[ULTRASTRUCTURE OF MOTOR NEURONS AND SYNAPSES IN THE OCULOMOTOR NERVE NUCLEI IN MICE].

Motor neuron and synapse ultrastructure in the somatic area of oculomotor nerve nuclei (cranial nerve III nuclei) was studied in C57 Black/6 mice. It was shown that that axodendritic and axosomatic synapses were characterized by rounded subjunctional bodies, located at the postsynaptic side of the synaptic contact at some distance from postsynaptic density. At the site of the subjunctional densities, the synaptic gap was expanded to 30 µm. In the same synapse, the synaptic gap could be reduced twice or more. Axosomatic and axodendritic synapses occured on spines. On the soma and dendrites of motoneurons, en passant type of synapses were found. No gap junctions were observed.

Ultrastructural changes in myotendinous nerve endings induced by injection of botulinum toxin into the extraocular muscle.

BACKGROUND: The myotendinous nerve endings located in the extraocular muscles are considered as proprioceptors. The proprioception of extraocular muscles can be altered if botuminium toxin, which is widely used for the treatment of strabismus, damages the endings. The purpose of this study is to investigate the ultrastructural changes in myotendinous nerve endings after injection of botulinum toxin into the extraocular muscles. METHODS: Under general anesthesia, 5 IU of botulinum toxin (0.1 ml) were injected into the superior and medial rectus muscles of one eye in each of 12 cats, and 0.1 ml of normal saline was injected into the muscles of the other eye. The myotendinous junction was harvested in four cats each at 1, 4, and 12 weeks after injection and examined using electron microscopy. The myotendinous junctions of four normal uninjected cats were also examined as a control group. RESULTS: There were no morphological differences between the control group and saline injection group. In the botox injection group, separation of the myelin sheath and an increase in neurofilaments in axons of myelinated and unmyelinated nerve fibers were observed 1 week after injection and persisted until 12 weeks. Around the terminal nerve endings, incomplete Schwann cells with axonal exposure were observed, and fibroblast-like changes in Schwann cells were detected. These findings were not observed in the control group. CONCLUSIONS: Injection of botulinum toxin into the extraocular muscle induced ultrastructural changes in the myotendinous nerve endings. The possibility that those changes might diminish the proprioceptive abilities of the extraocular muscle should be considered when botulinum toxin is used for the treatment of strabismus.

Ultrastructural changes of myotendinous nerve endings following recession or resection procedures of extraocular muscle surgeries in cats.

To verify the postoperative ultrastructural changes of the myotendinous nerve endings of feline extraocular muscles, which are known as proprioceptors. Sixteen recti of four cats were used and divided into three groups. In group A, eight lateral recti were recessed. In group B, four medial recti were resected by 10 mm from insertion to include the myotendinous junction. In group C, four medial recti were resected by 4 mm of muscle bellies only, without disturbing the myotendinous junction. Four weeks after surgery, specimens were examined with electron microscopy. In group A, overall neural structures were well maintained with slight axonal degeneration. In group B, only muscle fibers were observed without any regeneration of neural sprouts. In group C, axonal disintegration and shrinkage were evident. These results indicate that myotendinous nerve endings can be damaged in strabismus surgery, and that resection was more invasive than recession in disrupting myotendinous nerve endings.

Axon-myelin relationships in rat cranial nerves III, IV, and VI: a morphometric study of large- and small-fibre classes.

The primary objectives of this study were to determine (1) if quantitative axon-myelin relationships are similar for large- and for small-fibre classes within individual nerves and (2) if the same axon-myelin relationships hold for equivalent fibre classes in closely similar nerves. The oculomotor, trochlear, and abducent nerves of the rat were examined since they each contain distinct large- and small-fibre classes and are similar in a wide range of anatomical and developmental respects. Accordingly, morphometric analyses of axon-myelin relationships were performed separately on large and small fibres of each of the three nerves. Within each nerve, the setting of the relationship between the two parameters was found to be different for the two fibre classes: Scatterplots relating sheath thickness to axon perimeter for large fibres were shifted upwards relative to those for small fibres. These differences were also reflected in the positions of the regression lines fitted to the plots and in the g-ratios. Significant differences were found between nerves in relation to their large fibres: Those of the abducent nerve had significantly thicker sheaths, those of the oculomotor nerve had significantly smaller axon perimeters, and the myelin sheath-axon perimeter relationship of the abducent nerve differed significantly from that of the other two. This study therefore shows that morphometric axon-myelin relationships may differ significantly between equivalent fibre classes of nerves that are closely similar in respect of morphological class, central origin, peripheral distribution, developmental environment, and function.

