Presynaptic and postsynaptic organelles of synapses formed in cultures of previously dissociated mouse spinal cord

Summary This study describes some of the ultrastructural features of presynaptic and postsynaptic organelles at synapses developed in cultures of previously dissociated mouse spinal cord cells. Particular attention was paid to the agranular reticulum which is well developed at many presynaptic and postsynaptic sites, either in the form of simple tubules or cisternae, or more complex networks and often closely associated with mitochondria. In addition, the disposition of microtubules at and close to synaptic specializations is described. These and other features of synaptic zones, such as granular vesicles in presynaptic sites, are discussed in relation to cultures developed on feeder layers and synapses in vivo, and in relations to possible degenerative and regenerative events in the cell cultures.     

Studies on synaptic transmission in spinal cord cultures: a comparison of postsynaptic actions of classical neurotransmitters with the peptides

The postsynaptic action of the classical neurotransmitter noradrenaline (NA), the reversal potential of the excitatory postsynaptic potential (EPSP) and the effects of divalent cations on EPSPs in dissociated spinal cord cultures are described. In co-cultures of locus coeruleus explant and spinal cord cells, it was found that NA could mimic the response evoked by stimulation of the explant on the spinal cord cells surrounding the explants. Both depolarization and hyperpolarization responses were observed. On a few occasions, a biphasic response consisting of a hyperpolarization followed by a depolarization was observed. The depolarizing response was associated with an increase in input resistance of the membrane. This would suggest that NA may have a facilitatory effect on synaptic transmission. The depolarizations were antagonized by the α-antagonist piperoxane, and were not affected by the β-antagonist propranolol at the concentrations tested, indicating that the receptor mediating these responses is of the α-type. The reversal potential for dorsal root ganglion and spinal cord cells was +8±3.2 mV (mean±s.e.m.), and that for spinal cord and spinal cord cells was ?4±4.3 mV (mean±s.e.m.). These values are different from those previously reported for glutamate in spinal cord cultures. The effects of high and low concentrations of calcium ions on quantal output and mean quantal amplitude or quantal size of the EPSP were further examined. As expected, the cation had an effect mainly on the release process: increasing the concentration of calcium increased the amount of neurotransmitter released, while reducing the concentration of calcium reduced release. Quantal size was slightly or not affected by alteration of external calcium. In comparing the postsynaptic actions of classical neurotransmitters to those of peptides, there is apparently no evidence that the actions of the two groups of agents on central neurons are different. It appears, however, that the peptides generally elicit responses at lower concentrations than the classical neurotransmitters. Further experimentation is required to fully elucidate the actions of peptides on mammalian central neurons.     

Noradrenergic axon terminals in the substantia gelatinosa of the rat spinal cord

Summary The noradrenergic terminals in the substantia gelatinosa of the dorsal horn of the cervical spinal cord of the rat were investigated by means of the histofluorescence technique and electron-microscopic cytochemistry using the glyoxylic acid-KMnO₄ fixation technique. In accordance with the topographical distribution of fluorescent catecholaminergic fibers, noradrenergic terminals containing small granular vesicles were frequently observed electron microscopically in the outer layer of the substantia gelatinosa. These terminals were most frequently found to appose without showing typical synaptic features, small-caliber dendrites, spine apparatus, and rarely, large caliber dendrites. Only in a few cases, the noradrenergic terminals exhibited typical synaptic contacts with dendritic elements of small size. In addition, noradrenergic terminals apposed non-noradrenergic terminals containing small agranular vesicles. In rats bearing surgical lesions of the dorsal roots, no noradrenergic terminal were found in contact with the degenerated axon terminals in the substantia gelatinosa. These findings suggest that the noradrenergic afferents to the substantia gelatinosa may exert their influence on sensory transmission via dorsal horn cells.     

