Long-term acrylamide intoxication induces atrophy of dorsal root ganglion A-cells and of myelinated sensory axons

We have examined the effects of acrylamide on primary sensory nerve cell bodies and their myelinated axons in chronic acrylamide intoxication. The numbers and sizes of dorsal root ganglion cell bodies (L5) and myelinated nerve fibers were estimated with sterelogical techniques in severely disabled rats which had been treated with 33.3 mg/kg acrylamide twice a week for 7.5 weeks. There was no loss of dorsal root ganglion cells or myelinated nerve fibers in the roots, the sciatic nerve, sural nerve, and a tibial nerve branch. The mean perikaryal volume of A-cells was reduced by 20% (2P < 0.001) from 50000 m³ in controls (CV = 0.13) to 40000 m³ (0.12), whereas B-cell volume was unchanged. All size-frequency distribution curves of myelinated axon area of peripheral nerves and sensory roots were shifted to the left towards smaller values in rats exposed to acrylamide. In the L5 sensory root 3 mm from the ganglion, there was a significant reduction of mean cross sectional area of myelinated axons by 14% (2P < 0.05) from 7.6 m² (0.11) in controls to 6.5 m² (0.13) in intoxicated rats. The mean cross sectional area of myelinated sural nerve axons was reduced by 22% (2P < 0.001) from 8.6 m² (0.08) in controls to 6.7 m² (0.17) in intoxicated rats. We conclude that chronic intoxication with acrylamide leads to selective atrophy of type A dorsal root ganglion cell bodies and simultaneous atrophy along their peripheral axons, whereas neuronal B-cell bodies and motor axons are spared. It is suggested that the neuronal atrophy might well represent a defect of neurofilament synthesis and transport.     

Co-localization of the β-subtype of protein kinase C and phosphorylation-dependent immunoreactivity of neurofilaments in intact, decentralized and axotomized rat peripheral neurons

Summary The localizations of protein kinase C--immunoreactivity and phosphorylation-dependent immunoreactivity of neurofilaments were compared in rat dorsal root, hypogastric, and superior cervical ganglia. In all the ganglia studied, protein kinase C- and phosphorylation-dependent immunoreactivity of neurofilaments were co-localized in nerve fibres, and no fibres with only protein kinase C--immunoreactivity or phosphorylation-dependent immunoreactivity of neurofilaments were observed. Most intense perikaryal protein kinase c- and phosphorylation-dependent neurofilament-staining were seen in large dorsal root ganglion neurons, whereas in the superior cervical ganglion only very faint protein-kinase C- and no phosphorylation-dependent staining was seen in the neuronal cell bodies. Both decentralization and axotomy of the superior cervical ganglion induced an accumulation of protein-kinase C--immunoreactivity and phosphorylation-dependent immunoreactivity of neurofilaments in the majority of neuronal perikarya. The accumulation was first observed at 1–2 days postoperation and it persisted up to 6–10 days postoperation. In strongly labelled decentralized neuronal perikarya, precipitation of immunoreactivity was seen near the cell and nuclear membranes, whereas in axotomized neurons, immunoreactivity was often concentrated as a unipolar clump in the cytoplasm. The results show that protein kinase C--immunoreactivity and phosphorylation-dependent immunoreactivity of neurofilaments are colocalized in intact rat peripheral ganglia and that both accumulate transiently in cell bodies of the superior cervical ganglion after decentralization and axotomy.     

Ultrastructural synaptology of Clarke's column

Summary An electron microscopic analysis of the synapses in Clarke's column of the cat, using fresh short term degeneration, long term degeneration for detection of the persisting elements, and a newly devised two-step degeneration technique using fresh short term degeneration for further analysis of the persisting synaptic elements in chronic degeneration cases. — Three kinds of synaptic terminals can be identified in the lower part (L₃ segmental level) of Clarke's column: (1) very large (so-called giant) axon terminals with spheric vesicles of primary muscle afferents establishing multiple (climbing-type) contacts mainly with the large dorsal spinocerebellar tract neurons; (2) small bouton-type terminals having spheric synaptic vesicles and originating mainly from spinal interneurons, localized below L₄ in the lumbar enlargement and contacting small distal dendrites and cell bodies of small cells; (3) two types of nerve terminals, originating from local neurons, and characterized by flattened vesicles. One type of (3) contacts mainly the large Clarke neurons directly, whereas the other establishes axo-axonic synapses with the giant terminals (1) of primary muscle affer ents. The possible functional significance of these findings is discussed.     

