New Cerebello-Cortical Pathway Involved in Higher-Order Oculomotor Control.

The cerebellum and the basal ganglia play an important role in the control of voluntary eye movement associated with complex behavior, but little is known about how cerebellar projections project to cortical eye movement areas. Here we used retrograde transneuronal transport of rabies virus to identify neurons in the cerebellar nuclei that project via the thalamus to supplementary eye field (SEF) of the frontal cortex of macaques. After rabies injections into the SEF, many neurons in the restricted region, the ventral aspects of the dentate nucleus (DN), the caudal pole of the DN, and the posterior interpositus nucleus (PIN) were labeled disynaptically via the thalamus, whereas no neuron labeling was found in the anterior interpositus nucleus (AIN). The distribution of the labeled neurons was dorsoventrally different from that of DN and PIN neurons labeled from the motor cortex. In the basal ganglia, a large number of labeled neurons were confined to the dorsomedial portion of the internal segment of the globus pallidus (GPi) as more neurons were labeled in the inner portion of the GPi (GPii) than in the outer portion of the GPi (GPio). This is the first evidence of a projection between cerebellum/basal ganglia and the SEF that could enable the cerebellum to modulate the cognitive control of voluntary eye movement.

Glutamate modulates the firing rate in oculomotor nucleus motoneurons as a function of the recruitment threshold current.

Studies in alert preparations have demonstrated that ocular motoneurons exhibit a phasic­tonic firing rate related to eye velocity and position, respectively. The slopes of these relationships are higher in motoneurons with higher recruitment threshold and have been proposed to depend upon synaptic input. To investigate this hypothesis, motoneurons of the rat oculomotor nucleus were recorded in a brain slice preparation in control conditions and during glutamate (5 µm) application to the bath. Glutamate did not affect membrane potential or input resistance, but produced a decrease in rheobase and depolarization voltage as a function of the current needed for generating a maintained repetitive discharge (recruitment threshold current). In addition, glutamate compressed the range of recruitment threshold current (0.1­0.4 nA) as compared to the control (0.15­0.7 nA). Glutamate exposed motoneurons showed an increase in the tonic frequency gain and the peak frequency. Such increments depended on the recruitment threshold current and the last recruited motoneurons almost doubled the tonic frequency gain (35.2 vs. 57.9 spikes s(−1) nA(−1)) and the peak frequency (52.4 vs. 102.6 spikes s(−1)). Finally, glutamate increased the spike frequency adaptation due to a significant increase in the phasic firing component as compared to the tonic one. In conclusion, glutamate modulates tonic and phasic discharge properties as a function of the recruitment threshold current and, presumably, motoneuron size. These findings contribute to understand the link between cellular functions and motoneuron discharge during oculomotor behaviour.

The differentiation of the newborn nerve cells in oculomotor nuclear after oculomotor nerve injury.

Oculomotor nerve injury is a common complication of cranial trauma and craniotomy. For a long time, it has been generally considered that the oculomotor nerve is unable to regenerate and recover functionally after injury. With the development of neuroradiology, microsurgery and neurohistology, it has been reported that the injured oculomotor nerve could be repaired by operation. However, the mechanisms of neural regeneration of the injured oculomotor nerve remain obscure. Therefore, by investigating the differentiation of the newborn nerve cells in oculomotor nuclear after oculomotor nerve injury, the mechanisms of the neural regeneration of the injured oculomotor nerve was studied in the present paper. After animal model establishment, we found that the function of the injured oculomotor nerve could recover at some degree without treatment, at fourth week after the nerve injury. This result confirms that the injured oculomotor nerve per se has the potential to regenerate and repair. At the present study, by BredU stain, BrdU labeling cells were observed in oculomotor nuclear at the fourth week post-operatively. It indicated that the oculomotor nuclear per se has the ability of generating the cells, which will regenerate and differentiate after the nerve injury, without stimulation by exogenous agents. Immunofluorescence double staining was used in this study to show the differentiation of the newborn cells in oculomotor nuclear after oculomotor nerve injury. It is found that they could differentiate into neural progenitor cells, neuronal cells and neuroglial cells. It is suggested that the different differentiation of cells may play a role in the nerve regeneration procedure.

Accessory optic projections upon oculomotor nuclei and vestibulocerebellum.

