Neuromuscular transmission in cultures of adult human and rodent skeletal muscle after innervation in vitro by fetal rodent spinal cord

Electrophysiologic analyses have been carried out on in vitro-coupled explants of fetal rodent spinal cord and adult skeletal muscle of human as well as rodent origin. The studies demonstrate that characteristic neuromuscular transmission can develop and be maintained in these unusual tissue combinations during long-term culture. After coupling periods of 2–7 weeks in vitro, selective stimulation of spinal cord evokes widespread coordinated contractions in the muscle tissue. Simultaneous microelectrode recordings of cord and muscle responses to local cord, or ventral root, stimuli show that muscle action potentials (and contractions) generally occur with latencies of several msec after onset of cord discharges. Similar temporal relations are often seen during spontaneous rhythmic discharges of the coupled cord and muscle tissues. Long series of repetitive discharges, at 2–5 sec intervals, may occur synchronously between these cord and muscle explants, in response to single cord (or dorsal-root ganglion) stimuli, and they may also appear spontaneously. d-Tubocurarine (1–10 μg/ml) selectively and reversibly blocks neuromuscular transmission in these cultures. Eserine accelerates recovery of normal function. Spontaneous repetitive fibrillations of many of the cultured muscle fibers are observed sporadically, and these contractions often continue unabated after block of neuromusclar transmission by d-tubocurarine. Many of the fibers which show asynchronous fibrillations are probably not innervated (as in denervated muscle in situ). In some cases, however, extracellular as well as intracellular recordings indicate that similar fibrillations may also occur in fibers which are clearly innervated. Repetitive cord and muscle discharges are greatly augmented after introduction of strychnine. Complex rhythmic oscillatory (ca. 10/sec) afterdischarges generated in strychninized cord explants lead to similarly patterned muscle discharges (and contractions), which may also occur, at, times, in normal medium.     

Astrocyte death in organotypic cultures of fetal spinal cord from rats with unilateral micromelia

Growth and differentiation of neurons and glia in spinal cord explants of 16 days old rat fetuses with teratogen-induced left-sided micromelia were studied. Progressive destruction of astrocytes that differentiate in interstitial zone of cultures was observed in 37% of explants of the left side, while the development was normal in cultures of the right side. Possible mechanisms leading to destruction of astrocytes in cultures of spinal cord regions that innervate anomalous limbs are discussed.     

Effects of succinylcholine in an organotypic spinal cord-skeletal muscle coculture of embryonic mice

Intoxication with organophosphorus compounds is an important clinical problem worldwide. Although the core treatments – atropine, oximes and diazepam – are defined, high case fatalities were reported for intoxication with organophosphorus insecticides. In particular the role of oximes is not completely understood since they might benefit only patients poisoned by specific pesticides or patients with moderate poisoning and few randomised trials of such poisoning have been performed. This justifies the need for new in vitro test-systems like cocultures of spinal cord and muscle tissue, which have been recently introduced. However this test-system is not yet fully characterized. In order to estimate the applicability of cocultures of spinal cord and muscle tissue their sensitivity to succinylcholine (di-acetylcholine), a depolarizing muscle relaxant in clinical use, was tested.     

Development and differentiation of fetal rat sensory ganglia and spinal cord segments in vitro