Plasma membrane structure at the axon hillock, initial segment and cell body of frog dorsal root ganglion cells

Summary Analysis of the plasmalemma of frog dorsal root ganglion cells by freeze-fracture demonstrates regional differences in the distribution of intramembranous particles. Although P-face particles are distributed rather uniformly, the E-face particle concentration at the cell body (300 m^–2) is much lower than that at the axon hillock (900 m^–2), proximal initial segment (1000 m^–2), or intermediate portion of the initial segment (800 m^–2). The particle concentrations in the latter regions approach that at the node of Ranvier and, moreover, particle size analysis reveals that the E-face particles, like those at the node, include a large number that are 10 nm or more in diameter. Thin sections reveal patches of a dense undercoating on the cytoplasmic surface of the axolemma in some regions of the initial segment but not the axon hillock. It is concluded from these results that the axon hillock and the initial segment of dorsal root ganglion cells have some of the structural characteristics of the node of Ranvier.     

An electron microscopic study of the nodose (inferior vagal) ganglion cells in the monkey

Summary The present study described the normal ultrastructure of the monkey nodose ganglion cells. Furthermore, experimental monkeys were subjected to supranodose vagotomy in order to ascertain if the parent cell bodies would undergo degeneration following severance of their central processes. In the normal materials, most of the ganglion cells possessed a single neurite. However, occasional cells bearing more than one process in a sectioned profile were observed. The neurites, ranging between 2–4 m in diameter, displayed a relatively regular contour. Their cytoplasm contained parallel arrays of microtubules, ribosomes, endoplasmic reticulum and slender mitochondria. The electron density of some of these neurites was abnormally high. Embedded in these darkened neurites were a variable number of swollen mitochondria characterized by disrupted cristae. Axon terminals containing round agranular and a few large dense cored vesicles formed synaptic contacts primarily with the neurites of some of the ganglion cells. Three days after supranodose vagotomy, darkened neurites were more commonly observed but their incidence was comparable to that of the normal ganglion in longer survival animals. Another reactive change was the appearance of axon terminals undergoing various degrees of degeneration. There was no evidence of cell death in the duration studied.It was concluded from this study that the occasional darkened neurites from the normal ganglion cells was probably undergoing spontaneous degeneration which appeared to be accentuated when their central process was severed by supranodose vagotomy. The degeneration of axon terminals associated with some of the ganglion cells following the vagotomy suggested that they were derived from vagal descending fibres which were undergoing anterograde degeneration. The presence of synapses on some of the ganglion cells was also discussed and the possibility considered that the latter may represent aberrant or displaced autonomic neurons.     

Lability in the responses of cells in the auditory cortex of squirrel monkeys to species-specific vocalizations

Summary The activity of 28 cells located mainly in the secondary auditory cortex (A II) of awake squirrel-monkeys, was extracellularly recorded for periods of up to 6 h. Seven different species-specific vocalizations, which were repeatedly presented to the monkey, were used as auditory stimuli. Twenty-six cells responded, at least once, to one or more vocalizations; 22 cells revealed some change in their response (pattern or strength) to at least one vocalization (change in response). Twenty-one cells exhibited a change in the number and/or type of vocalization to which they responded during the recording period (change in selectivity). At some time during the recording period all the responding cells exhibited a change in response and/or a change in selectivity (change in responsiveness). A change in response of a cell to a vocalization did not necessarily exclude a change in selectivity, associated with the same vocalization, later in time and vice-versa. A change in responsiveness to one vocalization was not necessarily correlated with changes in responsiveness to other vocalizations.     

Effects of alcohols and volatile anesthetics on S-potential producing cells and on neurons

Summary Comparative studies were carried out regarding the effects of alcohols (ethyl- and methyl-alcohols) and of volatile anesthetics (ethyl-ether, Fluothane and chloroform) on the plasma membrane potential recorded from controller cells (S-potential producing cells, isolated fish retina) and from conductor cells (spike producing neurons, isolated frog dorsal root ganglion). The results clearly demonstrate that alcohols and volatile anesthetics in low concentrations suppress controller cell function, whereas the photoreceptor, conductor cells and synaptic transmission have a great resistance to these agents. The depolarizing component of the C-type S-potentials was selectively depressed by ethanol. A fundamental difference was found between the effects of ethanol and of ether on the controller cell membrane behavior.     

