Carbonic anhydrase activity in myelin fractions from rat optic nerves.

The carbonic anhydrase activity of myelin fractions isolated from the optic nerves of adult and immature (20-day-old) rats was examined. The specific activity in both total homogenate and myelin fractions was about 2-fold higher in adult than in immature animals and at both ages, the activity in the homogenate was higher than in myelin. After subfractionation by zonal gradient centrifugation, it was shown that carbonic anhydrase activity was greatest in the heaviest myelin particles at both ages. These data are consistent with the hypothesis that a small proportion of the total enzyme activity is localised in myelin.     

Cell kinetics and cell death in the optic nerve of the myelin deficient rat

Summary The optic nerves of myelin deficient (md) and normal rats were studied by light microscopic thymidine autoradiography and electron microscopy during the first postnatal month. Mean total glial cell counts were similar at most ages in the md and normal optic nerves, but were significantly increased in the md nerves at 18 days of age, and significantly reduced at 30 days, compared to the controls. Labelling indices in the md optic nerves were significantly higher than control values at 16–25 days. Oligodendrocytes, astrocytes, microgliacytes and unclassified cells were labelled in both normal and md rats. The mutants showed higher percentages of labelled astroglia and microglia, and the labelled oligodendroglia appeared immature when compared to the normal animals. Md optic nerves showed significantly higher counts of necrotic cells than controls at 14–30 days. Electron microscopy revealed the presence of abnormal oligodendrocytes, characterized by distended rough endoplasmic reticulum and a dilated perinuclear envelope, from 6 days of age in the mutant. These cells appear to degenerate and die. These results suggest a defect in the md rat oligodendrocyte, resulting in a protracted uptake of thymidine and increased cell death. Investigation of the link between these observations and the proposed myelin proteolipid protein defect in the md rat may reveal more about the role of myelin proteins in the CNS, and the specific cellular defect(s) in this mutant.     

Low myelin basic protein levels and normal myelin in peripheral nerves of myelin deficient mutant mice (MLD)

In myelin deficient mutant mice peripheral nerves appeared normally myelinated and the myelin sheath showed the typical alternation of minor and major dense lines. The protein analysis of myelin and direct determinations of myelin basic protein revealed very low amounts of this component in sciatic nerves from mutants. The other peripheral nervous system myelin proteins and the myelin enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase were not affected.These results suggest that myelin basic proteins do not play an important role in the formation of the major dense line in peripheral nervous system myelin.     

Microenvironmental changes during axonal regrowth in the optic nerve of the myelin deficient rat. Immunocytochemical and ultrastructural observations

Summary Lesion-induced regenerative sprouting of CNS axons is accompanied by reactions of the supporting glia and vascular and connective tissue which may influence the extent of regeneration. In a previous report, it was shown that after crush injury, the amyelinated optic nerve of the myelin deficient (md) mutant rat contains greater numbers of regrowing axons proximal to the site of crush than that of normally myelinated littermates. The present study was designed to compare the response of the microenvironment, i.e. glial cells and vascular and connective tissue, in md and normally myelinated optic nerves 2, 4 and 6 days after crush injury. In unoperated normal optic nerves monoclonal antibodies to the HNK-1 carbohydrate labelled astrocytic processes at the ultrastructural level whereas in unoperated md mutants HNK-1 staining was restricted to axonal surfaces. Immunoreactivity with monoclonal antibodies to stage-specific embryonic antigen-1 (SSEA-1) was confined to astrocytic surfaces in both md and wildtype animals. After axotomy of md optic nerves regrowing axons were more numerous in the proximal site of the crush and extended further into the lesion than in wildtype animals. In both md and wildtype rats regrowing axons were HNK-1-positive. In md rats strong reaction with antibodies to laminin and fibronectin was only seen in 6-day-old lesions of md rats whereas immunoreactivity was less distinct in operated littermate controls. Immunolabelling was obviously associated with blood vessels, since crush lesions in both md and wildtype rats were Schwann cell-free as assessed by electron microscopy and immunocytochemistry. In both operated md and normal littermates crush lesions contained degenerating astrocytes as well as reactive astrocytes in which the intermediate filaments of the perikarya failed to stain immunocytochemically for GFAP, vimentin, desmin, and a common determinant of intermediate filaments. In contrast, reactive astrocytes in the lesion site of normally myelinated rats expressed the SSEA-1 antigen intracytoplasmically whereas in md mutants astrocytes were completely SSEA-1-negative. Infiltration of crush lesions by macrophages was less extensive in md rats than in normal littermates. However the overall content of macrophages in the peritoneal cavity was also reduced. The present study demonstrates that (1) md optic nerves lack HNK-1-reactive astrocytes; (2) in the axotomized wildtype optic nerve impaired axonal regrowth may be associated with distinct immuno-phenotypes of the supporting glial cells, i.e. SSEA-1-positive astrocytes; (3) laminin and fibronectin seem not to be essential for improved axonal regrowth in md rats.     