Summary Fetal spinal ganglia and spinal cord segments with adhering spinal ganglia were explanted on collagen-coated coverslips. They were investigated with enzyme histochemical methods for the existence of hydrolases and dehydrogenases up to 54 days of cultivation.Alkaline phosphatase was located in arachnoid cells and in mantle cells (satellite cells). Butyrylcholinesterase and alpha-glycerophosphate-menadione-tetrazolium reductase were found in mantle cells. Acetylcholinesterase and indoxylesterase were active in the whole neuron; acid phosphatase and alpha-naphthylacetate esterase were restricted to the perikarya.During the process of cell differentiation in vitro alkaline phosphatase decreased in mantle cells. Acid phosphatase became diminished distinctly in spinal ganglion cells. The other neuronal enzymatic activities remained unaltered during the whole period of cultivation. Proliferated Schwann cells were conspicuous by their activity for butyrylcholinesterase. In newly formed myelin sheaths arylsulphatase was active. Lactate dehydrogenase was contained in the perineurium which had developed. Cultures of long duration could contain cytological formations which were reminiscent of sensory end-organs with respect to their enzyme patterns.The enzyme activities of nervous tissues in vitro in their approximation to the situation in situ are discussed.A preliminary report was presented at the 17th Meeting of the Association of German Neuropathologists and Neuroanatomists, in Freiburg, September 1972.The skilled technical assistance of Miss Johanna Sixel and Miss Charlotte Beyer is gratefully acknowledged.     

Action of steroid hormones on growth and differentiation of CNS and spinal cord organotypic cultures

Summary During the prenatal period, gonadal steroid environment induces dramatic sexually dimorphic changes in the nervous system.We have usedin vitro methods to study the mechanism and timing of hormonal influences on neuronal sprouting and myelination during the prenatal period.Organotypic cultures of hypothalamus and lumbar spinal cord (SC) slices from rat fetuses were grown on plasma clot or in hyaluronic acid and exposed to estogen (17 estradiol) and testosterone (T) during cultivation. Both steroid hormones were active: 17 estradiol enhanced sprouting of hypothalamic neuronal fibers and increased the amount of synapses. In SC cultures T induced regeneration of thick nerve processes and an early onset of myelination, mainly of peripheral myelin.     

Development of tolerance to opiates and opioid peptides in organotypic cultures of mouse spinal cord

Following chronic exposure of organotypic explants of mouse spinal cord with attached dorsal root ganglia (DRG) to low levels of morphine (1 μM) for 2–3 days (at 35°C), the initial opiate-depressant effects on sensory-evoked dorsal-horn network responses disappeared and characteristic dorsal cord responses could then be evoked by DRG stimuli in the presence of morphine — even after acute increases in concentration (up to 100-fold). Similar tolerance developed after chronic exposure of cord-DRG explants to low concentrations (10 nM) of an enkephalin analog (Sandoz FK 33-824). The latter cultures showed cross-tolerance to met-enkephalin and opiates; dorsal cord responses could still be evoked even after acute exposure to high levels of morphine. Morphine-tolerant cultures also showed cross-tolerance to met-enkephalin and to the Sandoz opioid peptide (FK 33-824). The tolerant state did not develop if the cultures were incubated at lower temperature, ca. 20°C, during exposure to 1 μM morphine for as long as 7 days. The data indicate that a temperature-dependent metabolic change occurs in these neurons after chronic exposure to morphine at 35°C leading to a sustained decrease in sensitivity to opiate-depressant effects. Enhanced dorsal-horn responses in tolerant cultures suggested development of “dependence” as well as tolerance to opiates in these isolated cord-DRG tissues.     

Innervation of heart and alary muscles in Sphinx ligustri L. (Lepidoptera)

Summary The origin and orientation of the heart nerves in Sphinx ligustri and Ephestia kuehniella were investigated by scanning electron microscopy using a special technique which involved pinning the dissected specimens on a stabilizing metal pad. The heart and alary muscles in Sphinx particularly their caudal extremity were also examined by transmission electron microscopy. The alary muscles form an incomplete sheath around the heart with a mainly longitudinal fibre orientation, e.i. antagonistically to the fibres of the heart itself. The heart and alary muscles are multiterminally innervated by branches of the transverse segmental nerves. All branches contain a single electron lucent axon; the thickest branches also possess several neurosecretory axons. Swellings of the segmental nerves may indicate the position of nerve cell bodies. There are no lateral heart nerves. Only one type of neuromuscular junction is abundant in the alary muscles but less frequently found in the heart. The terminals originate from the central axon only. They are capped by glial cells, which interdigitate with the muscle cells. They penetrate into the T-system toward the Z-discs and form a complex intercellular space system. Exocytosis of dense-cored vesicles into this perisynaptic reticulum seems likely. Sites of neurohaemal release are distributed along the nerve branches and special nerve endings occur at the level of the ostia. The possible nervous influence upon heart activity is discussed.The transmission electron microscopic part of this investigation was supported by a research scholarship from the Deutsche Forschungsgemeinschaft     