The ansa cervicalis and the infrahyoid muscles of the rat

Summary Perikarya of motoneurons and spinal ganglion cells attributed to infrahyoid muscle nerves of the rat were labelled by retrogradely transported horseradish peroxidase (HRP). For the differentiation of motor and sensory axons cross sections of the nerves were stained for acetylcholinesterase. Numbers and diameter distributions of perikarya and myelinated axons were determined.Motoneuronal perikarya innervating the infrahyoid muscles are located from the transition zone brain stem/spinal cord to the segment C 3. They are found mostly in the medial part of the Rexed laminae VII and VIII at the level of C 1 and C 2 and more ventrolaterally in C 3 and are therefore located to a large extent in areas until now not recognized to contain motoneurons. Our results provide evidence for a somatotopic organization of the motoneurons in the upper cervical spinal cord.The diameter distributions of motoneuronal perikarya and axons are in most cases bimodal, the two modes corresponding to -and -motoneurons. In relation to the diameters of their perikarya -axons are significantly thicker than -axons. In contrast to the motoneurons no clear correlation could be established between the sizes of perikarya of spinal ganglion cells and their peripheral processes.This work was partly supported by the Hartmann Müller-Stiftung Zürich     

Establishment of immortalized Schwann cells from Sandhoff mice and corrective effect of recombinant human β-hexosaminidase A on the accumulated GM2 ganglioside

We have established spontaneously immortalized Schwann cell lines from dorsal root ganglia and peripheral nerves of Sandhoff mice. One of the cell lines exhibited genetically and biochemically distinct features of Sandhoff Schwann cells. The enzyme activities toward 4-methylumbelliferyl N-acetyl--D-glucosamine (-hexosaminidases A, B, and S) and 4-methylumbelliferyl N-acetyl--D-glucosamine-6-sulfate (-hexosaminidases A and S) were decreased, and GM2 ganglioside accumulated in lysosomes of the cells. Incorporation of recombinant human -hexosaminidase isozymes expressed in Chinese hamster ovary cells into the cultured Sandhoff Schwann cells via cation-independent mannose 6-phosphate receptors was found, and the incorporated -hexosaminidase A degraded the accumulated GM2 ganglioside. The established Sandhoff Schwann cell line is useful for investigation and development of therapies for Sandhoff disease.     

Size classes of ganglion cells in the central yellow field of the pigeon retina

Summary Ganglion cells in the central yellow field of the pigeon retina were identified by retrograde transport of horseradish peroxidase from the optic tectum. The soma size range was from 5 to 16 m, with the mean at 7.7 m. At least 85% of the cells in the ganglion cell layer are true orthotopic ganglion cells, tightly packed in a non-random array.     

Immunohistochemical studies on S-100 cells in the anterior pituitary gland of Sprague Dawley rats and spontaneous dwarf rats

The S-100 cells in the pituitary glands of adult male Sprague Dawley rats (SDs) and spontaneous dwarf rats (SDRs) were immunohistochemically examined using anti-S-100 and anti-S-100 monoclonal antibodies. The immunoreactive cells against S-100 protein were divided into three subtypes on the basis of their immunore-activity against subunits of S-100 protein: S-100 dominant type (the -type cell), S-100 dominant type (the \-type cell) and immunoreactive against both S-100 and S-100 (the -type cell). In the SD, -type cells represented 26% of the total S-100 immunoreactive cells (S-100 cells) and were localized in the peripheral area of the ventral region of the pituitary gland. This type of cell was observed forming clusters, with more abundant cytoplasm than the -type cell. The proportion of -type cells was 53%. They were diffusely distributed throughout the gland, and their processes were thicker than those of the -type cell. In the SDR, the proportion of -type cells was 55%, and they were observed throughout the gland. In contrast, -type cells totalled 12% and were localized in small areas of the central and peripheral region of the gland. The proportion of -type cells was 21% in the SD and 33% in the SDR and they were observed forming small clusters in both animal groups. The proportion of -type cells compared with the total of S-100-immunoreactive cells was significantly higher (P < 0.05) in the SDR than in the SD, while the proportion of -type cells was markedly lower (P < 0.05).     