Displaced retinal ganglion cells in birds are the sole source of the retinal projection onto the nucleus of the basal optic root, the main component of the accessory optic system. This nucleus has direct bilateral axonal projections onto the oculomotor nuclear complex, the trochlear nucleus- and folia IXc,d and paraflocculus of the vestibulocerebellum. The cerebellar projection terminates within a superficial band of the granule cell layer adjacent to the Purkinje cell layer as a mossy fiber system. This bisynaptic projection onto oculomotor neurons and the cerebellum may play a functionally distinct and specific role in oculomotor reflexes.

The Amniote Oculomotor Complex.

The oculomotor (OM) complex is a combination of somatic and parasympatethic neurons. The correct development and wiring of this cranial pair is essential to perform basic functions: eyeball and eyelid movements, pupillary constriction, and lens accommodation. The improper formation or function of this nucleus leads pathologies such as strabismus. We describe the OM organization and function in different vertebrate brains, including chick, mouse, and human. The morphological localization is detailed, as well as the spatial relation with the trochlear nucleus in order to adjust some misleading anatomical topographic descriptions. We detailed the signaling processes needed for the specification of the OM neurons. The transcriptional programs driven the specification and differentiation of these neurons are partially determined. We summarized recent genetic studies that have led to the identification of guidance mechanisms involved in the migration, axon pathfinding, and targeting of the OM neurons. Finally, we overviewed the pathology associated to genetic malformations in the OM development and related clinical alterations. Anat Rec, 302:446-451, 2019. © 2018 Wiley Periodicals, Inc.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice.

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Motor axon subpopulations respond differentially to the chemorepellents netrin-1 and semaphorin D.

During development, growing motor axons are excluded from the ventral midline of the neural tube by diffusible chemorepellents emanating from this region. Molecular candidates for this chemorepellent activity include semaphorin D and netrin-1; the latter is known to repel trochlear motor axons. Qualitatively or quantitatively different responses to these molecules might underlie the initial deflection from the midline and subsequent segregation of motor axon trajectories. To test this idea, we have cocultured cell aggregates secreting netrin-1 or semaphorin D at a distance from tissue explants containing different motor neuron subpopulations, in collagen gels. Cranial motor axons that project dorsally in vivo such as those of the trigeminal, facial, and glossopharyngeal nuclei were repelled by both netrin-1 and semaphorin D. By contrast, ventrally projecting spinal motor axons and abducens axons were not affected by netrin-1. Spinal and abducens motor neurons also responded to semaphorin D. The ventrally projecting axons of oculomotor neurons were not repelled by netrin-1 or semaphorin D. Differential responsiveness to netrin-1 and semaphorin D could thus contribute to the generation of dorsal and ventral motor axon pathways during development.

Encoding of error and learning to correct that error by the Purkinje cells of the cerebellum.

The primary output cells of the cerebellar cortex, Purkinje cells, make kinematic predictions about ongoing movements via high-frequency simple spikes, but receive sensory error information about that movement via low-frequency complex spikes (CS). How is the vector space of sensory errors encoded by this low-frequency signal? Here we measured Purkinje cell activity in the oculomotor vermis of animals during saccades, then followed the chain of events from experience of visual error, generation of CS, modulation of simple spikes, and ultimately change in motor output. We found that while error direction affected the probability of CS, error magnitude altered its temporal distribution. Production of CS changed the simple spikes on the next trial, but regardless of the actual visual error, this change biased the movement only along a vector that was parallel to the Purkinje cell's preferred error. From these results, we inferred the anatomy of a sensory-to-motor adaptive controller that transformed visual error vectors into motor-corrections.

Anatomical and pharmacological relationship between acetylcholine and nitric oxide in the prepositus hypoglossi nucleus of the cat: functional implications for eye-movement control.

