[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.

Human CFEOM1 mutations attenuate KIF21A autoinhibition and cause oculomotor axon stalling.

The ocular motility disorder "Congenital fibrosis of the extraocular muscles type 1" (CFEOM1) results from heterozygous mutations altering the motor and third coiled-coil stalk of the anterograde kinesin, KIF21A. We demonstrate that Kif21a knockin mice harboring the most common human mutation develop CFEOM. The developing axons of the oculomotor nerve's superior division stall in the proximal nerve; the growth cones enlarge, extend excessive filopodia, and assume random trajectories. Inferior division axons reach the orbit but branch ectopically. We establish a gain-of-function mechanism and find that human motor or stalk mutations attenuate Kif21a autoinhibition, providing in vivo evidence for mammalian kinesin autoregulation. We identify Map1b as a Kif21a-interacting protein and report that Map1b⁻/⁻ mice develop CFEOM. The interaction between Kif21a and Map1b is likely to play a critical role in the pathogenesis of CFEOM1 and highlights a selective vulnerability of the developing oculomotor nerve to perturbations of the axon cytoskeleton.

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.

Morphologic indication for proprioception in the human ciliary muscle.

PURPOSE: To search for proprioceptive nerve terminals in human ciliary muscle. METHODS: In 48 human donor eyes, histologic and ultrathin sections cut in different planes and wholemounts of the ciliary muscle were studied. Immunohistochemical staining with antibodies against pan-neuronal antigens and antigens reported as markers for sensory terminals in other organs was performed. RESULTS: Among the markers for proprioceptive terminals, only calretinin was present in the ciliary body. Calretinin-immunoreactive (IR) nerve terminals surrounded the posterior and reticular ciliary muscle tips and their elastic tendons. Terminals in that region contained mitochondria and neurofilaments. At the anterior tips larger terminals with numerous membrane-filled vesicles were located between the muscle fibers. The most elaborate network of calretinin-IR nerve fibers was present in the ground plate covering the circular muscle portion. Here calretinin-IR neurons with morphologic features of mechanoreception were present. Within the circular muscle portion numerous calretinin-IR ganglion cells were found. Their processes were connected to the calretinin-IR network but also surrounded ciliary muscle cells and NADPH-diaphorase-positive ganglion cells. CONCLUSIONS: These morphologic findings indicate that there are proprioreceptors in the ciliary muscle that morphologically and presumably functionally differ at different locations. At the posterior muscle tips, the receptors could measure stretch of the tendons, whereas the large receptor organs located at the anterior muscle tips morphologically resemble mechanoreceptors measuring shear stress. The presence of the numerous intrinsic nerve cells indicates that contraction of the circular muscle portion can be modulated locally via a self-contained reflex arc.

Ultrastructural and molecular biologic comparison of classic proprioceptors and palisade endings in sheep extraocular muscles.

PURPOSE: To analyze and compare the structural and molecular features of classic proprioceptors like muscle spindles and Golgi tendon organs (GTOs) and putative proprioceptors (palisade endings) in sheep extraocular muscle (EOMs). METHODS: The EOMs of four sheep were analyzed. Frozen sections or wholemount preparations of the samples were immunohistochemically labeled and analyzed by confocal laser scanning microscopy. Triple labeling with different combinations of antibodies against neurofilament, synaptophysin, and choline acetyltransferase (ChAT), as well as alpha-bungarotoxin and phalloidin, was performed. Microscopic anatomy of the nerve end organs was analyzed by transmission electron microscopy. RESULTS: The microscopic anatomy demonstrated that muscle spindles and GTOs had a perineural capsule and palisade endings a connective tissue capsule. Sensory nerve terminals in muscle spindles and GTOs contained only a few vesicles, whereas palisade nerve terminals were full of clear vesicles. Likewise, motor terminals in the muscle spindles' polar regions were full of clear vesicles. Immunohistochemistry showed that sensory nerve fibers as well as their sensory nerve terminals in muscle spindles and GTOs were ChAT-negative. Palisade endings were supplied by ChAT-positive nerve fibers, and the palisade complexes including palisade nerve terminals were also ChAT-immunoreactive. Motor terminals in muscle spindles were ChAT and alpha-bungarotoxin positive. CONCLUSIONS: The present study demonstrated in sheep EOMs that palisade endings are innervated by cholinergic axons exhibiting characteristics typical of motoneurons, whereas muscle spindles (except the polar regions) and GTOs are supplied by noncholinergic axons. These results raise the question of whether palisade endings are candidates for proprioceptors in EOMs.

Age-related immunohistochemical and ultrastructural changes in rat oculomotor nerve.

