free medium. These action potentials are abolished by 30 mM-Ca2+ or mM-Co2+, and increase with Sr concentration. 5. TTX-resistant Ba spikes can similarly be elicited in R2 axon. These action potentials are reduced by Ca, Co, or Cd, and enhanced by raising the Ba concentration. 6. The Vm of Na spikes in the absence of Ca is greater in the axon than in the soma of R2, whereas the Vm of divalent spikes is greater in the soma. 7. During repetitive stimulation the axon spikes incrase in duration. This broadening is inhibited by replacing Ca in the bath with Mn or by the addition of 30 mM-CoCl2, and is enhanced by raising the Ca concentration by 30 mM. 8. The action potention of R2 axon has a mixed Na/Ca dependency. The density of Ca current may be greater in the some than in the axon of this cell.

Action Potentials↗
PubMed1982

Somatic motor axons can innervate autonomic neurones in the frog heart.

1. The effects of sympathetic and parasympathetic stimulation on the heart rate in frogs were tested after hearts were reinnervated with a somatic motor nerve. When frogs were vagotomized and hypoglossal axons were redirected to the heart for 8 or more weeks, stimulating the redirected hypoglossus nerve produced a parasympathetic-like inhibition of the heart. Stimulating sympathetic rami of the anastomosed hypoglossus nerve produced cardiac acceleration.2. Individual parasympathetic neurones received synaptic input from hypoglossal terminals. The excitatory post-synaptic potentials evoked by hypoglossal stimulation were much smaller than those evoked by vagal stimulation in control or vagal-reinnervated ganglia. However, hypoglossal axons innervated most (71%) of the ganglion cells and this level of innervation persisted for at least 60 weeks.3. Hypoglossal axons formed networks of varicose terminals within cardiac ganglia and established axo-axonic synapses with parasympathetic neurones. Hypoglossal terminals did not reinnervate the neuronal perikarya, in contrast to vagal axons in control or vagal-reinnervated ganglia.4. Axo-axonic synapses from redirected hypoglossal axons were identified in cardiac ganglia by bathing isolated hearts in horseradish peroxidase (HRP) and stimulating the redirected nerve. Electron micrographs showed that axo-axonic synapses contained HRP-labelled presynaptic vesicles.5. The source of foreign innervation in experimental cardiac ganglia was confirmed to be hypoglossal motoneurones (a), by comparing the conduction velocity of the redirected presynaptic axons (1.32 m/sec) with regenerating vagal preganglionic fibres (< 0.3 m/sec), and (b), by retrograde HRP-labelling of large motoneurones in the hypoglossal nucleus after applying peroxidase to the axons which had grown into the heart.

Animals↗
PubMed2006

Herpes simplex virus capsids are transported in neuronal axons without an envelope containing the viral glycoproteins.

Electron micrographic studies of neuronal axons have produced contradictory conclusions on how alphaherpesviruses are transported from neuron cell bodies to axon termini. Some reports have described unenveloped capsids transported on axonal microtubules with separate transport of viral glycoproteins within membrane vesicles. Others have observed enveloped virions in proximal and distal axons. We characterized transport of herpes simplex virus (HSV) in human and rat neurons by staining permeabilized neurons with capsid- and glycoprotein-specific antibodies. Deconvolution microscopy was used to view 200-nm sections of axons. HSV glycoproteins were very rarely associated with capsids (3 to 5%) and vice versa. Instances of glycoprotein/capsid overlap frequently involved nonconcentric puncta and regions of axons with dense viral protein concentrations. Similarly, HSV capsids expressing a VP26-green fluorescent protein fusion protein (VP26/GFP) did not stain with antiglycoprotein antibodies. Live-cell imaging experiments with VP26/GFP-labeled capsids demonstrated that capsids moved in a saltatory fashion, and very few stalled for more than 1 to 2 min. To determine if capsids could be transported down axons without glycoproteins, neurons were treated with brefeldin A (BFA). However, BFA blocked both capsid and glycoprotein transport. Glycoproteins were transported into and down axons normally when neurons were infected with an HSV mutant that produces immature capsids that are retained in the nucleus. We concluded that HSV capsids are transported in axons without an envelope containing viral glycoproteins, with glycoproteins transported separately and assembling with capsids at axon termini.

Animals↗
PubMed1999

Anterograde transport of herpes simplex virus proteins in axons of peripheral human fetal neurons: an immunoelectron microscopy study.

