Structural changes in locust leg muscle fibres in response to tenotomy and joint immobilization

Tenotomy and immobilization at minimum physiological length resulted in degenerative changes in the ultrastructure of the locust metathoracic flexor tibiae muscle.Degeneration was observed in some muscle fibres 9 days after tenolomy and was progressive, affecting an increasing proportion of fibres at longer times after operation. Changes included disruption and loss of myofilaments, Z-disk streaming, degeneration of sarcoplasmic reticulum, formation of large membranous complexes and changes in number and appearance of mitochondria. Different muscle fibres followed different degenerative pathways. By 73 days very few intact muscle fibres remained.Joint immobilization resulted in similar but much less severe progressive degeneration. Degenerate fibres were largely localized in the distal and medial parts of the muscle. Both immobilized and tenotomized muscles were invaded by large numbers of fat body cells in the later stages of degeneration.Nerve terminals on degenerate tenotomized and immobilized muscle fibres were ultrastructurally normal. Tendinous cells attaching degenerate muscle fibres to the cuticle themselves degenerated.     

Selective sympathetic denervation induced by 6-hydroxy-dopamine in the small intestine.

The structure of the nerve fibres in the intact and neurally isolated intestine of the cat was studied after 6-OHDA treatment by fluorescence and electron microscopy. A gradual disappearance of green fluorescence in the monoaminergic fibers was observed. Degeneration of nerve fibres containing 30--60 nm agranular and 30--60 nm densecore (granular) vesicles could be seen in all layers of the small intestine. Sporadically, degeneration of the presynaptic elements occurred also in the synapses of myenteric ganglia. A large number of degenerating fibres could be observed in close relation to blood vessels. These were thought to be monoaminergic because 6-OHDA selectively affected their terminals. In the chronically isolated small intestine degenerated nerve processes could not be observed following 6-OHDA treatment. It is concluded that the intrinsic nerve elements do not contain monoamines. Accordingly, the previously observed yellow fluorescence of the nerve elements of the isolated small intestine might be due to some other kind of neurotransmitter, possibly tryptamine.     

Scanning electron microscopic observations of the ependymal cell and the subependymal layer of the third brain ventricle in the rat

This investigation was undertaken to clarify the three dimensional ultrastructure of the subependymal layer in relation with the ependymal cell layer in rat brain using the scanning electron microscope (SEM). The subependymal layer existing below the ependyma of the third ventricle in the brain of mature albino rats was examined with S E M. The hypothalamus freshly excised after median sagittal section was treated by collagenase with or without trypsin for a short while to remove the ependymal cells at the ventricular wall. After the enzymatic pretreatment of the specimen, many ependymal cells were removed and the subependymal layer was partially exposed. Most of the ciliated ependymal cells remaining at the ventricular wall extended long, single basal processes which then penetrated into the subependymal layer. The subependymal layer was composed of a delicate framework of thin processes of glial cells, ependymal cells and, in addition nerve cells. Scattered among the neuropil just beneath the ependymal cell layer, there were relatively small, globular subependymal cells. Occasionally, there were large bundles of unmyelinated nerve fibres in the subependymal layer. The individual nerve fibres distinctly showed many axonal varicosities within the fibres. Intermingled with the nerve fibres, glial processes of various forms were present. The structure of the ependymal cells and the subependymal layer was compared with the findings already reported in the studies using light and transmission electron microscope.     

The laminar organization of optic nerve fibres in the tectum of goldfish.