During ageing process, multiple changes occur on nervous tissue composed of cells and extracellular matrix. Changes on nervous tissues are usually known as degenerative changes on axon structure and connective tissue covering the nerve such as a decrease in the number of fibre or general structural changes. For this purpose, we have studied age-dependent ultrastructural changes in the rat oculomotor nerve with electron microscopy and also demonstrated collagen structure of the neural sheaths with immunohistochemical techniques. This study was conducted in Gazi University Faculty of Medicine, Department of Anatomy with a total of nine Wistar albino rats. We observed strong collagen type I immunoreactivity in endoneurium and slight to moderate reactivity in fibroblast cytoplasm in 3-month- and 12-month-old groups and mild reactivity in 24-month-old group. Collagen type IV immunoreactivity was stronger in endoneurium and perineurium in the 3-month- and 12-month-old groups compared with collagen type I and fibroblast cytoplasm showed a very strong reactivity. On the other hand, in the 24-month-old group, there was slight reactivity in endoneurium and a strong reactivity in perineurium. NGF staining showed moderate to strong reactivity on Schwann cells of the 3-month-old group. The immunoreactivity decreased in the 12-month- and 24-month-old groups. In the 3-month-old rat group, Schwann cell cytoplasm, mitochondrial structure and neurofilaments were normal. In the 12-month-old group, there were no changes in organelle distribution, mitochondrial structure and neurofilaments, but there was an increase in the connective tissue. An inconsiderable number of degenerated myelinated nerves were observed. We detected an important decrease in the collagen type I immunoreactivity, which could suggest that the endoneurium, perineurium and epineurium are less resistant to the age-related collagen loss and that the peripheral nerve is protected by a weaker barrier in the old group. The collagen type IV immunoreactivity was significantly decreased with age. NGF synthesis decreases with age because of Schwann cell structural degeneration or for different reasons. Thus, this could explain the diminished capacity of regeneration and damage of the myelination of the peripheral nerve.

Structural and ultrastructural study of the intracranial portion of the oculomotor, trochlear and abducent nerves in dog.

The right intracranial portion of the oculomotor, trochlear and abducent nerves were removed from six adult German shepherd dogs and analysed by light and electron microscopy. In all cases the nerve sectional area was calculated. Unmyelinated and myelinated fibres were analysed and number, diameter and cross-sectional area were calculated. In myelinated fibres, also calculated were the corresponding axon area and diameter, and myelin sheath thickness. The mean number of myelinated fibres was 8543.50 +/- 1231.85 being the unmyelinated 1402 +/- 241.58 in the oculomotor nerve; 1509 +/- 223.17 and 287.67 +/- 72.28 in the trochlear nerve and 2473.00 +/- 211.41 and 231.25 +/- 92.67 respectively in the abducent. The mean diameter was 10.23 +/- 0.68 microm in myelinated and 0.43 +/- 0.21 for unmyelinated in oculomotor nerve, 10.53 +/- 0.55 microm and 0.33 +/- 0.04 for the trochlear, and 10.45 +/- 1.27 microm and 0.47 +/- 0.09 in the abducent nerve respectively. This study reveals that oculomotor, trochlear and abducent nerves of the dog show structural and ultra-structural features similar to the same nerves in other species.

Ultrastructural study of extraocular muscle tendon axonal profiles in infantile and intermittent exotropia.

PURPOSE: To investigate the ultrastructures of tendon axonal profiles in infantile and intermittent exotropia. Tendon axonal profiles are composed of myotendinous nerve endings that are presumed to serve as sensorial receptors in ocular proprioception. METHODS: The study subjects included 10 patients with exotropia who had undergone surgery in one eye (recession and resection). They were divided into two equal groups. Five patients with infantile exotropia that had developed at under 12 months of age were allocated to group A. Another five, with intermittent exotropia that had developed at over 12 months of age, were allocated to group B. In all patients, medial recti were resected by 3-4 mm in order to obtain tissue samples, which were then examined under an electron microscope. RESULTS: In group A, we noted many axonal degenerative findings, such as the retraction of axons from myelin sheaths with considerable shrinkage, axonal disintegration, and Schwann cell proliferation. On the other hand, we identified three unique findings in group B: intact axons with incomplete Schwann cell wrapping; intact Schwann cells not associated with axons, and disorganized Schwann cells with shrunken axons. CONCLUSION: Different patterns of tendon axonal profiles were seen in association with the two types of exotropia. These differences may be related to the pathogenesis of these exotropia types.

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.

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.

Time-dependent neural activations related to recognition of people's names in emotional and neutral face-name associative learning: an fMRI study.

Previous data have indicated that the left anterior temporal lobe contributes to the retrieval of familiar people's names, and that the extended network including the bilateral anterior temporal lobe plays an important role in the retrieval of newly learned people's names. However, there has been no direct evidence for time-dependent change in brain activation in face-name associations. In addition, previous studies have demonstrated that emotional information such as emotional faces may contribute to the organization of long-lasting episodic memory. In the present study, we investigated the activations related to the recognition of people's names in the context of emotional and neutral face-name associative learning. Before fMRI scanning, subjects learned face-name associations that included emotionally positive and neutral facial expressions. In immediate (5 min later) and delayed (2 weeks later) recognition with fMRI scanning, subjects were presented with studied faces with two names, and were asked to choose the correct associative name learned previously. Recognition-related activations were identified in the anterior part of bilateral temporal lobe for immediate recognition and only in the left temporal lobe for delayed recognition. Further analysis confirmed the time-dependent change in activation of the right anterior temporal lobe. Activation related to the processing of faces with positive expressions were observed in the left periamygdaloid area and temporal pole, although emotional information did not have an influence on task performance in this study. These findings suggest that the neural network involving the bilateral temporal lobe contributes to the retrieval of newly learned people's names, and that the left temporal lobe has a crucial and stable role in retrieval of people's names from faces, whereas the role of the right temporal lobe in retrieval of people's names may decrease with the time course.