Herpes simplex virus (HSV) reactivates from latency in the neurons of dorsal root ganglia (DRG) and is subsequently transported anterogradely along the axon to be shed at the skin or mucosa. Although we have previously shown that only unenveloped nucleocapsids are present in axons during anterograde transport, the mode of transport of tegument proteins and glycoproteins is not known. We used a two-chamber culture model with human fetal DRG cultivated in an inner chamber, allowing axons to grow out and penetrate an agarose barrier and interact with autologous epidermal cells in the outer chamber. After HSV infection of the DRG, anterograde transport of viral components could be examined in the axons in the outer chamber at different time points by electron and immunoelectron microscopy (IEM). In the axons, unenveloped nucleocapsids or focal collections of gold immunolabel for nucleocapsid (VP5) and/or tegument (VP16) were detected. VP5 and VP16 usually colocalized in both scanning and transmission IEM. In contrast, immunolabel for glycoproteins gB, gC, and gD was diffusely distributed in axons and was rarely associated with VP5 or VP16. In longitudinal sections of axons, immunolabel for glycoprotein was arrayed along the membranes of axonal vesicles. These findings provide evidence that in DRG axons, virus nucleocapsids coated with tegument proteins are transported separately from glycoproteins and suggest that final assembly of enveloped virus occurs at the axon terminus.

Axons↗
PubMed2006

Axon loss is an important determinant of weakness in multifocal motor neuropathy.

BACKGROUND: Multifocal motor neuropathy (MMN) is characterised by asymmetrical weakness and muscle atrophy, in the arms more than the legs, without sensory loss. Despite a beneficial response to treatment with intravenous immunoglobulins (IVIg), weakness is slowly progressive. Histopathological studies in MMN revealed features of demyelination and axon loss. It is unknown to what extent demyelination and axon loss contribute to weakness. Unlike demyelination, axon loss has not been studied systematically in MMN. Aims/ METHODS: To assess the independent determinants of weakness in MMN, 20 patients with MMN on IVIg treatment were investigated. Using a standardised examination in each patient, muscle strength was determined in 10 muscles. In the innervating nerve of each muscle, axon loss was assessed by concentric needle electromyography, and conduction block or demyelinative slowing by motor nerve conduction studies. Multivariate analysis was used to assess independent determinants of weakness. RESULTS: Needle electromyography abnormalities compatible with axon loss were found in 61% of all muscles. Axon loss, and not conduction block or demyelinative slowing, was the most significant independent determinant of weakness in corresponding muscles. Furthermore, axon loss and conduction block were independently associated with each other. CONCLUSION: Axon loss occurs frequently in MMN and pathogenic mechanisms leading to axonal degeneration may play an important role in the outcome of the neurological deficit in patients with MMN. Therapeutic strategies aimed at prevention and reduction of axon loss, such as early initiation of treatment or additional (neuroprotective) agents, should be considered in the treatment of patients with MMN.

Adult↗
PubMed2001

Differences in accommodative properties of median and peroneal motor axons.

OBJECTIVES: To investigate whether accommodation to depolarising and hyperpolarising currents differs for motor axons of human upper and lower limb nerves. METHODS: The threshold tracking technique was used to measure threshold electrotonus for median and peroneal motor axons. The threshold current that produced a compound muscle action potential 50% of maximum was measured, and membrane potential was altered using subthreshold polarising currents of 330 ms duration but of variable intensity, from +40% (depolarising) to -100% (hyperpolarising) of the unconditioned threshold. RESULTS: The maximal threshold changes (the peak of the S1 phase of threshold electrotonus) were significantly greater in median axons for both depolarising and hyperpolarising currents. The subsequent phases of accommodation to depolarising currents (S2) and to hyperpolarising currents (S3) were also significantly greater in median axons. These findings raised the possibility that greater accommodation (S2 and S3) in median axons resulted from greater changes in membrane potential. However, regression of S2 against S1 to depolarising currents disclosed significantly greater accommodation (27.8%) for median axons, suggesting that slow K(+) conductances may be more prominent on median than peroneal axons. By contrast, the relation between S3 and S1 to hyperpolarising currents was similar for the two nerves, suggesting that the difference in inward rectification was merely because the conductance varies with the extent of hyperpolarisation. CONCLUSIONS: Slow K(+) conductances are more prominent for median motor axons than for peroneal axons. It would therefore be expected that axons innervating the lower limbs have less protection from depolarising stress and could develop ectopic activity more readily.