Anatomical and physiological characteristics of vestibular neurons mediating the vertical vestibulo-ocular reflexes of the squirrel monkey.

The morphology of 35 vestibular neurons whose firing rate was related to vertical eye movements was studied by injection of horseradish peroxidase intracellularly into physiologically identified vestibular axons in alert squirrel monkeys. The intracellularly injected cells were readily classified into four main groups. One group of cells, down position-vestibular-pause neurons (down PVPs; N = 12), increased their firing rate during downward eye positions, paused during saccades, and were located in the medial vestibular nucleus (MV) and the adjacent ventrolateral vestibular nucleus (VLV). They had axons that crossed the midline and ascended in the medial longitudinal fasciculus (MLF) to terminate in the trochlear nucleus, the lateral aspect of the caudal oculomotor nucleus, and the dorsal aspect of the rostral oculomotor nucleus. A second group of cells (N = 15) were also located in the MV and VLV, but increased their firing rate during upward eye positions, and paused during saccades. These cells had axons that crossed the midline and ascended in the contralateral MLF to terminate in the medial aspect of the oculomotor nucleus. A third group of cells (N = 4) were located in the superior vestibular nucleus, generated bursts of spikes during upward saccades, and increased their tonic firing rate during upward eye positions. These cells had axons that ascended laterally to the ipsilateral MLF to terminate in regions of the trochlear and oculomotor nuclei similar to those in which down PVPs terminated. A fourth group of cells (N = 4), located in the VLV, had axons that projected to the spinal cord, although they had firing rates that were significantly correlated with vertical eye position. Electrical stimulation of the vestibular nerve evoked spikes at monosynaptic latencies in each of the above classes of cells, six of which were injected with horseradish peroxidase. Each group of cells had collateral projections to other areas of the brainstem. Some of the neurons that projected to the contralateral trochlear and oculomotor nuclei had collaterals that crossed the midline to terminate in the oculomotor nucleus ipsilateral to the soma, and some gave rise to small collaterals that terminated in the abducens nucleus. Other areas of the brainstem that received collateral inputs from neurons projecting to oculomotor and trochlear nuclei included the interstitial nucleus of Cajal, the caudal part of the dorsal raphe nucleus, the nucleus raphe obscurus, Roller's nucleus, the intermediate and caudal interstitial nuclei of the MLF, and the nucleus prepositus.

Morphology and distribution of serotoninergic and oculomotor internuclear neurons in the cat midbrain.

Serotoninergic fibers have been reported in both the abducens and facial nuclei of the cat. Furthermore, serotoninergic dorsal raphe and oculomotor internuclear neurons occupy similar locations in the periaqueductal gray overlying the oculomotor and trochlear motor nuclei. To resolve the issue of whether these two populations of neurons overlap, serotoninergic fibers were assayed in the abducens and facial nucleus; then the morphologies and distributions of identified serotoninergic neurons and oculomotor internuclear neurons were determined. Both the abducens and facial nuclei contained varicosities labelled with antibody to serotonin, but a much higher density of immunoreactive fibers was present in the latter, especially in its medial aspect. Distinct synaptic profiles labelled with antibodies to serotonin were observed in both nuclei. In both cases, terminal profiles contained numerous small, predominantly spheroidal, synaptic vesicles as well as a few, large, dense-core vesicles. These profiles made synaptic contacts onto dendritic and, in the facial nucleus, somatic profiles that occasionally displayed asymmetric, postsynaptic, membrane densifications. Following injection of horseradish peroxidase into either the abducens or facial nuclei, double-label immunohistochemical techniques demonstrated that the serotoninergic and oculomotor internuclear neurons form two distinct cell populations. The immunoreactive serotoninergic cells were distributed within the dorsal raphe nucleus, predominantly caudal to the retrogradely labelled oculomotor internuclear neurons. The latter were located in the oculomotor nucleus along its dorsal border and in the adjacent supraoculomotor area. Intracellular injection of horseradish peroxidase revealed that oculomotor internuclear neurons have multipolar somata with up to ten long, tapering dendrites that bifurcate approximately five times. Their dendritic fields were generally contained within the nucleus and adjacent supraoculomotor area. In contrast, putative serotoninergic neurons were often spindle-shaped and exhibited far fewer primary dendrites. Many of these long, narrow, sparsely branched dendrites crossed the midline and extended to the surface of the cerebral aqueduct. In the vicinity of the aqueduct they branched repeatedly to form a dendritic thicket. The axons of the intracellularly stained serotoninergic neurons emerged either from the somata or the end of a process with dendritic morphology, and in some cases they produced axon collaterals within the periaqueductal gray. Thus the oculomotor internuclear and serotoninergic populations differ in both distribution and morphology.(ABSTRACT TRUNCATED AT 400 WORDS)

