Ultrastructural Characterization of the Lower Motor System in a Mouse Model of Krabbe Disease.

Manipulating transmitter release at the neuromuscular junction of neonatal rats alters the expression of ChAT and GAP-43 in motoneurons

Considerable knowledge concerning developmental cell death has come from the study of somatic motor neurons (SMNs), but a related set of spinal neurons, the autonomic motor neurons (AMNs), have been studied less extensively in this respect. In the present study, we used three different approaches to determine the amount of AMN cell death during normal development in the rat. First, target dependency was studied in organotypic slice cultures, and it was found that AMNs survived for at least 12 days after removal of their postsynaptic targets. No factors were added to the serum-free medium to substitute for the ablated targets, indicating that AMNs were able to survive without target-derived trophic factors. Such target-independent survival is not characteristic of neurons that undergo typical developmental cell death. Second, AMNs were counted in double-stained choline acetyltransferase immunocytochemical and NADPH diaphorase histochemical preparations at ages (postnatal days 4-22) encompassing the period when AMN postsynaptic target cells undergo developmental death. Neuron numbers were essentially identical at all ages examined, indicating that no AMN cell death occurred postnatally. Finally, from embryonic day 13 to postnatal day 22, animals were analyzed by using terminal transferase-mediated nick-end labeling to identify dying cells. Many fewer labeled cells were observed among AMNs than among SMNs. Thus, all three approaches indicated that there is a significant SMN/AMN difference in developmental cell death. The phenotypic trait(s) that underlies this difference may also be important in the relative resistance of AMNs to pathological conditions that induce death of SMNs, e.g., those involved in amyotrophic lateral sclerosis and excitotoxicity.

Neuroprotection by Gene Therapy Targeting Mutant SOD1 in Individual Pools of Motor Neurons Does not Translate Into Therapeutic Benefit in fALS Mice

Glial-Derived Prodegenerative Signaling in the Drosophila Neuromuscular System

Degeneration of motor nerve terminals occurs in amyotrophic lateral sclerosis (ALS) patients as well as in mouse models of ALS, leading to progressive paralysis. What causes a motor nerve terminal to degenerate remains unknown. Here we report on transgenic mice expressing a ubiquitinated synaptic vesicle protein (Ub(G76V)-GFP-Syb2) that develop progressive degeneration of motor nerve terminals. These mice may serve as a model for further elucidating the underlying cellular and molecular mechanisms of presynaptic nerve terminal degeneration.

Neuromuscular junctions in the extensor digitorum longus muscle of control and dystrophic (dy) mice at stages ranging from 30 to 120 postnatal days were examined by scanning and transmission electron microscopy (SEM and TEM). In control mice, the neuromuscular junctions consisted of thin ramifying nerve endings and of complex labyrinthine gutters with a great number of slit-like junctional folds. The subneural apparatuses of dy mice on the 30th and the 60th postnatal days were composed of undeveloped gutters or a number of cup-like depressions containing a few pit-like or slit-like junctional folds. In dystrophic neuromuscular junctions on the 120th postnatal day, subneural apparatuses were classified into two types: one had a cluster of continuous cup-like depressions with or without slit-like junctional folds, and the other consisted of discrete depressions containing slit-like junctional folds. Moreover, a small number of dystrophic muscle fibers were endowed with two subneural apparatuses which were 20 to 30 microns away from each other. Small round pits (0.5-1.0 microns in diameter) in some cup-like depressions were occasionally protruded by short processes of terminal axons. The present findings suggest that the age-dependent postsynaptic structural changes of dystrophic dy mice induce a complex morphological reorganization of presynaptic nerve endings. This might help the nerve endings compensate for the functional deterioration of dystrophic muscle fibers.

CNS-targeted Viral Delivery of G-CSF in an Animal Model for ALS: Improved Efficacy and Preservation of the Neuromuscular Unit