The morphological correlates of X- and Y-like retinal ganglion cells in the retina of monkeys

Summary The morphology of the ganglion cells of the monkey's retina was revealed by filling the cells with horseradish peroxidase from their cut axons in the optic nerve. This procedure gave much more consistent results than the Golgi method, was much quicker and filled dendrites just as extensively. Quantitative measures of the dendritic tree of two types of ganglion cell, P and P, suggest that they correspond to the physiologically defined Y- and X-cells, respectively.Supported by MRC grant G971/397/B     

Fluorescence histochemical and electron-microscopical observations on the innervation of the atrial myocardium of the adult human heart

Summary The existence of both adrenergic and cholinergic innervation of the atrial myocardium of the adult human heart was demonstrated by means of fluorescence induced by formaldehyde or glyoxylic acid and by electron microscopy.The adrenergic fluorescing axons (1) followed the course of blood vessels as typical perivascular nerve plexuses, and (2) formed a three-dimensional fairly dense nerve net obviously not related to the blood vessels. The varicosities frequently came into close apposition on myocardial cells.Several types of nerve terminals were differentiated at electron microscopy: (1) an adrenergic type containing small (diameter 450–700 Å) dense-cored vesicles and usually (in various proportions) small empty and/or large (900–1500 Å) dense-cored vesicles, (2) a cholinergic type containing small (ca. 500 Å) empty vesicles and occasionally also some large (mean diameter ca. 1200 Å) dense-cored vesicles, (3) a pale type containing only a few or no vesicles, (4) a disintegrated type containing degenerated mitochondria, autophagic vacuoles, and occasional normal-looking mitochondria, (5) nerve terminals containing a large number of mitochondria in addition to varying vesicle populations, and (6) a (possibly baroreceptive type of) nerve terminal containing myelinlike lamellated structures. The disintegrated and the pale types of nerve terminals possibly represent different stages of axonal degeneration, or may correspond to diminution in the transmitter substance concentration under certain pathophysiologic conditions, respectively. Nerve terminals crowded with mitochondria may be sensory and involved in mechano-or chemoreceptive functions.In preliminary experiments convincing evidence was obtained that the glyoxylic acid-induced fluorescence histochemical method will be suitable for comparative studies on (human) clinical specimens, e.g., for analyzing the degree of the functional activity of the intrinsic adrenergic innervation of the myocardium under various pathophysiologic conditions. The modification which appeared most appropriate for such studies is described in detail, and is proposed for use as a standard method in other similar or related studies on human clinical series. The essential criteria for analyzing the specimens at fluorescence microscopy are suggested as well.     

Localization of tissue transglutaminase and N (epsilon)-(gamma) -glutamyl lysine in duodenal cucosa during the development of mucosal atrophy in coeliac disease

Expression and transamidation activity of tissue transglutaminase (tTG) may be involved in the morphological modifications leading to the mucosal atrophy observed in coeliac disease (CD). We aimed to investigate the localization of tTG within the duodenal mucosa during the development of villous atrophy. The localization and level of expression of N-(-glutamyl) lysine isopeptides which could reflect the transamidation activity of tTG were also analyzed. tTG and N-(-glutamyl) lysine were localized using an immunohistochemical technique on duodenal biopsies obtained from 75 patients with CD and 51 subjects with normal mucosa (control group). The number of cases displaying tTG-expressing cells in the basement membrane and lamina propria was significantly higher in CD patients than in the control group. Moreover, the intensity of tTG staining in these areas was higher in CD. In contrast, the number of biopsies with tTG-expressing enterocytes was significantly lower in CD than in the control group. There was no difference in N-(-glutamyl) lysine between the two populations. Tissue transglutaminase was differently expressed in the various areas of the mucosa according to the stage of atrophy, whereas the localization and the intensity of the labelling of N-(-glutamyl) lysine isopeptides did not show any modification. The preferential localization in the basement membrane and lamina propria may reflect the involvement of tTG in the development of mucosal atrophy in CD.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Co-induction of neuronal interferon-gamma and nitric oxide synthase in rat motor neurons after axotomy: a role in nerve repair or death?