Abducens internuclear and ascending tract of deiters inputs to medial rectus motoneurons in the cat oculomotor nucleus: synaptic organization.

Abducens internuclear and ascending tract of Deiters (ATD) inputs to medial rectus motoneurons in the oculomotor nucleus are important for conjugate horizontal movements. In the present study, the organization of these separate populations of neurons and their synaptic connections with medial rectus motoneurons in the cat oculomotor nucleus have been examined by light and electron microscopy by using retrograde and anterograde axonal tracers. Consistent with the patterns of retrograde horseradish peroxidase labeling, the abducens internuclear projection is predominantly, if not exclusively, contralateral, whereas the ATD projection is exclusively ipsilateral, as demonstrated by anterograde autoradiographic and biocytin labeling. Both populations of synaptic endings contain spheroidal synaptic vesicles and establish synaptic contacts with modest postsynaptic densifications. In addition, ATD synaptic endings frequently are associated with subjunctional dense bodies and subsurface cisternae. The two populations of excitatory inputs differ, however, in their soma-dendritic distribution. The majority of abducens internuclear synaptic endings contact distal dendrites, whereas the majority of ATD synaptic endings contact proximal dendrites or somata. Abducens internuclear synaptic endings furthermore have a higher density of mitochondria than ATD synaptic endings. The more proximal location of ATD synaptic endings is consistent with the faster rise time and earlier reversal to polarizing currents of ATD excitatory postsynaptic potentials in comparison to those evoked by the abducens internuclear pathway as determined electrophysiologically. Given the differences in the physiologic signals conveyed by the abducens internuclear (eye velocity and eye position) and ATD (head velocity) pathways, the findings in this study suggest that the soma-dendritic stratification of the two inputs to medial rectus motoneurons may provide a means for the separate control of visuomotor and vestibular functions, respectively.

Monoamine innervation of the oculomotor nucleus in the rat. A radioautographic study.

The serotonin and noradrenaline innervations of the rat oculomotor nucleus were examined by high resolution radioautography after in vivo labeling with tritiated 5-hydroxytryptamine and dopamine, respectively. Noradrenaline as well as serotonin endings (axonal varicosities) pervaded the entire nucleus, but the latter were at least six times more numerous (1.3 X 10(6) per mm3 of tissue) and were often found in the immediate vicinity of neuronal somata and proximal dendrites. The axon terminals of both types were of similar size and exhibited some large dense-cored vesicles in association with aggregated small and clear vesicles. The dense-cored vesicles were, however, more frequent and the content in clear vesicles more pleomorphic in serotonin than noradrenaline endings. In single thin sections, the proportion of noradrenaline and serotonin profiles exhibiting a synaptic junction was relatively small (15%). These were either symmetrical or asymmetrical when made on dendritic branches but invariably symmetrical on spines. In addition, a significant number of serotonin terminals were seen in close apposition or synaptic contact with neuronal perikarya and large dendrites, allowing for a direct, "proximal" action of serotonin. Moreover, many such terminals appeared to be coupled with unlabeled endings of another category, characterized by dispersed, uniformly round and clear synaptic vesicles, providing an alternate route for a proximal effect of serotonin in the oculomotor nucleus. In line with previous investigations on other motor nuclei, these data support the likelihood of a close involvement of both noradrenaline and serotonin in the control of motoneuronal activity.

Grafting of a new target prevents synapse loss in abducens internuclear neurons induced by axotomy.