Action Potentials↗
PubMed1987

Properties of single Na+ channels in cut-open Myxicola giant axons.

Time- and voltage-dependent behavior of the Na+ conductance in dialyzed intact Myxicola axons was compared with cut-open axons subjected to loose-patch clamp of the interior and to axons where Gigaseals were formed after brief enzyme digestion. Voltage and time dependence of activation, inactivation, and reactivation were identical in whole-axons and loose-patch preparations. Single channels observed in patch-clamp axons had a conductance of 18.3 +/- 2.3 pS and a mean open time of 0.84 +/- 0.12 ms. The time-dependence of Na+ currents found by averaging patch-clamp records was similar to intact axons, as was the voltage dependence of activation. Steady-state inactivation in patch-clamped axons was shifted by an average of 15 mV from that seen in loose-patch or intact axons. Substitution of D2O for H2O decreased single channel conductance by 24 +/- 6% in patch-clamped axons compared with 28 +/- 4% in intact axons, slowed inactivation by 58 +/- 8% compared with 49 +/- 6%, and increased mean open time by 52 +/- 7%. The results confirm observations on macroscopic channel behavior in Myxicola and resemble that seen in other excitable tissues.

Animals↗
PubMed1995

The role of the floor plate in axon guidance.

The data reviewed above have implicated the floor plate in directing axonal growth towards the midline, in directing the behavior of axons at the midline, and finally, in directing the longitudinal growth of axons alongside the midline. In the case of growth to the midline, there is clear evidence for the existence of two types of cues that collaborate to direct growth--short-range cues that can direct axons along the edge of the spinal cord and a long-range chemoattractant secreted by the floor plate cells whose main function may be to direct later-extending commissural axons that must migrate through the complex environment of the developing motor column. Determining the precise contribution of these cues will require identifying them and perturbing them in vivo. The cues that direct growth along the edge are unknown; their identification in the spinal cord would be of quite general significance, since the growth of axons parallel to (but not in contact with) the pial surface is a widespread feature of early axon growth at all axial levels of the neural tube. A strong candidate for the long-range chemoattractant is netrin-1, a homologue of the UNC-6 protein of C. elegans and a distant relative of laminin, which is expressed by floor plate cells and which can both promote and orient commissural axon outgrowth. Netrin-1 may also influence growth of other populations of neurons that exhibit stereotyped behaviors near the ventral midline. Much less is known about the exact role of the floor plate in directing axon growth at the midline, though it is clearly required for accurate guidance. In the absence of the floor plate, a range of errors has been found, the most prominent of which are aberrant midline crossings and errors in longitudinal growth near the ventral midline. The severity of these errors varies with species, which could result from either the variable importance of the floor plate in the different species or the fact that, so far, quite different manipulations of the ventral midline region have been performed in different species. The most specific perturbation of the ventral midline occurs in the zebrafish cyc-1 mutant, where the selective loss of the floor plate leads to stereotyped misrouting events. Perhaps surprisingly, virtually all axons that grow to the midline turn longitudinally (although sometimes in the wrong direction).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2005

Vascular endothelial-derived semaphorin 3 inhibits sympathetic axon growth.

Vascular sympathetic innervation is an important determinant of blood pressure and blood flow. The mechanisms that determine vascular sympathetic innervation are not well understood. Recent studies indicate that vascular endothelial cells (EC) express semaphorin 3A, a repulsive axon guidance cue. This suggests that EC would inhibit the growth of axons to blood vessels. The present study tests this hypothesis. RT-PCR and Western analyses confirmed that rat aortic vascular ECs expressed semaphorin 3A as well as other class 3 semaphorins (sema 3s). To determine the effects of EC-derived sema 3 on sympathetic axons, axon outgrowth was assessed in cultures of neonatal sympathetic ganglia grown for 72 h in the absence and presence of vascular EC. Nerve growth factor-induced axon growth in the presence of ECs was 50 +/- 4% (P < 0.05) of growth in the absence of ECs. ECs did not inhibit axon growth in the presence of an antibody that neutralized the activity of sema 3 (P > 0.05). RT-PCR and Western analyses also indicated that sema 3s were expressed in ECs of intact arteries. To assess the function of sema 3s in arteries, sympathetic ganglia were grown in the presence of arteries for 72 h, and the percentage of axons that grew toward the artery was determined: 44 +/- 4% of axons grew toward neonatal carotid arteries. Neutralization of sema 3s or removal of EC increased the percentage of axons that grew toward the artery (71 +/- 8% and 72 +/- 8%, respectively). These data indicate that vascular EC-derived sema 3s inhibit sympathetic axon growth and may thus be a determinant of vascular sympathetic innervation.