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.

[Morphology of oculomotor muscles and their nervous apparatus in lateral amyotrophic sclerosis in conditions during long-term artificial lung ventilation]. / Morfologiia glazodvigatel'nykh myshts i ikh nervnogo apparata pri bokovom amiotroficheskom skleroze, protekavshem v usloviiakh dlitel'noi iskusstvennoi ventiliatsii legkikh.

Light and electron microscopy, histochemical study of acetylcholinesterase (ACE) were used in examination of the state and innervation of ocular muscles in autopsy material from a patient who died of amyotrophic sclerosis (ATS). The patient had lived 14 years under artificial lung ventilation, ATS was diagnosed 22 years before the death. Light microscopy demonstrated the intactness of the muscle fibers and the presence of three types characteristic of the extraocular muscles: thin, granular and rough. Besides typical differences structural changes were observed in some muscle fibers of the myopathic character and inclusions not limited by membrane having filiform or granular structure. Ocular muscles had an intensive innervation. Nervous fiber terminals revealed by a reaction for ACE were represented by single motor plaques and multiple cluster-like endings. Ultrastructurally, nervous endings of two types differed by terminals and fold expression on the postsynaptic membrane. There were no pathological changes in axons and myelin. Thus, ocular muscles were not affected as well as their nervous apparatus at completion of AMS, this indicating the noninvolvement of this muscular allotype in a specific degenerative process.

Congenital spongiform change in the brain stem nuclei of a domestic kitten.

This report describes a novel spongiform change in the brain stem nuclei of a 9-month-old mixed breed kitten with neurological signs. Histologically, vacuoles were found in perineuronal spaces and neuropil, with mild to moderate astrocytosis in the brain stem nuclei. Vacuoles were not observed in the cytoplasm of neurons and no evidence of neuronal loss was found. Ultrastructurally, there were intramyelinic vacuoles with separation of lamellae at intraperiod lines and larger spaces formed by coalescence of ruptured vacuoles. Immunohistochemically, abnormal accumulation of prion protein (PrP) was not detected in the brain stem lesions. This is, to our knowledge, the first report of a feline spongiform change localized in the brain stem nuclei.

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.

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.

Motonuclear changes after cranial nerve injury and regeneration.

Little is known about the mechanisms at play in nerve regeneration after nerve injury. Personal studies are reported regarding motonuclear changes after regeneration of injured cranial nerves, in particular of the facial and oculomotor nerves, as well as the influence that the natural molecule acetyl-L-carnitine (ALC) has on post-axotomy cranial nerve motoneuron degeneration after facial and vagus nerve lesions. Adult and newborn animal models were used. Massive motoneuron response after nerve section and reconstruction was observed in the motonuclei of all nerves studied. ALC showed to have significant neuroprotective effects on the degeneration of axotomized motoneurons. Complex quantitative, morphological and somatotopic nuclear changes occurred that sustain new hypotheses regarding the capacities of motoneurons to regenerate and the possibilities of new neuron proliferation. The particularities of such observations are described and discussed.

Pathway of the primary afferent nerve fibres serving proprioception in monkey extraocular muscles.

Following intracranial section of either the oculomotor or ophthalmic nerve, Wallerian degeneration studies revealed 1.38-3.7% of nerve fibres in the nerves to the inferior and superior rectus muscles were of ophthalmic nerve origin; more than half of them were unmyelinated. The results of the two experiments were complementary. The proportion of fibres identified as sensory is substantially smaller than the 10%, estimated in other studies, required to serve muscle receptors. These results indicate, contrary to some reports, that a substantial majority of proprioceptive fibres are conducted from extraocular muscles to the brainstem in the motor nerves and that their somata are not housed in the trigeminal ganglion.

Morphologic changes in the vestibular nerves and nuclei after labyrinthectomy in the cat: a case for the neurotrophin hypothesis in vestibular compensation.

Morphological changes in the vestibular nerves and superior vestibulocular neurons (SVON) after unilateral labyrinthectomy in cats revealed a progressive loss of axons in the ipsilateral vestibular nerve (35%) and synaptic profiles (SP) on ipsilateral SVON (60%) up to a 1-year survival period. Although the ipsilateral vestibular nerve showed further degeneration (45%) at 2 years post ablation, the number of SP on ipsilateral SVON increased to 60% of normal (40% loss). These SP likely represent sprouting from crossing commissural or cerebellar pathways. Contralateral vestibular nerves at 1 and 2 years post ablation revealed normal numbers and size spectrum, but the number of SP contacting the contralateral SVON at 8 weeks, 1 and 2 years paralleled the levels of SP found on ipsilateral SVON. The symmetry in adjustment of SP on the SVON of both sides of the brainstem after ablation may be explained by the neurotrophin hypothesis.