The loss of afferent synaptic boutons is a prominent alteration induced by axotomy on adult central neurons. In this work we attempted to prove whether synapse loss could be reverted by reconnection with a new target. We severed the medial longitudinal fascicle of adult cats and then transplanted embryonic cerebellar primordia at the lesion site immediately after lesion. As previously shown, the transected axons from abducens internuclear neurons penetrate and reinnervate the graft [J Comp Neurol 444 (2002) 324]. By immunocytochemistry and electron microscopy we studied the synaptology of abducens internuclear neurons under three conditions: control, axotomy and transplant (2 months of survival time). Semithin sections of the abducens nucleus were immunostained against calretinin, to identify abducens internuclear neurons, and either synaptophysin (SF), to label synaptic terminals, or glial fibrillary acidic protein (GFAP) to detect the astrocytic reaction. Optical and linear density of SF and GFAP immunostaining were measured. Data revealed a significant decrease in the density of SF-labeled terminals with a parallel increase in GFAP-immunoreactive elements after axotomy. On the contrary, in the transplant group, the density of SF-labeled terminals was found similar to control, and the astrocytic reaction induced by lesion was significantly reduced. At the ultrastructural level, synaptic coverage and linear density of boutons were measured around the somata of abducens internuclear neurons. Whereas a significant reduction in both parameters was found after axotomy, cells of the transplant group received a normal density of synaptic endings. The ratio between F- and S-type boutons was found similar in the three groups. Therefore, these findings indicate that the grafting of a new target can prevent the loss of afferent synaptic boutons produced by the axotomy.

Innervated myotendinous cylinders in human extraocular muscles.

PURPOSE: To analyze palisade endings and their end organs, the so-called innervated myotendinous cylinders (IMCs), of human extraocular muscle (EOM) in more detail and to clarify with the help of double-fluorescent labeling and electron microscopy whether terminals in IMCs are sensory, serving proprioception. METHODS: EOMs obtained from a donated cadaver (66 years) and distal parts of EOMs from multiorgan donors (35, 53 years) were processed for double-fluorescent labeling. Antibodies against the protein gene product 9.5 and alpha-bungarotoxin labeling were used on cryostat sections of distal myotendons. EOMs from multiorgan donors (2, 17 years) were prepared for electron microscopy. RESULTS: Palisade endings investing muscle fiber tips established contacts with tendon fibrils and the muscle fiber attached. Alpha-bungarotoxin bound to myoneural contacts but not to axonal varicosities in the tendon compartment. Ultrastructural analysis revealed that palisade endings form IMCs, which were associated exclusively with multiply innervated global layer muscle fibers. IMCs consisted of a muscle fiber tendon junction, tightly enclosed by fibrocytes, and a supplying axon with preterminals and terminals. Terminals contained mitochondria, few neurotubuli, few neurofilaments, and accumulations of clear vesicles of uniform size. A basal lamina always intervened between axolemma and tendon fibrils as well as between axolemma and muscle fiber cell membrane. CONCLUSIONS: Palisade endings of human EOM form IMCs as in cat, monkey, and sheep. In contrast to animals, myoneural contacts in human IMCs are almost certainly motor, whereas terminals contacting tendon fibrils are arguably sensory. Thus, IMCs might be best described as "propriocept-effectors."

Calcium-containing endosomes at oculomotor terminals in animal models of ALS.

Altered calcium homeostasis has been demonstrated in human spinal cord motor axon terminals of ALS patients, in spinal motor neurons of mutant SOD transgenic mice and following injection of ALS immunoglobulins. In all three paradigms oculomotor neurons are relatively spared. To explore mechanisms of selective resistance, we applied similar calcium localization techniques to terminals of oculomotor neurons in the two animal models. In both cases large vacuoles, which connect with the extracellular space, accumulated the majority of intracellular calcium, while terminals of vulnerable neurons (e.g. innervating interosseus muscle), which possess no such vacuoles, displayed evenly distributed calcium. These relatively unique membrane enveloped structures may permit neurons to control their cytoplasmic Ca2+ concentration and contribute to selective resistance.

Sequential double labelling with different fluorescent dyes coupled to dextran amines as a tool to estimate the accuracy of tracer application and of regeneration.