Animals↗
PubMed1994

Spontaneous firing patterns and axonal projections of single corticostriatal neurons in the rat medial agranular cortex.

1. Spontaneous fluctuations of membrane potential, patterns of spontaneous firing, dendritic branching patterns, and intracortical and striatal axonal arborizations were compared for two types of corticostriatal neurons in the medial agranular cortex of urethan-anesthetized rats: 1) pyramidal tract (PT) cells identified by antidromic activation from the medullary pyramid and 2) crossed corticostriatal (CST) neurons identified by antidromic activation from the contralateral neostriatum. The ipsilateral corticostriatal projections of intracellularly stained PT neurons as well as contralateral corticostriatal neurons were confirmed after labeling by intracellular injection of biocytin. 2. All well-stained PT neurons had intracortical and intrastriatal collaterals. The more common type (6 of 8) was a large, deep layer V neuron that had an extensive intracortical axon arborization but a limited axon arborization in the neostriatum. The less common type of PT neuron (2 of 8) was a medium-sized, superficial layer V neuron that had a limited intracortical axon arborization but a larger and more dense intrastriatal axonal arborization. Both subclasses of PT neurons had anatomic and physiological properties associated with slow PT cells in cats and monkeys and conduction velocities < 10 m/s. All of the PT cells but one were regular spiking cells. The exception cell fired intrinsic bursts. 3. Intracellularly stained CST neurons were located in the superficial half of layer V and the deep part of layer III. Their layer I apical dendrites were few and sparsely branched. Their axons gave rise to an extensive arbor of local axon collaterals that distributed in the region of the parent neuron, frequently extending throughout the more superficial layers, including layer I. Axon collaterals were also traced to the corpus callosum, as expected from their contralateral projections, and they contributed axon collaterals to the ipsilateral neostriatum. In the neostriatum, these axons formed extended arborizations sparsely occupying a large volume of striatal tissue. All CST neurons were regular spiking cells. 4. Both types of cells displayed spontaneous membrane fluctuations consisting of a polarized state (-60 to -90 mV) that was interrupted by 0.1- to 3.0-s periods of depolarization (-55 to -45 mV) accompanied by action potentials. The membrane potential was relatively constant in each state, and transitions between the depolarized and hyperpolarized states were sometimes periodic with a frequency of 0.3-1.5 Hz. A much faster (30-45 Hz) subthreshold oscillation of the membrane potential was observed only in the depolarized state and triggered action potentials that locked to the depolarizing peaks of this rhythm.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1994

Spinothalamic and spinohypothalamic tract neurons in the cervical enlargement of rats. III. Locations of antidromically identified axons in the cervical cord white matter.

1. Fifty-five neurons in the cervical enlargement (C6-C8) of urethan-anesthetized rats were antidromically activated from the contralateral posterior diencephalon. In all cases, antidromic thresholds were < or = 30 microA. The locations of the axons of these neurons within the white matter of segments C2-C6 were determined by tracking systematically using a second antidromic stimulating electrode. 2. The recording locations of 51 neurons were marked and recovered. Twenty neurons were recorded in the superficial dorsal horn (SDH) and 31 were in the deep dorsal horn (DDH). Eighty-three lowest threshold points for antidromic activation within the white matter of segments C2-C6 were determined for these 51 neurons. The mean antidromic threshold at these points was 9.5 +/- 0.5 (SE) microA. For 26 neurons, the lowest threshold point for antidromic activation was determined at one segmental level. We also attempted to determine whether individual axons maintained their position as they ascended through the cervical cord white matter. In 25 cases, lowest threshold points were determined at two or more segmental levels. 3. In segments C5-C6, 88% (7/8) of the lowest threshold points of the examined axons were located in the contralateral ventral funiculus, indicating that the majority of examined axons crossed the midline within one or two segments. 4. In segments C3-C4, 32% (14/44) of all examined axons were found in the dorsal lateral funiculus (DLF) and 66% (29/44) were within the ventral quadrant [ventral lateral funiculus (VLF) and ventral funiculus (VF)]. Sixty-nine percent (11/16) of the axons of neurons recorded in the SDH were located in the contralateral DLF and 31% (5/16) were located in the ventral quadrant (VQ). In contrast, only 11% (3/28) of the axons of neurons recorded in the DDH were located in the contralateral DLF and 86% (24/28) were located in the VQ. Therefore, in segments C3-C4, the locations of axons differed significantly. Those from neurons recorded in the SDH were located primarily in the DLF and those from neurons recorded in the DDH were located principally in the VQ. 5. In segment C2, 74% (23/31) of all examined axons were found in the DLF, 23% (7/31) were in the VQ, and 3% (1/31) were in the dorsal horn. Thus, the percentage of all examined axons in the DLF in C2 was approximately 2.5 times greater than it was in C3-C4.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1996