Multiple sclerosis cerebrospinal fluid produces myelin lesions in tadpole optic nerves.

To investigate the myelinotoxicity of cerebropsinal fluid in multiple sclerosis, we used an in vivo model of the myelinated central-nervous-system tract of tadpoles for quantitative double-blind tests of 46 cerebrospinal-fluid samples. Groups of xenopus tadpoles were injected with cerebrospinal fluid near the optic nerve. Forty-eight hours later, whole mounts of optic nerves were prepared, and a differential interference microscope was used to count myelin lesions. Cerebrospinal-fluid samples from 60 per cent of the patients with an acute attack of definite multiple sclerosis had myelinotoxic activity. This activity correlated best with the severity and duration of the disease, rather than with gamma-globulin or total protein concentrations. Activity was negative in 85 per cent of cerebrospinal-fluid samples from a control group with other neurologic diseases. This assay is a useful method for investigating myelinotoxic factors of cerebrospinal fluid in patients with multiple sclerosis, but was not helpful diagnostically.     

Nicotinic acetylcholine receptors in mouse and rat optic nerves

Receptor-mediated calcium signaling in axons of mouse and rat optic nerves was examined by selectively staining the axonal population with a calcium indicator. Nicotine (1-50 microM) induced an axonal calcium elevation that was eliminated when calcium was removed from the bath, suggesting that nicotine induces calcium influx into axons. The nicotine response was blocked by d-tubocurarine and mecamylamine but not alpha-bungarotoxin, indicating the presence of calcium permeable, non-alpha7 nicotinic acetylcholine receptor (nAChR) subtype. Agonist efficacy order for eliciting the axonal nAChR calcium response was cytisine approximately nicotine > acetylcholine. The nicotine-mediated calcium response was attenuated during the process of normal myelination, decreasing by approximately 10-fold from P1 (premyelinated) to P30 (myelinated). Nicotine also caused a rapid reduction in the compound action potential in neonatal optic nerves, consistent with a shunting of the membrane after opening of the nonspecific cationic nicotinic channels. Voltagegated calcium channels contributed little to the axonal calcium elevation during nAChR activation. During repetitive stimulations, the compound action potential in neonatal mouse optic nerves underwent a gradual reduction in amplitude that could be partially prevented by d-tubocurarine, suggesting an activity-dependent release of acetylcholine that activates axonal AChRs. We conclude that mammalian optic nerve axons express nAChRs and suggest that these receptors are activated in an activity-dependent fashion during optic nerve development to modulate axon excitability and biology.     

Changes in axon birefringence during the action potential

1. Observations have been made on the changes in optical retardation accompanying the passage of impulses along crab leg nerves and squid giant axons.2. The nerves were mounted on the stage of a polarizing microscope, at 45 degrees to the planes of polarization and analysis, brightly illuminated with white light. During the nerve impulse the intensity of the light passing the analyser decreased temporarily by 1 part in 10(3)-10(6). Signal-averaging techniques were used to obtain an acceptable ratio of signal to noise.3. The changes in light intensity recorded under these conditions were shown to arise almost entirely from alterations in retardation, with little or no interference from scattering, absorption, linear dichroism or optical rotation effects; the occurrence of stimulus and coupling artifacts was also ruled out.4. In the squid giant axon, the retardation change was shown to be located in a thin cylinder immediately surrounding the axoplasm, and to have a radially oriented optic axis.5. The time course of the decrease in optical retardation was very similar to that of the action potential recorded with an intracellular electrode, suggesting that the retardation closely followed the electrical potential across the membrane.     