We present a technique to estimate the accuracy of a given application procedure for neuronal tracers. In a second series of animals we used this technique for the estimation of successful regeneration of peripheral nerves. Dextran amine coupled to rhodamine was applied to the cut trochlar nerve in Xenopus tadpoles. To assess the accuracy of tracer application, experiments were done in which a second dye, dextran amine coupled to fluorescein, was applied after 1 day proximal to the first dye. More then 90% of all trochlear motoneurons were doubly labelled after this procedure. Their total numbers were not significantly different from numbers obtained after single labelling with HRP in a comparable age group. To assess success of regeneration after 5 and 8 days, the second application of fluorescein dextran amine was distal to the first application side. Statistically significant differences suggest incomplete regeneration of many neurons. After 42 days the numbers of singly and doubly labelled motoneurons was in the same proportion as before regeneration. This suggests that about 90% of the surviving motoneurons had successfully regenerated back to the periphery.

Microtubules have special physical associations with smooth endoplasmic reticula and mitochondria in axons.

Ultrastructural morphometry was used to document the non-random spatial distributions of organelles within the compact myelinated region of avian oculomotor axons. These regions contain large numbers of loosely packed neurofilaments (NFs) (241/microns 2) and only a relatively small number of microtubules (MTs) (4/microns 2), mitochondria (0.6/microns 2), and smooth endoplasmic reticulum (SER) (1.6/microns 2). Random co-occurrences between the relatively sparsely distributed MTs, mitochondria, and SER are probably infrequent in these axons. The actual co-occurrences of MTs, mitochondria, and SER with MTs were counted and compared to the co-occurrences expected in a random Poisson distribution. At long distances (200 nm), the co-occurrences were random. At shorter distances (40 nm and less), MTs were still randomly associated with other MTs. However, at these shorter distances, the spatial associations of mitochondria with MTs and of SER with MTs were not random; such preferential stable associations may be produced by specific MT associated cross-bridging proteins. In axons, MTs tend to be clustered together, giving the appearance of MT bundles. We propose that the MT-MT bundling is an indirect result of MT concentration along the continuous intra-axonal SER network, to which the MTs are apparently tied directly by dynamic molecular cross-bridges.

Parasympathetic nuclei.

A series of experiments in monkeys using the fluorescent tracer substances Fast Blue and Nuclear yellow as well as wheat germ agglutinin-horseradish peroxidase injected into the ciliary ganglion has demonstrated labeling in 3 distinct regions of the mesencephalon: (1) anterior median nucleus; (2) Edinger-Westphal nucleus; and (3) the nucleus of Perlia. Further it was shown that the caudal extent of the Edinger-Westphal nucleus reaches the level of the central caudal nucleus of the somatic complex and that the lateral visceral column divided into a major and accessory column at the junction of the middle and posterior one-third of the somatic complex.

Radioautographic identification of [3H]glutamic acid labeled nerve endings in the cat oculomotor nucleus.

Slices of the cat third oculomotor nucleus were incubated in vitro with [3H]glutamic acid. Electron microscopic radioautographs revealed that glutamate had been taken by small nerve endings distributed on the oculomotor motoneuron distal dendrites. In contrast, there was no uptake in the other types of terminals. The labeled terminals seem to correspond to the excitatory vestibulo-oculomotor nerve endings and different correlations suggest their glutamergic nature.

The peripheral and central projections of the Edinger-Westphal nucleus in the rat. A light and electron microscopic tracing study.