Potentials in the tectum of large (12--20 cm) goldfish, evoked by stimulation of the optic nerve, were recorded extracellularly with double-barrelled electrodes (d.c., saline and a.c., Woods metal--Pt). Four fibre groups (E, M1, M2, M3) were recorded at latencies of approximately 2, 3, 5 and 8 ms after stimulation (conduction velocities of approximately 7, 5, 3 and 2 m/s). The same four groups were recorded from the optic nerve when the tectum was stimulated. The fastest fibre groups (E) did not give rise to a postsynaptic wave. Fibre groups M1, M2 and M3 gave rise to postsynaptic potentials which, following computation of their second spatial derivatives with depth, were found to have current sinks at depths of approximately 100-150 micrometers, 150--200 micrometers and 250--350 micrometers respectively. Thus the fastest conducting retinotectal fibres make their synapses most superficially, the opposite of the arrangement in the frog tectum. These postsynaptic waves fatigued at repetitive stimulus rates of 20--50 per second, and in twin pulses at interstimulus intervals of 10--15 ms; and they were reversibly blocked by topical application of pentobarbitol. The fibre potentials, however, were virtually undecremented under these conditions. To compare these electrophysiological findings with the anatomy, the cobalt procedure was used to visualize the profiles of the optic fibres in the various tectal laminae. A thick dense projection filled the superficial grey and white (s.g.w.) layer, and there was a thin satellite band just superficial to it. In addition, there were two deeper bands of sparse innervation, in the middle of the central grey zone (c.g.) and in the deep white (d.w.) layer. These bands were associated with the field potential sinks through lesions made with recording electrodes. The two deep bands correspond to the M3 fibre group. The dense s.g.w. innervation contains both the M1 and M2 fibre groups, the M1 just superficial to the M2. The fastest fibre group, E, which had no postsynaptic wave associated with it, persisted at least six weeks after retinal removal, and probably represents efferent cells with fibres projecting back through the optic nerve to the retina. Filled cell profiles could not be positively identified with the cobalt technique, but could be seen with the HRP technique, when the optic afferents were first allowed to degenerate. The filled cells were the pyramidals of the s.g.w. layer.     

The intrinsic, association and commissural connections of area 17 on the visual cortex.

An experimental neurohistological study has been made of the intrinsic connections of the cortex of area 17 of the monkey, of the commissural connections of the visual cortex of the cat and monkey and of the association fibres passing into area 17 of the cat. In light microscopic studies the axonal degeneration method of Nauta has been used, and the site and mode of termination of the degenerating fibres has also been determined with the electron microscope...     

Some Golgi data on visual cortex of the rhesus monkey.

The rhesus monkey's visual cortex was studied on Golgi material. The terminal arborization of the geniculate fibres and non-specific vertical fibres have been analysed. The interneurons (intrinsic neurons) of the area described in detail and classified on the basis of their axonal and dendrite arborizations. The stellate neurons in layer IV are discussed.     

Cdk5 is required for multipolar-to-bipolar transition during radial neuronal migration and proper dendrite development of pyramidal neurons in the cerebral cortex

The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When we introduced GFP into migrating neurons at E14.5 by in utero electroporation, we observed migrating neurons in wild-type but not in Cdk5(-/-) embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons.     

Ultrastructural characteristics of the 1st cortical layer of the cingulate area of the brain in rats

The limiting membrane, participating in formation of the liquor- and hematoencephalic barrier on the brain surface, and specialized intercellular contacts are considered as factors regulating entrance of substances from liquor and blood into the intercellular medium. Heteromorphism of the cingulate cortex first layer takes place: in the 1-b sublayer a large amount of myelinated fibres is revealed, in the 1-c sublayer numerous longitudinal sections of dendrites are seen, they arrange perpendicularly or tangentially to the brain surface. In the 1-a sublayer, more often than in others, multifocal axodendritic synapses are observed they are of convergent or divergent types, several synapses occur. In the first layer of the cingulate cortex, axonal profiles are revealed, they resemble axons of the neurosecretory cells described in the literature. This cannot exclude any hormonal mechanisms affecting the neurons of the cingulate cortex. Numerous synaptic contacts, formed by afferent fibers that come from different brain structures, with processes of the first layer cells and ascending dendrites of deeper layers are considered as a morphological substrate of integrative and modulating activity of the first layer.     