Distribution of sensory neurones of the pudendal nerve in the dorsal root ganglia and their projection to the spinal cord

Summary The morphology and distribution of the sensory neurones of the pudendal nerve within the spinal ganglia of rats were investigated by use of horseradish peroxidase (HRP). The labelling was visualized in diaminobenzidine (DAB) or tetramethyl-benzidine (TMB)-stained sections. Injection of HRP directly into the pudendal nerve labelled perikarya predominantly in the sixth lumbar DRG (L6). Following injection of HRP into the scrotal skin, however, additional cells were labelled in L5 and SI. Labelling was invariably unilateral. Approximately equal numbers of small (<30 m) and large neurones (>40 m) were labelled following subcutaneous injections although injections into the nerve marked twice as many small cells as large cells. This suggests that, in the rat, most of the small-diameter fibres within the pudendal nerve ascend through L6. Although a cluster of neurones was observed in one experiment, the remaining 25 experiments did not reveal any somatotopic arrangement since the labelled perikarya were distributed evenly throughout the ganglion. Similar numbers of retrogradely labelled neurones (somatopetal transport of the tracer) were observed in both DAB- and TMB-stained sections, although TMB allowed the demonstration of anterograde (somatofugal) HRP transport by terminal labelling in the superficial laminae of the lumbar spinal cord, extending into laminae II–IV.Partially supported by grants from the DFG to HWK (Ko 758/1)     

The on/off of Pax6 controls the tempo of neuronal differentiation in the developing spinal cord

During neurogenesis, complex networks of genes act sequentially to control neuronal differentiation. In the neural tube, the expression of Pax6, a paired-box-containing gene, just precedes the appearance of the first post-mitotic neurons. So far, its only reported function in the spinal cord is in specifying subsets of neurons. Here we address its possible function in controlling the balance between proliferation and commitment of neural progenitors. We report that increasing Pax6 level is sufficient to push neural progenitors toward cell cycle exit and neuronal commitment via Neurogenin 2 (Ngn2) upregulation. However, neuronal precursors maintaining Pax6(On) fail to perform neuronal differentiation. Conversely, turning off Pax6 function in these precursors is sufficient to provoke premature differentiation and the number of differentiated neurons depends of the amount of Pax6 protein. Moreover, we found that Pax6 expression involves negative feedback regulation by Ngn2 and this repression is critical for the proneural activity of Ngn2. We present a model in which the level of Pax6 activity first conditions the moment when a given progenitor will leave the cell cycle and second, the moment when a selected neuronal precursor will irreversibly differentiate.     

Atrial natriuretic factor-like immunoreactivity in spinal cord and in primary sensory neurons of spinal and trigeminal ganglia of guinea-pig: correlation with tachykinin immunoreactivity*

Summary Atrial natriuretic factor (ANF) is a cardiac hormone with various functions in body homeostasis. It is also processed in the brain and in the peripheral nervous system where it appears to play a role as a neuromodulator. Little is known about the presence of ANF throughout the spinal cord of the guinea-pig. We therefore examined the distribution of ANF and its possible interrelation with primary sensory afferents in this species. Using enzyme- and fluorescence-immunohistochemistry on deparaffinized sections, ANF-like immunoreactivity was found to be present in nerve fibers in laminae I/II of the spinal cord and in neurons of spinal and trigeminal ganglia. Tachykinins and ANF coexisted in very few fibers of the spinal cord but did not coexist in primary sensory spinal or trigeminal neurons. Our results indicate that spinal ANF-immunoreactive fibers are of dual origin, primary sensory and non-primary sensory. The possibly heterogeneous source of the non-primary sensory ANF, its possible coexistence with other co-transmitters and functional implications are discussed.Preliminary aspects of this study have been reported at the 2nd World Congress of Neuroscience in Budapest (Weihe et al. 1987) and at the Versammlung der Anatomischen Gesellschaft in Zürich (Nohr et al. 1989)     

Detection of phosphorylated mitogen-activated protein kinase in the developing spinal cord of the mouse embryo