Ectopic adenoviral vector-directed expression of Sema3A in organotypic spinal cord explants inhibits growth of primary sensory afferents

Sema3A (Sema III, SemD, collapsin-1) can induce neuronal growth cone collapse and axon repulsion of distinct neuronal populations. To study Sema3A function in patterning afferent projections into the developing spinal cord, we employed the recombinant adenoviral vector technique in embryonic rat spinal cord slices. Virus solution was injected in the dorsal aspect of organotypic spinal cord cultures with segmentally attached dorsal root ganglia (sc-DRG). In cultures grown in the presence of nerve growth factor (NGF), injected either with the control virus AdCMVLacZ or with vehicle only, afferent innervation patterns were similar to those of control. However, unilateral injection of AdCMVSema3A/AdCMVLacZ in sc-DRG slices revealed a strong inhibitory effect on NGF-dependent sensory afferent growth. Ectopic Sema3A in the dorsal spinal cord, the target area of NGF-responsive DRG fibers in vivo, created an exclusion zone for these fibers and as a result they failed to reach and innervate their appropriate target zones. Taken together, gain of Sema3A function in the dorsal aspect of sc-DRG cultures revealed a dominant inhibitory effect on NGF-dependent, nociceptive sensory DRG afferents, an observation in line with the model proposed by E. K. Messersmith et al. (1995, Neuron 14, 949-959), suggesting that Sema3A secreted by spinal cord cells can act to repel central sensory fibers during the formation of lamina-specific connections in the spinal cord.     

Factors released by ciliary neurons and spinal cord explants induce acetylcholine receptor mRNA expression in cultured muscle cells

The nuclei of cultured noninnervated muscle cells are heterogeneous with respect to production of mRNA for the nicotinic acetylcholine receptor (AChR). Some nuclei actively express AChR mRNA while others have a low level of activity or are inactive. To determine if innervation, or a factor released by neurons, influences nuclear expression of AChR mRNA, we examined mRNA at a single cell level via in situ hybridization and autoradiography with an alpha-subunit AChR genomic probe. Four days after plating, we co-cultured chicken primary muscle cells with spinal cord explants, ciliary neurons, or dorsal root ganglia (DRG) cells. In situ hybridization of the spinal-cord and muscle-cell co-cultures with the AChR alpha-subunit probe revealed a high density of silver grains on muscle cells, which were within two explant diameters of the spinal cord explant, and a graded decrease in silver grain density as the distance from the explant increased, as well as the appearance of a strikingly nonhomogenous distribution of active and inactive muscle cell nuclei. When ciliary neurons were uniformly distributed over the muscle cells, a high level of AChR mRNA was induced, but no gradients appeared. Neither an increased mRNA level nor a gradient was observed when DRG cells were co-cultured with muscle cells. When ciliary neurons are cultured within Costar permeable inserts, which prevent any contact between the neurons and the underlying muscle cells, AChR messenger RNA is still induced, showing that diffusible factors are responsible. Our results indicate that molecules released by cholinergic neurons regulate the expression of AChR mRNA in the myotubes and raise the possibility that AChR expression depends on both neuronal signals and on intracellular information from the muscle cell.     