Objective To investigate whether tissue plasminogen activator(tPA),type Ⅰ plasminogen activator inhibitor(PAI-1)and neuroserpin(NSP)are involved in neurodegeneration following sciatic nerve transection in rats.Methods Fifty-six male Sprague Dawley rats were randomly divided into normal group and sciatic nerve transection group.Spinal cord from level L4 to L6 was removed at several time points after sciatic nerve transection for Nissl staining and transmission electron microscopy to evaluate neurodegenerative changes and neuronal death.The dynamic expression of tPA,NSP and PAI-1 were examined by immunohistochemical staining and semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR).Results Statistically significant neuron loss in spinal anterior horn of the injured side was found 7 days following sciatic nerve transection.The neuron survival rate at 21 days after the operation was 61.6％.Furthermore transmission electron microscopy showed a considerable number of apoptotic neurons and glia cells at various phases in the anterior horn from 7 days postoperatively.Furthermore,a few apoptotic cells could be detected in the posterior horn at 14 days after the operation as well.The results of immunohistochemistry showed that tPA was up-regulated in laminae Ⅴ to Ⅸ of the injury side by 1 day post-lesion(P ＜ 0.05)and peaked at 7 days.After that,tPA protein was down-regulated.But until the time point of 21 days,the level of tPA had not returned to the normal level(P ＞ 0.05).However,PAI-1 could be detected in neither normal nor experimental spinal cord.Semi RT-PCR revealed a change tendency of tPA mRNA that was similar to tPA protein but just a little earlier than the latter.Meanwhile,NSP mRNA was up-regulated by 1 day and stayed at a higher level for two weeks.It returned to normal level at 21 days.Conclusion The 50％ motor neuron loss in the ipsilateral spinal cord anterior horn after sciatic nerve transection was related to the upregulation of synthesis and release of tPA from neurons and microglia in the grey matter.Meanwhile the axotomy stimulated upregulation of tPA inhibitor NSP which may play a neuroprotective role. Key words: Sciatic nerve; Spinal cord; Neurons; Degeneration; Tissue plasminogen activator

Proteomics uncovers thus far unknown age and sex differences in sciatic nerve and skin of naïve mice, highlighting the importance of adequate age matching and parallel investigation of male and female mice in biomedical studies.

Effects of isepamicin sulfate (HAPA-B), a new aminoglycoside antibiotic, on the neuromuscular transmission were studied in rats and compared with those of amikacin (AMK) or other aminoglycoside antibiotics. The HAPA-B, as well as other aminoglycoside antibiotics, depressed the twitch response of diaphragm to phrenic nerve stimulation in vitro. The depression effects of different drugs were compared and graded in the order of strengths of blocking action as: netilmicin (NTL) greater than gentamicin (GM) greater than streptomycin (SM) greater than kanamycin (KM) greater than AMK greater than HAPA-B. The IC50 (concentration which inhibited the response by 50%) of HAPA-B was 3.6 X 10(-3) g/ml. The neuromuscular blockade produced by HAPA-B was reversed by CaCl2, KCl or caffeine but not by neostigmine. D-Tubocurarine or MgCl2 augmented the neuromuscular effects of HAPA-B. Intramuscular (400 mg/kg) and intravenous (100 mg/kg) injections of HAPA-B did not affect the twitch response of gastrocnemius muscle to sciatic nerve stimulation in situ. Intravenous injection of 200 mg/kg caused death in some rats and depression of the twitch response in others. Intravenous AMK produced no significant effect on the twitch response at 50 mg/kg and caused death at 100 mg/kg. GM and SM caused death or significant degree of depression of the twitch response at intravenous doses of 50 mg/kg. In experiments of intravenous drug infusion for 60 minutes, the twitch response was depressed by HAPA-B at 400 mg/kg/hr and by AMK at 200 and 400 mg/kg/hr. In conclusion, HAPA-B has a neuromuscular blocking action presumably at the nerve terminal. However, its action was the weakest among the aminoglycoside antibiotics tested.

We have used in situ hybridization with a digoxigenin-labelled probe for tenascin-C mRNA and immunocytochemistry with antibodies against tenascin-C, glial fibrillary acidic protein, OX-42 and the 200 kDa neurofilament protein to study the expression, distribution and cellular relationships of tenascin-C mRNA and protein in the developing (postnatal) and adult spinal cord of rat, and the effects thereon of dorsal root, ventral root and sciatic nerve injuries. The most interesting finding was that on postnatal day 7 (P7), P14 and in the adult, but not on P0 or P3, a group of neurons in the lumbar ventral horn expressed the tenascin-C mRNA gene. They represented about 5% of ventral horn neurons in the adult and were among the smaller such neurons. Since 40-60% of such cells were lost at P13 following sciatic nerve crush on P0, some were almost certainly motor neurons. In addition, we found that at P0 and P3, mRNA-containing glial cells were widespread in grey and white matter but sparse in the developing dorsal columns; tenascin-C immunofluorescence showed a similar distribution. By P7 there were fewer mRNA-containing cells in the ventral horns and in the area of the dorsal columns containing the developing corticospinal tract where immunofluorescence was also weak. At P14 there were no glial-like mRNA-containing cells in the grey matter; such cells were confined to the periphery of the lateral and ventral white columns but were present throughout the dorsal columns where tenascin-C immunofluorescence was also strong. No glial-like mRNA-containing cells were present in the adult lumbar spinal cord and tenascin-C immunofluorescence was confirmed to irregular patches in the ventral horn, especially around immunonegative cell bodies of small neurons, a zone around the central canal, and a thin zone adjacent to the glia limitans. Thus the expression of tenascin-C is differentially developmentally regulated in the grey matter and in different parts of the white matter. Three days after injury of dorsal roots L4-6, many cells containing tenascin-C mRNA, some identified as glial fibrillary acidic protein-positive astrocytes, were present in the ipsilateral dorsal column, but were rare after longer survivals. Immunoreactivity, however, was elevated in the ipsilateral dorsal column at 3 days, remained high for several months and disappeared at 6.5 months. Dorsal root injury had no effect on tenascin-C mRNA or protein in the grey matter. Sciatic nerve or ventral root injury had no effect on these molecules in any part of the spinal cord.