Global understanding of the proteome is a major research topic. The comprehensive visualization of the distribution of proteins in vivo or the construction of in situ protein atlases may be a valuable strategy for proteomic researchers. Information about the distribution of various proteins under physiological and pathological conditions should be extremely valuable for the basic and clinical sciences.The mitogen-activated protein kinase (MAPK) cascade plays an essential role in intracellular signaling in organisms. This cascade also regulates biological processes involving development, differentiation, and proliferation. Phosphorylation and dephosphorylation are integral reactions in regulating the activity of MAPKs. Changes in the phosphorylation state of MAPKs are rapid and reversible; therefore, the localizations of physiologically phosphorylated MAPKs in vivo are difficult to accurately detect. Furthermore, phosphorylated MAPKs are likely to change phosphorylated states through commonly used experimental manipulations.In the present study, as a step toward the construction of in situ phosphoprotein atlases, we attempted to detect physiologically phosphorylated MAPKs in vivo in developing spinal cords of mice. We previously reported an improved immunohistochemical method for detecting unstable phosphorylated MAPKs. The distribution patterns of phosphorylated MAPKs in the spinal cords of embryonic mice from embryonic day 13 (E13) to E17 were observed with an improved immunohistochemical method. Phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated c-Jun N-terminal kinase 1/2 (p-JNK1/2) were strongly observed in the marginal layer and the dorsal horn from E13 to E17. Our results suggest that p-ERK1/2 and p-JNK1/2 play critical roles in the developing spinal cord. Constructing phosphoprotein atlases will be possible in the future if this work is systematically developed on a larger scale than we presented here.     

Effects of combining methylprednisolone with rolipram on functional recovery in adult rats following spinal cord injury

Methylprednisolone (MP) has been widely used as a standard therapeutic agent for the treatment of spinal cord injury (SCI). Because of its controversial beneficial effects, the combination of MP and other pharmacological agents aimed at enhancing functional recovery is desirable. The phosphodiesterase 4 (PDE4) inhibitor rolipram has been implicated in promotion of regeneration due to elevating cAMP. In the present study, we sought to determine the effects of MP and rolipram, administered in combination, after spinal cord injury (SCI) in adult rats. Here we show that in vitro administration of rolipram and MP significantly increased neuron survival and promoted neurite outgrowth of neurons on the inhibitory substrate CSPGs by upregulation of MMP-2 expression; in vivo administration of rolipram and MP inhibited CSPG expression and increase CSPG digestion after rat SCI. Rolipram and MP combining treatment promoted significant neuroprotection through reduced motoneuron death, minimized lesion cavity, and increased regeneration of lesioned corticospinal tract (CST) axons beyond the lesion site after SCI. Enhanced functional recovery was also observed. Overall, our study strongly suggested that the combination treatment of MP and rolipram may represent a promising strategy for clinically applicable pharmacological therapy for rapid initiation of neuroprotection after SCI.     

Association of glial cells with the terminal parts of neurite bundles extending from chick spinal cord in vitro

Summary The terminal parts of radially directed neurite bundles growing out from chick embryo spinal cord in vitro have been examined by phase and electron-microscopy,A type of ending is described in which the terminal parts of the neurites are associated with a glial cell. The latter sends a single major process proximally towards the explant. Distally it is attached to the substrate, and the neurite ends are related to its dorsal (nonsubstrate) aspect. Appearances suggesting a mechanism of adhesion of neurites to each other and to the gial cell are described.Growth vesicles were found in both neurites and glia.It is suggested that movements of terminal glial cells may affect the pattern of outgrowth of their attached neurite bundles.We are grateful to the Medical Research Council for financial assistance, to Mr. A. Aldrich and Mr. D. Gunn for photography, to Mr. P. Howell and Miss 0. Chmyliwsky for technical assistance, and to Mrs. B. Fisher for valued secretarial help.     

Transneuronal degeneration in the Rolando substance of the primate spinal cord evoked by axotomy-induced transganglionic degenerative atrophy of central primary sensory terminals