The effects of paranodal myelin damage on action potential depend on axonal structure

Biophysical computational models of axons provide an important tool for quantifying the effects of injury and disease on signal conduction characteristics. Several studies have used generic models to study the average behavior of healthy and injured axons; however, few studies have included the effects of normal structural variation on the simulated axon’s response to injury. The effects of variations in physiological characteristics on axonal function were mapped by altering the structure of the nodal, paranodal, and juxtaparanodal regions across reported values in three different caliber axons (1, 2, and 5.7 μm). Myelin detachment and retraction were simulated to quantify the effects of each injury mechanism on signal conduction. Conduction velocity was most affected by axonal fiber diameter (89%), while membrane potential amplitude was most affected by nodal length (86%) in healthy axons. Postinjury axonal functionality was most affected by myelin detachment in the paranodal and juxtaparanodal regions when retraction and detachment were modeled simultaneously. The efficacy of simulated potassium channel blockers on restoring membrane potential and velocity varied with axonal caliber and injury type. The structural characteristics of axons affect their functional response to myelin retraction and detachment and their subsequent response to potassium channel blocker treatment.     

Ultrastructure of the central nervous system in a myelin deficient rat

Summary Myelin deficiency (md) is a new mutant in the Wistar rat caused by an X-linked recessive lethal gene. One-half of the male offspring develop tremor and ataxia at 10–12 days of age and seizures at 16–21 days. Usually, the animals die 24–28 days postnatally unless survival is prolonged by anticonvulsants. Light microscopic examination of the C.N.S. shows a complete lack of myelin. The P.N.S. is normally myelinated, however.Frontal cortex, corpus callosum, optic nerves, cerebellum and spinal cord were studied routinely in affected animals aged 3–46 days. Abnormal males were identified three days after birth by the absence of myelinated axons from the ventral funiculus of the cervical cord. In mutants aged 3–16 days, axons had the usual ultrastructural features but were either entirely non-myelinated or, rarely, were invested by poorly organized, non-compacted, myelin-like loops of membranes, 2 to 4 in number. In mutants aged 17–20 days, axonal swellings appeared. These increased in number with longer survival times and contained large numbers of microtubules, neurofilaments, mitochondria and dense bodies. Normal C.N.S. myelin was not observed at any age.Two types of abnormal glial cell occur in md. The first, present in white matter at three days of age, is an abnormal oligodendrocyte. The cytoplasm contains dilatation of the rough-surfaced endoplasmic reticulum and the nuclear envelope is widened. A second cell-type, conspicuous by 10 days, has an electron-dense nucleus with prominently clumped chromatin and large cytoplasmic lipid droplets. This second cell type is believed to be a microgliacyte. The number of cytologically-normal oligodendrocytes decreases as mutants age while hypertrophied, filament-rich astrocytes occur in increasing numbers.The myelin defect in md C.N.S. is probably due to an abnormality of oligodendrocytes. Axonal alterations are probably secondary. Myelin deficiency resembles the murine mutant, Jimpy (jp), although ultrastructural changes in oligodendrocytes appear to be dis-similar and md, in contrast to jp, contains no normal-appearing C.N.S. myelin.     

The localization of axon branchings in two muscle nerves of the rat

Summary The distribution of fiber branching in the proximal intramuscular motor nerves of the gracilis and gastrocnemius muscles of the rat was studied in whole-mount preparations of teased nerves. Branchings of nerve fibers were clustered at fascicular divisions. Such concurrence of fascicular and fiber branchings determines the dispersion of the single muscle fibers belonging to a motor unit. The distribution of fiber branching reveals the wiring pattern of muscle fibers. These patterns differed for the gracilis and gastrocnemtus muscles in correspondence with their functional organization.This study was supported by grant Fr 609/1 from the Deutsche Forschungsgemeinschaft     

Oligodendrocyte survival and function in the long-lived strain of the myelin deficient rat

Summary This study has examined cellular and molecular aspects of glial cell function in a newly described long-lived myelin deficient rat mutant. In contrast to the shorter-lived mutants which died at 25–30 days, the longer-lived mutant rats lived to 75–80 days of age. Despite living longer, these mutants had a similar frequency of seizures to their younger counterparts. In the spinal cord and optic nerves of the older mutants, myelinated fibres in similar numbers to those seen in the younger myelin deficient rats were present. However, the total glial cell numbers were markedly reduced with few remaining normal appearing oligodendrocytes, and very few microglia compared to the younger mutants. In addition, little or no cell death or division was seen in the longer-lived rats. However, there was some evidence of ongoing myelination and the persistence of immature oligodendrocytes or their progenitors in the older mutant. There was some continued myelin gene expression, although this was at much reduced levels compared to normal, with proteolipid protein and myelin basic protein being most affected.In situ hybridization analysis for proteolipid protein mRNA showed that few proteolipid protein expressing oligodendrocytes remained in the 70–80-day-old mutant. Polymerase chain reaction analysis of exon 3 of the long-lived mutant revealed the same point mutation as described in the younger myelin deficient rat.     