The prepositus hypoglossi (PH) nucleus has been proposed as a pivotal structure for horizontal eye-position generation in the oculomotor system. Recent studies have revealed that acetylcholine (ACh) in the PH nucleus could mediate the persistent activity necessary for this process, although the origin of this ACh remains unknown. It is also known that nitric oxide (NO) in the PH nucleus plays an important role in the control of velocity balance, being involved in a negative feedback control of tonic signals arriving at the PH nucleus. As it could be expected that neurons taking part in eye-position generation must control their tonic background inputs, the existence of a relationship between nitrergic and cholinergic neurons is hypothesized. In the present study we analyzed the distribution, size, and morphology of choline acetyltransferase-positive neurons, and their relationship with neuronal nitric oxide synthase in the PH nucleus of the cat. As presumed, some 96% of cholinergic neurons were also nitrergic in the PH nucleus, suggesting that NO is regulating the level of ACh released by cholinergic PH neurons. Furthermore, we studied the alterations induced by muscarinic-receptor agonists and antagonists on spontaneous and vestibularly induced eye movements in the alert cat and compared them with those induced in previous studies by modification of NO levels in the same animal preparation. The results suggest that ACh is necessary for the generation of saccadic and vestibular eye-position signals, whereas the NO is stabilizing the eye-position generator by controlling background activity reaching cholinergic neurons in the PH nucleus.

The effects of unilateral eighth nerve block on fictive VOR in the turtle.

Multiunit activity during horizontal sinusoidal motion was recorded from pairs of oculomotor, trochlear, or abducens nerves of an in vitro turtle brainstem preparation that received inputs from intact semicircular canals. Responses of left oculomotor, right trochlear and right abducens nerves were approximately aligned with leftward head velocity, and that of the respective contralateral nerves were in-phase with rightward velocity. We examined the effect of sectioning or injecting lidocaine (1-2 microL of 0.5%) into the right vestibular nerve. Nerve block caused a striking phase shift in the evoked response of right oculomotor and left trochlear nerves, in which (rightward) control responses were replaced by a smaller-amplitude response to leftward table motion. Such "phase-reversed" responses were poorly defined in abducens nerve recordings. Frequency analysis demonstrated that this activity was advanced in phase relative to post-block responses of the respective contralateral nerves, which were in turn phase-advanced relative to pre-block controls. Phase differences were largest (approximately 10 degrees) at low frequencies (approximately 0.1 Hz) and statistically absent at 1 Hz. The phase-reversed responses were further investigated by eliminating individual canal input from the left labyrinth following right nVIII block, which indicated that the activation of the vertical canal afferents is the source of this activity.

Differential expression of the NMDA NR2B receptor subunit in motoneuron populations susceptible and resistant to amyotrophic lateral sclerosis.

We have compared the expression pattern of NMDA receptor subunits (NR1 and NR2A-D) and NR1 splice variants (NR1-1a/1b,-2a/2b,-3a/3b,-4a/4b) in motor neuron populations from adult Wistar rats that are vulnerable (hypoglossal, XII) or resistant (oculomotor, III) to death in amyotrophic lateral sclerosis (ALS). The major finding was higher levels of expression of the NR2B subunit in the hypoglossal nucleus. Quantitative real-time PCR showed that NR1 was expressed at a greater level than any of the NR2 subunits (>15 fold greater, P

Differential neuronal vulnerability identifies IGF-2 as a protective factor in ALS.

The fatal disease amyotrophic lateral sclerosis (ALS) is characterized by the loss of somatic motor neurons leading to muscle wasting and paralysis. However, motor neurons in the oculomotor nucleus, controlling eye movement, are for unknown reasons spared. We found that insulin-like growth factor 2 (IGF-2) was maintained in oculomotor neurons in ALS and thus could play a role in oculomotor resistance in this disease. We also showed that IGF-1 receptor (IGF-1R), which mediates survival pathways upon IGF binding, was highly expressed in oculomotor neurons and on extraocular muscle endplate. The addition of IGF-2 induced Akt phosphorylation, glycogen synthase kinase-3ß phosphorylation and ß-catenin levels while protecting ALS patient motor neurons. IGF-2 also rescued motor neurons derived from spinal muscular atrophy (SMA) patients from degeneration. Finally, AAV9::IGF-2 delivery to muscles of SOD1(G93A) ALS mice extended life-span by 10%, while preserving motor neurons and inducing motor axon regeneration. Thus, our studies demonstrate that oculomotor-specific expression can be utilized to identify candidates that protect vulnerable motor neurons from degeneration.

Visualization of cranial motor neurons in live transgenic zebrafish expressing green fluorescent protein under the control of the islet-1 promoter/enhancer.