Summary Transection of the sciatic nerve in Rhesus monkeys and the consequent transganglionic degenerative atrophy (TDA) of central terminals of primary afferents result in transneuronal degeneration of substantia gelatinosa (SG) cells. Severe degeneration is characterized by an increased electron density of the nucleus and by conspicuous shrinkage of the cytoplasm, mitochondrial swelling, dilation of cisterns of the rough-surfaced endoplasmic reticulum, accumulation of free ribosomes and an electron-dense material in the cytoplasm. In the mild form, dilation of cisternal elements of the endoplasmic reticulum, swollen mitochondria and accumulation of free ribosomes takes place. About 10% of SG cells in segment L₅ undergo the severe form whereas the rest shows signs of the mild form. Cytoplasmic alterations that occur during transneuronal degeneration seem to start at the level of subsurface cisterns. Dendrites and axons of transneuronally degenerating SG cells also show a conspicuous electron density. By analyzing the synaptic relationships of such darkened dendrites, connections in the upper dorsal horn can be deciphered. Modular units of the primary nociceptive analyzer that evaluate noxious and innocuous inputs on the basis of thin versus thick (AC/A) afferent activity and subjecting them to descending control appear to be recruited from structurally dispersed elements of synaptic glomeruli. These are arranged alongside dendritic processes of large antenna cells which relay impulses to projection cells of the spinothalamic tract.     

Between-day reliability of MyotonPRO for the non-invasive measurement of muscle material properties in the lower extremities of patients with a chronic spinal cord injury

Measuring the muscle properties of patients with spinal cord injuries (SCIs) is important to better understand their biomechanical features. In this study, we sought to evaluate the between-day reliability of MyotonPRO, a handheld device that can measure muscle mechanical properties, and assess whether it is reliable to measure muscle properties over time in patients with SCI. Thirteen men with complete SCIs (age 53.9 ± 6.3 years, height 171.0 ± 5.2 cm, weight 66.1 ± 5.8 kg), and injury levels ranging from L1 to T12, were enrolled. Oscillation frequency; logarithmic decrement; dynamic stiffness; mechanical stress relaxation time; and creep of the biceps femoris, medial and lateral gastrocnemius, rectus femoris, tibialis anterior, and Achilles tendon were measured on consecutive days using MyotonPRO. The intraclass coefficient for most muscles and the Achilles tendon ranged from 0.53 to 0.99 for all parameters. The percentage standard error of the measurement for many parameters in most muscles and the Achilles tendon was less than 10%. Bland-Altman analysis showed a high agreement for all mechanical properties. No significant differences were observed in any muscle or Achilles tendon properties between days (all p > 0.05). These results indicate that the MyotonPRO is reliable for between-day measurements of the mechanical properties of lower limb muscles and Achilles tendon in patients with SCI.     

Effect of maximal arm exercise on skin blood flux in the paralyzed lower limbs in persons with spinal cord injury

The purpose of the present study was to examine the effect of maximal arm exercise on the skin blood circulation of the paralyzed lower limbs in persons with spinal cord injury (PSCI). Eight male PSCI with complete lesions located between T3 and L1 performed graded maximal arm-cranking exercise (MACE) to exhaustion. The skin blood flux at the thigh (SBFT) and that at the calf (SBFC) were monitored using laser-Doppler flowmeter at rest and for 15 s immediately after the MACE. The subject's mean peak oxygen uptake and peak heart rate was 1.41 ± 0.22 1·min⁻¹ and 171.6 ± 19.2 beats·min⁻¹, respectively. No PSCI showed any increase in either SBFT or SBFC after the MACE, when compared with the values at rest. These results suggest that the blood circulation of the skin in the paralyzed lower limbs in PSCI is unaffected by the MACE.     

Immunohistochemical examination of intraspinal serotonin neurons and fibers in the chicken lumbar spinal cord and coexistence with Leu-enkephalin

Intraspinal serotonin-positive cells and fibers were examined in the chicken lumbar spinal cord following removal of descending serotonin fibers by spinal transection. Co-localization of Leu-enkephalin immunoreactivity in intraspinal serotonin cells was also examined using a double immunofluorescence labeling technique. By one or two weeks after spinal transection, virtually all supraspinal serotonin fibers were eliminated. Intraspinal serotonin cells were located ventral or ventrolateral to the central canal corresponding to laminae VII, VIII, and IX, and the anterior funiculus. Intraspinal serotonin cells sent fibers to (1) the pia mater on the ventral or ventrolateral surface of the spinal cord; (2) vessels in the spinal cord; (3) sympathetic preganglionic column of Terni; (4) other intraspinal serotonin neurons; (5) the central canal. Some 30%–50% of the intraspinal serotonin cells co-localized with Leu-enkephalin. Intraspinal serotonin fibers co-containing Leu-enkephalin were observed in the pia mater located on the most lateral surface of the spinal cord. Permanent address: This study was supported by Grant-in-Aid for Scientific Research on Priority Area from the Ministry of Education, Science and Culture, Japan.     