Effect of ethanol on action potential and ionic membrane currents in rat ventricular myocytes

Aim: Even though alcohol intoxication is often linked to arrhythmias, data describing ethanol effect on cardiac ionic channels are rare. In addition, ethanol is used as a solvent of hydrophobic compounds in experimental studies. We investigated changes of the action potential (AP) configuration and main ionic membrane currents in rat cardiomyocytes under 20–1500 mm ethanol. Methods: Experiments were performed on enzymatically isolated rat right ventricular myocytes using the whole cell patch-clamp technique at room temperature. Results: Ethanol reversibly decelerated the upstroke velocity and decreased AP amplitude and duration at 0.2 and 3 Hz. The fast sodium current I_Na, l -type calcium current I_Ca and transient outward potassium current I_to were reversibly inhibited in a concentration-dependent manner (50% inhibition at 446 ± 12, 553 ± 49 and 1954 ± 234 mm , respectively, with corresponding Hill coefficients 3.1 ± 0.3, 1.1 ± 0.2 and 0.9 ± 0.1). Suppression of I_Na and I_Ca magnitude was slightly voltage dependent. The effect on I_Ca and I_to was manifested mainly as an acceleration of their apparent inactivations with a decreased slow and fast time constant respectively. As a consequence of marked differences in n_H, sensitivity of the currents to ethanol at 10% inhibition decreases in the following order: I_Ca (75 mm , 3.5‰), I_to (170 mm , 7.8‰) and I_Na (220 mm , 10.1‰). Conclusion: Our results suggest a slight inhibition of all the currents at ethanol concentrations relevant to deep alcohol intoxication. The concentration dependence measured over a wide range may serve as a guideline when using ethanol as a solvent.     

Minisegments of newborn rat optic nerves in vitro: gliogenesis and myelination

Summary The question of whether the development of CNS glial cells requires the presence of axons or not can be studied with in vitro systems. In order to compare the differentiation of glial cells during development in vitro with that in situ, we have selected the optic nerve, which is anatomically as well as histotypically a well defined structure. For the in vitro investigations, small explants, called minisegments, of newborn rat optic nerves were cultivated taking four major conditions into account: (1) the regular size of the minisegments should guarantee a permanent exchange of the culture medium in order to avoid cell death, (2) neither mechanical nor enzymatic dissociation of the tissue were applied, (3) the minisegments were explanted into flasks without substrate for cell adhesion and (4) the minisegments were under constant gyratory agitation. The following in situ results were obtained: optic nerves of newborn rats are morphologically characterized by the presence of naked axons, astrocytes, glial precursors, and the absence of both differentiated oligodendrocytes and myelin. At postnatal day 5 myelin sheaths are still absent. Two weeks after birth, differentiated oligodendrocytes and microglial cells are present and numerous axons are surrounded by compact myelin. The in vitro experiments show the following main results, which were obtained after 14 h, 2 d, 5 d and 14 d in culture: during time in culture, the shape of minisegment of newborn rat optic nerves undergoes drastic changes, which indicate high cellular dynamics. After 14 h in vitro, axonal profiles, cells with pyknotic nuclei as well as clusters of astrocytes and glial precursors are present. After 2 days in culture the axonal profiles disappeared and the number of degenerating cells decreased drastically. Many large cells, probably phagocytes containing inclusions and more cells are differentiated. At the stage of 5 d in vitro 4 major types of cells can be distinguished: differentiated oligodendrocytes, which form compact and loose myelin, astrocytes, large and small glioblasts and phagocytes. Immunoprecipitates for myelin basic protein and/or myelin associated glycoprotein were found in oligodendrocytes, in their processes and associated to the myelin. Processes of some astrocytes showed immunoreactive products of glial fibrillary acidic protein. After two weeks in culture, the minisegments were mostly composed of astrocytes, whereas oligodendrocytes became rare and phagocytes disappeared. It can be concluded that CNS glial cells can attain their structural and immunocytochemical characteristics in the total absence of neuronal cell bodies and axons. However, it can be speculated that neurons (or neuronal factors) could regulate the number of astrocytes and oligodendrocytes and keep these glial cells in a physiological equilibrium.     