Evidence for GAP-43 within descending spinal axons in the North American opossum, Didelphis virginiana.

We have shown previously that GAP-43, a growth associated protein characteristically present in growing and regenerating axons, is relatively abundant in the spinal cord of adult opossums. In the present study, we combined the orthograde transport of the fluorescent marker Fluoro-Ruby with immunofluorescence for GAP-43 to determine if any of it is present within descending spinal axons. When Fluoro-Ruby was injected into the red nucleus and midbrain tegmentum, the medial pontine or medullary reticular formation, the medullary raphe or the lateral vestibular nucleus, axons were labeled in the expected areas of the spinal cord, but in most cases none showed evidence for GAP-43. In two of the four cases with rubral injections, however, a few labeled axons within the rubrospinal tract showed GAP-43 immunofluorescence, and in one case with an injection of the gigantocellular reticular nucleus and adjacent raphe, labeled axons within lamina IX immunostained for the protein. Since serotoninergic neurons are present within the gigantocellular reticular nucleus and adjacent raphe, and axons of the same phenotype are abundant within lamina IX, we asked whether serotoninergic axons contain GAP-43. When sections of the spinal cord were immunostained for both serotonin and GAP-43, many axons within lamina IX showed evidence for both substances. Such axons appeared to contact presumptive motoneurons. In cases with Fluoro-Ruby injections of the forelimb motor cortex, labeled axons were present within the pyramidal tract, and some of them showed evidence for GAP-43.

Animals↗
PubMed2003

Varicosities of intraretinal ganglion cell axons in human and nonhuman primates.

PURPOSE: To describe varicosities of intraretinal ganglion cell axons in the nerve fiber layer of human and nonhuman primate retinas. METHODS: Intraretinal ganglion cell axons of seven human donors (1-85 years old) and two nonhuman primates (Macaca mulatta, 15 and 17 years old) were immunohistochemically stained with an antibody of neurofilament on flatmounted retinas and examined with light microscopy. In addition, the axons within the retinal nerve fiber layer were examined with transmission electron microscopy in one human and one nonhuman retina. The variations of diameters of single axons were measured on transverse- and parallel-cut sections, and the frequency distributions of the diameters were statistically evaluated. RESULTS: Varicosities of the intraretinal ganglion cell axons were found throughout the retinas in both nonhuman primate and human eyes of all ages examined. The varicosities were rich in mitochondria and had desmosome- and hemidesmosome-like junctions with other axons and retinal glial cells. Measured on parallel-cut axons, the mean diameter (+/-SD) of varicosities was 2.7 +/- 0.9 micro m, whereas the mean diameter of intervaricosity regions was 0.7 +/- 0.3 micro m. The diameter distribution for transverse-cut axons was also bimodal, but the two peaks were much closer because the peak of the larger-diameter group decreased. CONCLUSIONS: The results demonstrated that intraretinal ganglion cell axons are predominantly varicose fibers in both human and nonhuman primates. Size variations exist within a single axon's diameter and thereby affect the patterns of diameter distribution seen in transverse-cut preparations. The mitochondria-rich varicosities and the presence of intercellular junctions suggest that the varicosities may be functional sites that serve local high-energy demands of unmyelinated fibers and signal transmission.

Adolescent↗
PubMedDate not supplied

Grafts of genetically modified Schwann cells to the spinal cord: survival, axon growth, and myelination.