Ultrastructural transformation of fast chicken muscle fibres induced by nerve cross-union

Summary The fast posterior latissimus dorsi (PLD) muscle of newly hatched chickens was transposed and cross-innervated by the slow-type nerve originally innervating the anterior latissimus dorsi (ALD) muscle. The innervation and the ultrastructure of the cross-innervated posterior latissimus dorsi (PLD-X) muscle was investigated from one week up to 18 months after the operation and compared with that of the control fast (PLD-C) and control slow (ALD-C) muscles. All nerve terminals in the PLD-X muscle were of the slow type. Yet the degree of ultrastructural transformation differed from fibre to fibre. Only about 30% of PLD-X fibres had transformed ultrastructure closely resembling the control slow fibres. In this group of maximally altered fibres, the myofibrils had large diameters, wide Z lines and indistinct M lines as the control slow fibres. The amount of mitochondria was increased to levels found in control slow fibres. The mean percentage of triads was also comparable to that of control slow fibres, being approximately by two thirds lower than in control fast fibres.The differences in the degree of ultrastructural transformation are presumably due to different plasticity of muscle cells at the time of cross-innervation. In the transposed PLD-X muscles large areas undergo degeneration and regeneration. It is suggested that an almost complete changeover of the fibre type is only brought about after cross-innervation of newly differentiating muscle cells, whereas partial alteration occurs after reinnervation of young myofibres.The skillful technical assistance of Dr. Z. Liková, Mrs. M. Sobotková, Ing. M. Doubek and Mr. H. Kunz is gratefully acknowledged.     

Local anaesthetic block protects against electrically-induced damage in peripheral nerve

This study is one of a series addressing the mechanisms involved in the production of neural damage caused by continuous, prolonged electrical stimulation of peripheral nerve. It has been previously shown that sustained, high frequency electrical stimulation of the cat's peroneal nerve may cause irreversible neural damage in the form of axonal degeneration of the large myelinated fibres. In this study we demonstrate that blocking the action potentials on most of the nerve fibres with local anaesthetics (10% procaine or 2% lidocaine) almost completely prevents the axonal degeneration. The abolition of axonal injury by local anaesthetic block strongly suggests that the electrically-induced damage is due to prolonged electrical excitation of axons. Furthermore, since less than complete suppression of the induced neural activity by local anaesthetic engenders essentially complete sparing of all axons, our results suggest that the damage to individual axons derives, at least in part, from stimulation-induced global changes in the nerve.     

Afferent and efferent connections of lateral geniculate body pars ventralis in albino rats

The afferent and efferent connections of the Corpus geniculatum laterale, pars ventralis (Cglv) of the albino rat were investigated lightmicroscopically by using either silver degeneration methods and the HRP-method as well. In contrast to the dorsal Cgl the results show remarkably different afferent connections in the ventral Cgl. The afferent fibres originate from the following areas: a) Retina: the terminal degeneration area of optic fibres includes the lateral part of the Cglv only. b) Regio praetectalis: degenerating fibres from these region can also be observed in the medial part of the Cglv. c) Colliculus superior: degenerating fibres terminate mainly in the lateral part of the Cglv. In the superior colliculus these fibres originate particularly from the cells of lamina III. d) Visual cortex: neurons of layer V of area 17 project mainly to the lateral half of the Cglv. There was no evidence for a projecting of the parastriate cortex (area 18 a) to the Cglv. The efferent fibres reach the following target areas: a) Nucleus lateralis posterior, Regio praetectalis and Colliculus superior. Evidence for a projection to the dorsal Cgl requires further investigation. b) Zona incerta, Formatio reticularis mesencephali and Nucleus medialis et lateralis pontis. Neurons in the medial half ot eh Cglv project to the pons region. c) Crossing the Commissura posterior and the Commissura suprachiasmatica, efferent fibres reach the contralateral Cglv, the Regio praetectalis and the Colliculus superior. The results obtained from the rat are compared with findings from other mammalian species. The functional importance of the Cglv in the visual processes is discussed taking into consideration the specific connections.     

The effect of destroying the whisker follicles in mice on the sensory nerve, the thalamocortical radiation and cortical barrel development.