Excitotoxic cell death induces delayed proliferation of endogenous neuroprogenitor cells in organotypic slice cultures of the rat spinal cord

The aim of the present report was to investigate whether, in the mammalian spinal cord, cell death induced by transient excitotoxic stress could trigger activation and proliferation of endogenous neuroprogenitor cells as a potential source of a lesion repair process and the underlying time course. Because it is difficult to address these issues in vivo, we used a validated model of spinal injury based on rat organotypic slice cultures that retain the fundamental tissue cytoarchitecture and replicate the main characteristics of experimental damage to the whole spinal cord. Excitotoxicity evoked by 1 h kainate application produced delayed neuronal death (40%) peaking after 1 day without further losses or destruction of white matter cells for up to 2 weeks. After 10 days, cultures released a significantly larger concentration of endogenous glutamate, suggesting functional network plasticity. Indeed, after 1 week the total number of cells had returned to untreated control level, indicating substantial cell proliferation. Activation of progenitor cells started early as they spread outside the central area, and persisted for 2 weeks. Although expression of the neuronal progenitor phenotype was observed at day 3, peaked at 1 week and tapered off at 2 weeks, very few cells matured to neurons. Astroglia precursors started proliferating later and matured at 2 weeks. These data show insult-related proliferation of endogenous spinal neuroprogenitors over a relatively brief time course, and delineate a narrow temporal window for future experimental attempts to drive neuronal maturation and for identifying the factors regulating this process.     

Morphogenesis of nuclear inclusions in the soleplate region of rat skeletal muscle fibers following chronic daily administration of neostigmine

Summary In the course of ultrastructural investigations of motor endplate pathology mediated by calcium ions, intranuclear sarcoplasmic inclusions, either membrane-free (true type) or membrane-delimited (false type), were observed during chronic daily high-dose exposure to the anticholinesterase neostigmine. At the stage in which subjunctional components, including soleplate nuclei, were severely damaged (day 7), the true nuclear inclusions were frequently associated with the disrupted nuclear envelope (fragmentation, vesiculation etc.) and nuclear pores. At a subsequent stage, in which muscle repair was accelerated and most soleplatenuclei were less severely affected (day 21), formation of the false inclusions in these nuclei was enhanced. Analysis of serial sections of the less severely affected nuclei, where only a true inclusion type was present, revealed no sign of invaginated nuclear envelopes or other membranes enclosing the inclusions. Our findings indicate that morphogenesis of true inclusions depends upon the severity of nuclear degeneration, i.e., in severely affected nuclei there is disruption in the nuclear envelope and/or nuclear pores, while in less severely affected nuclei, either a pinched-off invagination or diffusion of excessive sarcoplasmic proteins into the nucleus via nuclear pores occurs.     

S100β as an early biomarker of excitotoxic damage in spinal cord organotypic cultures

S100β is a cytoplasmic calcium‐binding protein mainly expressed by glia and considered to be a useful biomarker for brain or spinal cord injury. Indeed, clinical studies suggest that the S100β concentration in serum or cerebrospinal fluid may predict lesion outcome and prognosis. The relation of S100β levels to damage severity and its timecourse remains, however, unclear. This study used a validated in vitro model of spinal cord injury induced by kainate‐mediated excitotoxicity to investigate these issues. After 22 days in vitro, rat organotypic spinal cord slices were subjected to one transient application (1 h) of 1 or 100 μM kainate followed by washout. While the lower kainate concentration did not evoke neuronal loss or S100β increase, the larger concentration elicited 40% neuronal death, no change in glial number and a delayed, significant rise in extracellular S100β that peaked at 24 h. This increase was associated with a stronger expression of the S100β protein as indicated by western blotting and immunohistochemistry. Application of the microtubule disrupting agent colchicine did not change the rise in S100β induced by kainate, an effect blocked by the glutamate receptor antagonists CNQX and APV. Our data suggest that excitotoxicity was followed by release of S100β perhaps from a readily releasable pool through a mechanism independent of microtubule assembly. The raised extracellular level of S100β appeared to reflect glial reactivity to the kainate‐evoked lesion in accordance with the view that this protein may be involved in tissue protection and repair after acute injury.