Heterogeneity of astrocytic membranes in the optic nerve and spinal cord of the lizard,Anolis carolinensis

Summary The architecture of astrocytic membranes in the optic nerve and the spinal cord of the lizard, Anolis carolinensis, was investigated by use of the freeze-fracturing technique. Whereas astrocytes in mammals reveal so-called orthogonal arrays of particles (OAPs) in their membranes, astrocytes in lower vertebrates lack these structures. This study demonstrates for the first time OAPs in astrocytes from a submammalian species. They were found commonly in the optic nerve and less frequently in the spinal cord. However, the OAPs in astrocytes of spinal cord were confined to midtrunk levels; the astrocytes in the caudal spinal cord failed to reveal OAPs.Additionally, the ependymal cells around the central canal did not show any OAPs, either in the thoracic or in the caudal spinal cord. They were interconnected by gap and tight junctions, which were not intercalated with each other.The findings support our current working hypothesis according to which the presence and absence of OAPs in astrocytes may be correlated with regenerative incapability or capability of CNS-structures; i.e., whereas the thoracic spinal cord in Anolis carolinensis is known to be incapable of regeneration after injury, the caudal spinal cord is regenerative.     

Effect of arm cranking exercise on skin blood flow of lower limb in people with injuries to the spinal cord

The purpose of this study was to examine whether arm cranking exercise induces changes in skin blood flow in the paralyzed lower limbs of people with injuries to the spinal cord (PISC). Ten PISC with lesions located between Th5 and L5 and six control subjects performed arm cranking exercise for 6 min at three intensities, 10, 30 and 50 W, at a room temperature of 25°C. Oxygen uptake (Vo₂) and heart rate (HR) were measured for the last 2 min of each exercise period. The skin blood flow at the anterior thigh (BF_sk,t) was continuously monitored using laser Doppler flowmetry for the whole 6-min period and for the first 10 min of recovery following exercise. During exercise, the PISC showed lower Vo₂ and greater HR than the control subjects. No increase in BF_sk,t was found in six of the PISC with lesions at or above Th12, irrespective of the exercise intensity. On the other hand, in PISC with lesions at L1 or below, BF_sk,t increased significantly (P < 0.05) with an increase in Vo₂ and HR, although the BF_sk,t at a given Vo₂ and HR was lower than that in the control subjects. These results would suggest that arm exercise can promote the blood circulation in the skin of the lower limbs if the injury level is below L1.     

Ascending curbs of progressively higher height increases forward trunk flexion along with upper extremity mechanical and muscular demands in manual wheelchair users with a spinal cord injury

High upper extremity (U/E) demands are required when manual wheelchair users (MWUs) with spinal cord injury (SCI) ascend curbs; this may contribute to the risk of developing U/E musculoskeletal impairments. The aim of this study was to compare movement strategies (kinematics), mechanical loads (kinetics) and muscular demand (EMG) at the non-dominant U/E among 15 MWUs with SCI when ascending curbs of 4 cm (3 trials), 8 cm (3 trials) and 12 cm high (3 trials) from a starting line set 3 m before the curb. Biomechanical data was collected during three trials for each height. The curb ascent task was divided into three adjustment phases: caster pop, rear-wheel ascent and post-ascent. The greatest effort was generated by the shoulder flexors and internal rotators as well as the elbow flexors. A significant difference (p < 0.0167) between the curb heights was found for most outcome measures studied: movement excursion, net joint moments and muscular utilization ratio (MUR) of the main muscles increased with the higher curb heights, mainly around the shoulder joint. These results provide insight that aside from adhering to a highly structured training method for wheelchair curb ascent, rehabilitation professionals need to propose task-specific strength training programs based on the demands documented in this study and continue to advocate for physically accessible environments.