Electrolytic destruction of whisker follicles in mice on the day of birth has been found to cause degeneration in the sensory nerve fibres supplying the follicles. The severity of the degeneration has been assessed in animals between 2 and 20 days old by counting the total number of myelinated fibres in the maxillary nerves on both normal and lesioned sides. The degeneration is apparent after 2 days and by 20 days the nerve on the lesioned side contains only 38% of the normal fibre content. This degeneration has also been shown to involve the trigeminal root, central to the ganglion. In addition, the lesioning procedure modifies the terminations of thalamocortical fibres in the barrel region of the sensory cortex. These terminations are normally in clusters, each corresponding to a barrel, but, after lesioning the follicles, the terminals appear to be evenly distributed in layer IV and cortical barrel structures no longer develop. In postnatal mice, electrolytic destruction of whisker follicles had less effect upon maxillary nerve fibres and cortical barrels. The number of myelinated axons surviving until day 20 increased progressively with later lesioning to reach nearly 80% of the control level when lesions were made on day 10. Cortical barrels became secure earlier than the maxillary nerve, for a normal number of cortical barrels was present at day 12 when follicles were destroyed on day 4. The implications of these results for the formation of cortical barrels is discussed.     

Retrograde degeneration of the neurons of the cerebellar nuclei after partial removal of the associative cerebral cortex

Retrograde degeneration of the cerebellar nuclei cells has been studied after partial ablation of the associative parietal cerebral cortex in the cat. The material is stained after Nissl. Retrogradely degenerated and normal cells are counted. The "ghost-cells" in the cerebellar nuclei indicate that a direct axonal connection exists between some neurons and the cerebral cortex operated, while the cells that are at other stages of degeneration are, perhaps, connected with this part of the cortex by means of axonal collaterals.     

Distribution of afferents from the association nuclei of the thalamus in the projection and association cortex in cats

Patterns of distribution of terminal degeneration in the parietal cortex (field 7) and in the occipital cortex (field 17) were studied after ultrasonic destruction of the pulvinar by the Fink-Heimer and electron microscopy methods. Degenerating fibers and their terminals were observed in the parietal cortex within all the layers; the greatest amount of degeneration was found in the III--V layers. In the occipital cortex the fibers from the pulvinar end predominantly in the IV layer. Degenerating axons end on the dendritic spines and thin dendritic branches both in the parietal and occipital cortex.     

Effects of frontal motor cortex ablation on the ultrastructure of cat substantia nigra

3,4 and 5 days after the removal by suction of the left motor and premotor cortex in cats, the presence of ultrastructural changes in the substantia nigra was investigated. Whereas after 3 days dark bouton degeneration was rare, after 4- and 5-day survival it was regularly found for a distinct type of synapse containing in its bouton densely packed small round vesicles and possessing an asymmetric synaptic junction with a dendrite. Often these darkly degenerated boutons contained dense bodies which were also observed in the same type of synapse not yet exhibiting a dark axoplasm. Various inclusions, especially glycogen depots, were present in these boutons suggesting that they were in the process of degeneration. The glial reaction was comparatively severe. In addition, darkly shrunken dendrites contacted both, by intact and by altered boutons were frequently encountered as well as single degenerated neuronal perikarya. The nature of this effect, i.e. whether transneuronal or retrograde, could not be clarified. All these alterations were found bilaterally after the unilateral cortex ablation, and were confined to the substantia nigra pars compacta along its whole anterior-posterior extent. In the pars reticulata, solely traversing myelinated axons in the process of degeneration were observed. Thus, the results are in agreement with the older studies and with evidence from primates demonstrating that the substantia nigra receives a bilateral projection from the motor and premotor cortex.     

Experience with the use of horseradish peroxidase for light and electron microscope studies of interneuronal connections]

By the method based on a retrograde axonal transport of exogenous horseradish peroxidase (HRP), the origins of afferentation of the motor cortex of adult cats, kittens and albino rats were studied. HRP-positive neurons were found by light and electron microscopy in the somatosensory cortex (C1) of the ipsilateral hemisphere and in the portions of the cortex of the contralateral hemisphere which were symmetrical to the site of injection of HRP. The disposition of neurons, marked by HRP, in the Vth layer of the motor cortex suggest that these neurons may send their axons into the bundles of comissural fibres going to the motor cortex of the opposite hemisphere. This method considerably expands possibilities of revealing the origins of afferentation of the investigated portion of the nervous system and allows more complete and reliable investigation of interneuronal connections.     