Effects of reinnervation with normal and tetrodotoxin-inactive nerves on resting membrane potential of rat skeletal muscle

Resting membrane potentials (RMPs) have been recorded in vitro near the end-plate region of rat soleus muscles reinnervated with tetrodotoxin-inactive nerves and compared with those of denervated muscles whose reinnervation had been prevented. The two muscle groups exhibited the same low values of RMP typical of denervated muscles. In control muscles of rats in which impulse conduction was left unimpaired, reinnervation induced the expected increase in RMP values towards normal. It is suggested that, at least for this property, reinnervation restores to normal the muscle fibre membrane essentially through the return of activity.     

Characteristic variations of relative myelin sheath thickness in 11 nerves of the rat

Summary Computer-assisted measurements of relative myelin sheath thickness (the g ratio) were made in 11 peripheral nerves of the rat. The scatter diagrams showed nerve-specific variations in the distribution of relative myelin sheath thickness. Myelinated fibers of less than 3.5 m axon diameter had relatively thin myelin sheaths, particularly in the splanchnic, vagus and glossopharyngeal nerves. The oculomoter nerve had two fiber populations clearly set apart in terms of relative myelin sheath thickness. Thickly myelinated fibers were found in facial and hypoglossal nerves. No single functional modality was evident for the thinly myelinated fibersThis study was supported by grant 609 from the Deutsche Forschungsgemeinschaft     

Manic-depression neural conduction speeds and action potential event dyscorrelation.

In manic-depressive disorder, the monotonic, humor/receptor, excess/deficit, i.e. gain, theories have prevailed for many years, even though there is increasing evidence of a timing abnormality that may or may not be related to gain. In addition, the manic-depressive course is clearly multimodal and unpredictable except in revealing but rare cases, yet the system is real, and thus presumably deterministic, clearly suggesting a dynamic instability. A wide range of disciplines was reviewed by the author. Those data that were significantly different by standard tests were considered. However, averaged values were always questioned. During this review, higher water compartmentalization differentials were found in manic-depressive patients. Also indicated, event-related potential components differed in latency correlations, suggesting action potential firing event dyscorrelation. In conclusion, the manic-depressive action potential firing events may be dyscorrelated in a space-time reference frame.     

Temperature sensitivity of transduction and action potential conduction in a spider mechanoreceptor

Previous work has suggested that the activation energy of mechanotransduction is higher than expected from the simple electrochemistry of ion channels, but the temperature sensitivity of mechanically activated receptor current has not been measured directly before. We used the single-electrode voltage-clamp technique to measure receptor currents in sensory neurons of the VS-3 slit-sense organ in the spider, Cupiennius salei. Receptor currents were generated by deforming the cuticular slits. Conduction velocity in afferent axons from the same organ was also measured by recording action potentials at two locations in the leg during mechanical stimulation of the slits. Activation energies of mechanotransduction and conduction velocity were estimated by making the measurements at a range of temperatures. The mean activation energy for receptor current was 23.1 kcal/mol (96.6 kJ/mol), corresponding to a Q10 value of 3.2. Conduction velocity in the afferent axons was approximately equal to 5 m/s at room temperature and it was much less temperature sensitive, with an activation energy of 6.3 kcal/mol (26.3 kJ/mol), corresponding to a Q10 value of 1.5. These results provide the first direct measurements of the activation energy of mechanically activated currents and support previous suggestions that a high thermal energy barrier is involved in mechanotransduction.     

An axon pacemaker: diversity in the mechanism of generation and conduction of action potentials in snail neurons

One of the main principles in neuroscience is that in vertebrate motoneurons and certain interneurons the decision to initiate an action potential is made at the initial segment of the axon, the axon hillock [Kandel E. R. and Schwartz J. H. (1991) In Principles of Neural Science (eds Kandel E. R., Schwartz J. H. and Jessell T. M. ), pp. 166-167. Elsevier, New York]. The situation in invertebrate motoneurons is different. The axon has many arborizations near its soma, in the nearby neuropil, and many branches in the target region. The action potentials are generated in the region of the axon in the neuropil and in some cases the trigger zone can be found more than 1mm apart from the soma [Tauc C. (1962) Aplysia. J. gen. Physiol. 45, 1077-1097]. Thus, it is obvious that, in a neuron, the removal of the trigger zone would cease the spiking activity in the axon. The purpose of this work is to demonstrate that, in snails, there are axons of certain neurons, like neuron B2, which are able to maintain their firing activity after the removal of their cell body and the so-called trigger zone.