     

Innervation and acetylcholine sensitivity of skeletal muscle cells differentiated in vitro from chick embryo

Trypsin-dissociated myoblasts from leg muscle of 12-day chick embryos have been cultured in monolayers. After four days the muscle cultures have been confronted with fragments of the spinal cord of six-day chick embryos. Electrophysiological and morphological analysis demonstrate that characteristic neuromuscular transmission can develop in these cultures. Electrical stimulation of the cord fragment evokes contractions of innervated muscle fibers, from which end plate potentials and miniature end plate potentials with average frequency around one per second or more can be recorded. D-tubocurarine (1 μg/ml) suppresses reversibly these synaptic potentials. Non-innervated muscle fibers are sensitive to acetylcholine over all their surface, while innervated muscle fibers are sensitive at the regions where structures suggestive of motor end plate (“bulb-type”) are found. We can conclude that neuromuscular connections developed in vitro in our experiments are functional in respect of transmission of impulses but also in respect of neurotrophic influences for restriction of chemosensitivity.     

Comparison of callus induction and plant regeneration from different explants in triploid and tetraploid turf-type bermudagrasses

The turf-type bermudagrasses are genetically variable and do not respond uniformly to tissue culture and plant regeneration protocols. We evaluated the callus induction response of two explant types, young inflorescences and nodes, from multiple genotypes including triploid TifSport, TifEagle, and Tift97-4 and tetraploid Tift93-132, Tift93-135, Tift93-156 and Tift93-157 on MS medium supplemented with 1–1.5 mg l⁻¹ 2,4-D + 0.01 mg l⁻¹ BA + 1.16 g l⁻¹ proline. Four types of callus were observed. Type I was fluffy, soft, and white non-embryogenic callus, common to all cultures. Type II was globular, transparent, and hard, but sticky callus, which was pre-embryogenic and could be selected for subculture. Type III callus was transparent, compact, and embryogenic. Type IV callus was opaque white and compact. Both Type III and Type IV calluses were embryogenic and regenerative. A combination of gelling agents in the medium (2 g l⁻¹ Gelrite and 5 g l⁻¹ agar) improved callus quality and increased the rate of compact callus formation during subculture. Embryogenesis from compact callus was observed in TifEagle, TifSport and Tift93-132, and shoots were regenerated on MS medium with 0.1 mg l⁻¹ 2,4-D + 0.5–4.0 mg l⁻¹ BA. Low intensity light treatment (30 μmol m⁻2 s⁻¹ of cool white fluorescence) to callus before regeneration greatly enhanced regeneration frequency from 6.7% to 40% in recalcitrant TifSport.     

Forelimb muscle activity following nerve graft repair of ventral roots in the rat cervical spinal cord

Current research on the cellular mechanisms of nerve regeneration suggests the application of nerve growth factors at the repair sites to be beneficial. To test the effectiveness of this approach, we performed transections of the C6 and C7 ventral rootlets from their original sites in the spinal cord of 18 rats. We investigated the electrophysiological changes in three groups of rats operated on by different repair strategies. Six rats comprised the control group (G1). In the other 12 rats, 24 rootlets were implanted into the spinal cord by means of an intercostal nerve graft through the pia mater immediately after transection. Six rats (G2) had fibrin glue applied at the incision. The last 6 rats (G3) had grafts with acidic fibroblast growth factor (aFGF) added to the fibrin glue. The rats' functional recovery was evaluated electrophysiologically at 6 weeks and 6 months after the operation. Needle electromyography showed profound fibrillation potentials (Daube's scoring system) in the deltoid, biceps, and triceps of the operated forelimbs in all groups 6 weeks after the operation. After 6 months, there was a significant decrease in the amount of fibrillation potentials in all groups (G1, G2 and G3, p < 0.0001, 0.0001, 0.0009, respectively, generalized estimating equation, repeated measures) and a significantly high probability for motor units present in sampled muscles of G2 and G3 as compared to G1 (log odds ratio in G2 = 51.8316, G3 = 57.4262, generalized estimating equation). We conclude that several cervical roots can regenerate through intercostal nerve grafts applied using fibrin glue. Adding aFGF may increase the efficacy of sprouting.