Myofibril breakdown during spreading damage. II. A calcium-free medium]

Peculiarities of Zenker's degeneration (ZD) have been investigated in fast muscle fibres of the frog incubated in a Ringer solution free of Ca++ (R--Ca) with a normal or increased (by 100 mM) concentration of KCl. ZD in these solutions is distinguished by a 10--90 minutes delay of the appearance of the primary contraction knot and cessation of ZD development in the majority of fibres after formation of several (1--5) contraction knots. In the presence of 0.5 mM EDTA in R--Ca, after a few typical contraction knots are formed, fibres commonly fall into large fragments that retain cross-striation. Contracted or super-contracted state of sarcomeres in detached contraction knots and at the necrosis boundary, as well as an increasing lysis of contactile material and proliferation of fibre membrane structures in the region of ZD arrested boundary, are characteristic of ultrastructural changes during ZD in calcium-free solutions.     

Identification of early glial elements as the precursors of Bergmann-glia: a Golgi-analysis of the developing rat cerebellar cortex.

The developing rat cerebellar cortex was studied by the rapid Golgi procedure in 200 mu thick slices and in 1--2 mu thick semithin sections poststained with toluidine-blue. Glial cells having radial fibres directed towards the pial surface were found to be present continuously in the internal granular layer during cerebellar maturation. This cell type was identified as the developing Bergmann-glia.     

On the axonal migration of catecholamines in constricted sciatic nerve of the rat

Summary The radioautographic technique has been used to study the axonal migration of catecholamines in sympathetic fibres of the sciatic nerve of rats after ligature. A first series of experiments aimed at ascertaining the capacity of the proximal portion of adrenergic fibres to take up and store exogenous tritiated catecholamines (³H-DOPA; ³H-DA and ³H-NA) 3 to 22 hours after ligation. The results are qualitatively similar in rats pretreated and non-pretreated with IMAO, but the intensity of the radioautographic reaction is lower in the latter cases. Most of the labeled axons are filled mainly with vesicular and tubular profiles of endoplasmic reticular origin, large dense bodies (probably lysosomes) and/or mitochondria. The silver grains are generally superimposed on the vesicular and/or the tubular profiles, but in some cases on the large dense bodies, suggesting that exogenous catecholamines can be stored in lysosomes. The question whether SGV specifically store catecholamines also in the modified adrenergic fibres has been investigated in KMnO₄ fixed material. These results show that beside a large number of fibres in which there is a strict correlation between labeling and SGV, some fibres containing SGV do not retain the ³H-NA. Conversely some fibres which contain mainly agranular vesicles display radioautographic reaction. Therefore, in case of ligated fibres, SGV cannot be considered the specific organelles for storage of catecholamines.The axonal migration of labeled catecholamines has been studied in animals pretreated with IMAO. A moderate, but selective, labeling is present in the proximal portion of sciatic fibres of rats in which administration of labeled catecholamine preceeded of 2 hours the ligature and this was performed 22 hours before fixation.From these combined types of experiments, it is concluded that despite the presence of all the structures necessary for the storage of a high amount of catecholamines in the modified adrenergic fibres, only a small fraction of catecholamines accumulated above the ligature has been transported by axonal migration. Therefore, the axonal migration of catecholamines appears as an epiphenomenon related to the distal migration of enzymatic and storage proteins from the perikaryon.Part of this work was presented to the 84th Annual Session of American Association of Anatomists (Sotelo and Taxi, 1971).Some of the electron microscopic observations were made at the Unité 106 I.N.S.E.R.M.     

An improved cobalt labeling technique with complex compounds.

Results of axonal labelings with CoCl2, cobaltous lysine and cobaltic lysine complexes are compared on dorsal roots and nerves of the spinal cord and brain stem in the living frog. The most satisfactory staining of fibres and terminals is given by CoCl2; its application however, is limited by its rather short (6--10 mm) axonal transport. Cobaltous lysine is transported somewhat better, but it gives a poor fibre staining in the spinal cord. The axonal transport of cobaltic lysine is the best, covering a distance of 40--50 mm. Combination of cobaltic lysine with 2--5% dimethyl sulphoxide greatly enhances the axonal uptake of cobalt and extends the distance of transport to 70--80 mm. It is assumed that better transport of cobalt complexes is achieved by their less toxic effect on the nerve cell.