The glaucoma-associated gene, myocilin, is expressed in ocular and non-ocular tissues including the peripheral nervous system, but its functions in these tissues remain poorly understood. We demonstrate that in sciatic nerve, myocilin is expressed in Schwann cells with high concentrations at the nodes of Ranvier. There, myocilin interacts with gliomedin, neurofascin, and NrCAM, which are essential for node formation and function. Treatment of isolated dorsal root ganglion cultures with myocilin stimulates clustering of the nodal proteins neurofascin and sodium channel Nav1.2. Sciatic nerves of myocilin null mice express reduced levels of several myelin-associated and basal membrane proteins compared with those of wild-type littermates. They also demonstrate reduced myelin sheath thickness and partial disorganization of the nodes. Myocilin signaling through ErbB2/3 receptors may contribute to these observed effects. Myocilin binds to ErbB2/ErbB3, activates these receptors, and affects the downstream PI3K-AKT signaling pathway. These data implicate a role for myocilin in the development and/or maintenance of myelination and nodes of Ranvier in sciatic nerve.

Acute injury of skeletal muscle damages myofibers, microvessels and neuromuscular junctions (NMJs). Although myofiber regeneration is well‐characterized, little is known of concurrent events in microvessels and motor nerves during skeletal muscle regeneration. Because growth and patterning are coordinated between nerves and microvessels during development, we tested the hypothesis that effective microvascular regeneration is required for restoring neuromuscular structure and function post injury in the adult. To impair angiogenesis, mice were bred for endothelial cell (EC)‐specific deletion of ephrin‐B2. Male mice (3–6 months old; n=2–3/group) were injected with tamoxifen to delete ephrin‐B2 in ECs (EfnB2CKO; confirmed with PCR); uninjected mice of the same genotype were used as controls. The gluteus maximus (GM) or tibialis anterior (TA) muscle was injured by local injection of 1.2% BaCl2 and muscles were evaluated at 10 days post injury, by which time morphological and functional myofiber regeneration will have occurred. Intravascular injection of fluorescent wheat germ agglutinin (WGA; stains only perfused vessels) or immunolabeling for CD31 (stains all ECs) revealed microvessels of the GM in EfnB2CKO to be vacuolated with disorganized ECs vs. intact microvascular networks with aligned ECs in control mice. Using indirect stimulation via the sciatic nerve, isometric force production in the TA was 19±6 N/cm2 in EfnB2CKO vs. 33±3 N/cm2 in control mice; force produced with direct electrical field stimulation was similar between groups. Immunostaining for neurofilament‐heavy revealed reduced presynaptic coverage of NMJs in EfnB2CKO vs. control [60±9% vs. 74±10% of acetylcholine receptors (AChRs) per 200 μm2 NMJ area]; postsynaptic staining with α‐bungarotoxin revealed dispersed AChRs [200±118 vs. 92±43 fragments/200 μm2, respectively]. These preliminary findings indicate that the loss of ephrin‐B2 in ECs results in functional deficits for neuromuscular transmission and NMJ morphology in association with impaired microvascular regeneration following acute injury. We conclude that regeneration of intact NMJs with functional reinnervation of myofibers requires effective revascularization and recovery of the microcirculation following skeletal muscle injury.Support or Funding InformationSupport: APS Postdoctoral Fellowship (ABM), R01AR067450 (DDWC), R37HL041026 (SSS)

Synaptic vesicle pools at diaphragm neuromuscular junctions vary with motoneuron soma, not axon terminal, inactivity

The GTPase dMiro Is Required for Axonal Transport of Mitochondria to Drosophila Synapses