Summary Induction of an interferon-gamma-like molecule, previously isolated from neurons (N-IFN-), and of the neuronal isoform I of the synthetic enzyme of the free radical nitric oxide, nitric oxide synthase I, as well as of NADPH-diaphorase, were examined in axotomized dorsal motor vagal and hypoglossal neurons. Unilateral transection of the vagal and hypoglossal nerves was performed in the same rat and an induction of N-IFN- and nitric oxide synthase I immunostaining as well as NADPH-diaphorase histochemical positivity was observed in the ipsilateral motoneurons after 2–4 days. The immuno- and enzyme- histochemical positivities were much stronger in the dorsal motor vagal neurons than in hypoglossal neurons. Two and 4 weeks after axotomy N-IFN- immunoreactivity and NADPH-diaphorase positivity persisted in the former, but started to decrease in the latter neurons. Previous data have shown that 23 weeks after nerve transection the majority of the dorsal motor vagal neurons are lost, while the majority of the hypoglossal neurons survive. The high and persistent expression of N-IFN- and nitric oxide synthase I after axotomy in the dorsal motor vagal neurons, that are largely destined to die, indicates that the co-induction of these two molecules may be implicated in the pathogenesis of neuronal degeneration.     

Postsynaptic specializations at excitatory and inhibitory cholinergic synapses

Summary In serial sections of neurons in the paravertebral ganglia of the frog (Limnodynastes dumerili), the postsynaptic structures termed postsynaptic bar (PSB) and junctional subsurface organ (JSO) were never observed in the same ganglion cell. Further, PSBs were found mostly in small ganglion cells (less than 22 m), while JSOs were found mostly in large ganglion cells (up to 45 m). Between 10 and 22 PSBs were located at both spine and non-spinous somatic synapses of the smaller ganglion cells; while 8 to 16 JSOs were located largely in the axon hillock region of the larger ganglion cells.Based on these observations, it is suggested that the two ganglion cell populations represent the B and C cell types defined according to electrophysiological data. Further, since the nerve terminals adjacent to both these postsynaptic structures appear to be cholinergic according to their vesicular content, this provides some basis for suggesting that JSOs are associated with slow excitatory synapses, while PSBs are present at slow inhibitory synapses.     

Firing characteristics of vestibular nuclei neurons in the alert monkey after bilateral vestibular neurectomy

Summary After destruction of the peripheral vestibular system which is not activated by moving large-field visual stimulation, not only labyrinthine-ocular reflexes but also optokinetic-ocular responses related to the velocity storage mechanism are abolished. In the normal monkey optokinetic-ocular responses are reflected in sustained activity changes of central vestibular neurons within the vestibular nuclei. To account for the loss of optokinetic responses after labyrinthectomy, inactivation of central vestibular neurons consequent on the loss of primary vestibular activity is assumed to be of major importance. To test this hypothesis we recorded the neural activity within the vestibular nuclear complex in two chronically prepared Rhesus monkeys during a period from one up to 9 and 12 months after both vestibular nerves had been cut. The discharge characteristics of 829 cells were studied in relation to eye fixation, and to a moving small and large (optokinetic) visual stimulus producing smooth pursuit (SP) eye movements and optokinetic nystagmus (OKN). Units were grouped into different subclasses.After chronic bilateral vestibular neurectomy (BVN) we have found: (1) a rich variety of spontaneously active cells within the vestibular nuclear complex, which — as far as comparison before and after BVN is possible — belong to all subclasses of neurons functionally defined in normal monkey; and (2) no sustained activity changes which are related to the activation of the velocity storage mechanism; this is especially true for pure-vestibular, vestibular-pause and tonic-vestibular-pause cells in normal monkey which show a pure, pause and tonic-pause firing pattern after BVN. Neurons which are modulated by eye position are, however, modulated with the velocity of slow eye movements with comparable sensitivity during SP and OKN. Retinal slip is extremely rarely encoded. The results of the present study do not directly answer the question why the velocity storage mechanism is abolished after BVN but they suggest that only a small number of central vestibular cells may be inactivated by neurectomy.Supported by SNF grant no. 3.510-0.86