The peripheral and central efferent projections of the rostral part of the Edinger-Westphal nucleus in the rat were investigated at the light and electron microscopic level by means of iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin and retrograde tracer injections of Fast blue and Nuclear yellow into the facial nucleus and into the principal olive. Two pathways leaving the rostral part of the Edinger-Westphal nucleus were studied, a peripheral and a central descending pathway. Fluorescent experiments demonstrated that the central pathway fibers originated from distinct individual Edinger-Westphal neurons. These neurons were mainly distributed throughout the rostral part of the Edinger-Westphal nucleus and had fusiform cell bodies. The neurons rarely form collateral projections. The central descending pathway left the Edinger-Westphal nucleus medially and terminated bilaterally in the principal olive, in the subnuclei A, B and C of the inferior olive and ipsilaterally in the medial accessory olive. The central pathway also terminated contralaterally in the lateral parabrachial nucleus, the facial nucleus, the trigeminal brainstem nuclear complex, the lateral reticular nucleus and the rostroventral reticular nucleus. The projection to the facial nucleus provides evidence for the existence of a polysynaptic loop forming the central part of the corneal blink reflex. Projections from the Edinger-Westphal nucleus to the cerebellar cortex or the deep nuclei, as described in cat and primate, could not be confirmed. The peripheral pathway left the Edinger-Westphal nucleus ventrally and terminated on dendrites of ciliary ganglion cells, along smooth muscle cells of ciliary ganglion associated arterioles and in the proximity of ciliary ganglion associated venules. The central and peripheral terminals that originate in the Edinger-Westphal nucleus all had similar ultrastructural features: clear, round vesicles and electron dense mitochondria. The terminals originating from the central descending pathway were often found to be arranged in glomerular-like structures. The central and peripheral terminals made asymmetric synaptic membrane specializations (Gray type one), except terminals innervating the ciliary ganglion associated vessels, which showed no synaptic contacts.

Axon number in oculomotor nerves in Xenopus: removal of one eye primordium affects both sides.

Unilateral removal of one eye primordium from Xenopus laevis embryos affects the number of axons in both oculomotor nerves: on the side of the operation, we observed a substantial decrease, and on the contralateral side an increase of over 30%. Mechanisms that may be invoked to explain this striking increase are: sprouting, rerouting of axons, decreased death of the motoneurones involved, and abnormal survival of ephemeral axon collaterals.

Calcium ions trigger the expansion in bistable myelin.

Ultrastructural studies of nerve myelin emphasize its static aspects. However, current work indicates that the myelin multilayer is poised for a supramolecular transition from a compact to an expanded form. In teleosts, the membrane pair becomes 27 A thicker. The trigger for this expansion is physiological levels of Ca. Varying proportions of the myelin can be trapped in the expanded state by transferring an excised specimen from Ca-containing to Ca-free medium in order to prevent expansion of the remaining compact myelin. Images of the myelin multilayer show that the expansion occurs in the aqueous spaces between lipid bilayers. Previously we demonstrated the reversal of expansion by removing Ca under special conditions. These results together lead us to suggest that myelin may expand locally in normal nerve function, while wholesale expansion may be an early step in myelin breakdown in vivo.

Electron-microscopic observations on preterminal fibers in the oculomotor nucleus of the cat. With special reference to the relation between axon diameter and myelin thickness in mammalian gray matter.

Electron microscopy of myelinated fibers in main lateral cell groups of the cat oculomotor nucleus reveals the majority to have diameters of less than 3 mum, with more than one-half less than 1 mum in diameter. The values of the ratio g (axon diameter/total fiber diameter) range between 0.58 and 0.88, with a mean of 0.74. There is no definite relation between g and fiber diameter. The morphological data are consistent with physiological data on preterminal conduction velocities in the oculomotor nucleus, and extend the dimensional analysis of myelinated fibers in gray matter, which has previously been confined to lower vertebrates, to the mammalian central nervous system.

Clinicopathological study of the peripheral nervous system in Machado-Joseph disease.

Three cases of histologically-proven Machado-Joseph disease (MJD) (clinically, types II and III) are described with special reference to the peripheral nervous system. We systematically and quantitatively analyzed the myelinated fiber densities in various nerves of three MJD patients and compared the results with the patients' clinical features and the pathological findings for the central nervous system (CNS). The results obtained were (1) motor system: The number of intermediate gammamotoneuron fibers was decreased prominently in all three MJD patients. In the type III MJD cases, the number of large alpha-motoneuron fibers also were decreased. (2) Sensory system: it generally was less involved than the motor system at the root level; but the large fibers of the distal portion were vulnerable. (3) Oculomotor system: the number of large oculomotor fibers was decreased markedly in all three cases, but there was relative preservation of parasympathetic fibers. (4) Autonomic system: the preganglionic sympathetic fibers from the intermediate lateral nucleus generally were preserved. These results suggest that in MJD the vulnerability of the peripheral nerves reflects the degree of loss of their original neuronal cells and varies according to the clinical phenotype. The characteristic peripheral neuropathy of MJD may be the result of axonal degeneration due to perikaryal neuronal damage in the central nervous system.