Schwann cells naturally support axonal regeneration after injury in the peripheral nervous system, and have also shown a significant, albeit limited, ability to support axonal growth and remyelination after grafting to the central nervous system (CNS). It is possible that Schwann cell-induced axonal growth in the CNS could be substantially increased by genetic manipulation to secrete augmented amounts of neurotrophic factors. To test this hypothesis, cultured primary adult rat Schwann cells were genetically modified using retroviral vectors to produce and secrete high levels of human nerve growth factor (NGF). These cells were then grafted to the midthoracic spinal cords of adult rats. Findings were compared to animals that received grafts of nontransduced Schwann cells. Spinal cord lesions were not placed prior to grafting because the primary aim of this study was to examine features of grafted Schwann cell survival, growth, and effects on host axons. In vitro prior to grafting, Schwann cells secreted 1.5+/-0.1 ng human NGF/ml/10(6) cells/day. Schwann cell transplants readily survived for 2 wk to 1 yr after in vivo placement. Some NGF-transduced grafts slowly increased in size over time compared to nontransduced grafts; the latter remained stable in size. NGF-transduced transplants were densely penetrated by primary sensory nociceptive axons originating from the dorsolateral fasciculus of the spinal cord, whereas control grafts showed significantly fewer penetrating sensory axons. Over time, Schwann cell grafts also became penetrated by TH- and DBH-labeled axons of putative coerulospinal origin, unlike control cell grafts. Ultrastructurally, axons in both graft types were extensively myelinated by Schwann cells. Grafted animals showed no changes in gross locomotor function. In vivo expression of the human NGF transgene was demonstrated for periods of at least 6 m. These findings demonstrate that primary adult Schwann cells 1) can be transduced to secrete augmented levels of neurotrophic factors, 2) survive grafting to the CNS for prolonged time periods, 3) elicit robust growth of host neurotrophin-responsive axons, 4) myelinate CNS axons, and 5) express the transgene for prolonged time periods in vivo. Some grafts slowly enlarge over time, a feature that may be attributable to the propensity of Schwann cells to immortalize after multiple passages. Transduced Schwann cells merit further study as tools for promoting CNS regeneration.

Animals↗
PubMed2002

Astrocytes mediate cerebral cortical neuronal axon and dendrite growth, in part, by release of fibroblast growth factor.

Astrocytes occupy a central role in central nervous system (CNS) function. In particular astyrocytes can support neurite growth, in part, by release of diffusable factors. We therefore performed biochemical analysis of astrocyte conditioned medium to examine possible mechanisms of astrocyte mediated axon and dendrite growth in the mammalian CNS. Culture medium was conditioned on purified astrocyte monolayers derived from P3 rat cerebral cortex or on fibroblasts. Conditioned medium (CM) was subject to protein denaturation, molecular weight fractionation, and heparin affinity chromatography. E18 mouse cerebral cortical neurons were then cultured in the various media or directly on astrocyte monolayers and axon and dendrite growth from 50 neurons in each condition quantified after 3 DIV using double-labeled immunohistochemical techniques. Axon and dendrite growth was supported by astrocyte CM and both were significantly greater than process growth from neurons incubated in fibroblast CM. Protein denaturation significantly reduced astrocyte CM support of axon and dendrite growth. Following ultrafiltration and dialysis dendrite and axon growth was observed in the molecular weight fraction between 10 and 100 kDa. Axon growth also was observed in the CM molecular weight fraction greater than 100 kDa. Conditioned medium was eluted on a heparin column; when the bound fragment was reconstituted in chemically defined medium extensive dendrite and axon growth was observed. Since fibroblast growth factor (FGF) has these biochemical characteristics we added anti-bFGF neutralizing antibodies to astrocyte monolayers or CM; this significantly reduced astrocyte support of process growth. By contrast, the addition of heparin, which helps activate FGF receptors, to astrocyte CM further enhanced process growth. Western blot analysis confirmed that bFGF was present in astrocyte CM. We then examined axon and dendrite growth from cortical neurons after the addition of various growth factors to chemically defined medium. Axon and dendrite growth, similar to that found in astrocyte CM was observed after the addition of bFGF or aFGF. Astrocyte support of cerebral cortical neuron axon and dendrite growth in vitro may be explained, in part, by FGF release.

Animals↗