We generated germ line-transmitting transgenic zebrafish that express green fluorescent protein (GFP) in the cranial motor neurons. This was accomplished by fusing GFP sequences to Islet-1 promoter/enhancer sequences that were sufficient for neural-specific expression. The expression of GFP by the motor neurons in the transgenic fish enabled visualization of the cell bodies, main axons, and the peripheral branches within the muscles. GFP-labeled motor neurons could be followed at high resolution for at least up to day four, when most larval neural circuits become functional, and larvae begin to swim and capture prey. Using this line, we analyzed axonal outgrowth by the cranial motor neurons. Furthermore, by selective application of DiI to specific GFP-positive nerve branches, we showed that the two clusters of trigeminal motor neurons in rhombomeres 2 and 3 innervate different peripheral targets. This finding suggests that the trigeminal motor neurons in the two clusters adopt distinct fates. In future experiments, this transgenic line of zebrafish will allow for a genetic analysis of cranial motor neuron development.

In vivo analysis of Schwann cell programmed cell death in the embryonic chick: regulation by axons and glial growth factor.

The present study uses the embryonic chick to examine in vivo the mechanisms and regulation of Schwann cell programmed cell death (PCD) in spinal and cranial peripheral nerves. Schwann cells are highly dependent on the presence of axons for survival because the in ovo administration of NMDA, which excitotoxically eliminates motoneurons and their axons by necrosis, results in a significant increase in apoptotic Schwann cell death. Additionally, pharmacological and surgical manipulation of axon numbers also affects the relative amounts of Schwann cell PCD. Schwann cells undergoing both normal and induced PCD display an apoptotic-like cell death, using a caspase-dependent pathway. Furthermore, axon elimination results in upregulation of the p75 and platelet-derived growth factor receptors in mature Schwann cells within the degenerating ventral root. During early development, Schwann cells are also dependent on axon-derived mitogens; the loss of axons results in a decrease in Schwann cell proliferation. Axon removal during late embryonic stages, however, elicits an increase in proliferation, as is expected from these more differentiated Schwann cells. In rodents, Schwann cell survival is regulated by glial growth factor (GGF), a member of the neuregulin family of growth factors. GGF administration to chick embryos selectively rescued Schwann cells during both normal PCD and after the loss of axons, whereas other trophic factors tested had no effect on Schwann cell survival. In conclusion, avian Schwann cells exhibit many similarities to mammalian Schwann cells in terms of their dependence on axon-derived signals during early and later stages of development.

Intracellular calcium parallels motoneuron degeneration in SOD-1 mutant mice.

Transgenic mice with Cu,Zn superoxide dismutase (SOD-1) mutations provide a unique model to examine altered Ca homeostasis in selectively vulnerable and resistant motoneurons. In degenerating spinal motoneurons of G93 A SOD-1 mice, developing vacuoles were filled with calcium, while calcium was gradually depleted from the cytoplasm and intact mitochondria. In oculomotor neurons, no degenerative changes, vacuolization, or increased calcium were noted. Motor axon terminals of interosseus muscle gradually degenerated and intracellular calcium was depleted. Oculomotor terminals of mutant SOD-1 mice were smaller and exhibited no degenerative changes, but did exhibit unique membrane-enclosed organelles containing calcium. Spinal motoneurons of SOD-1 mice were shown to have fewer calcium binding proteins, such as parvalbumin, compared with oculomotor neurons. These data suggest that the SOD-1 mutation is associated with impaired calcium homeostasis in motoneurons in vivo, with increased likelihood of degeneration associated with higher levels of intracellular calcium and lower to absent levels of calbindin-D28K and/or parvalbumin, and decreased likelihood of degeneration associated with minimally changed calcium and ample calbindin-D28K and/or parvalbumin.

Oculomotor system of the weakly electric fish Gnathonemus petersii.

The peripheral and central aspects of the extraocular system were studied in the weakly electric fish Gnathonemus petersii. All six extraocular muscles show a similar composition of large and small fibers grouped characteristically in the proximal and distal regions respectively. The exit of the three extraocular nerves from the brain is similar to that in other vertebrates. However, the intracephalic and intracranial course of the trochlear nerve is unusual, partly because of the extraordinary hypertrophy of the cerebellum. The three nerves course rostrally on the ventral brain surface; the trochlear nerve penetrates the orbital cavity separately from the two other nerves. The fiber-diameter spectrum of each extraocular nerve is bimodal; unmyelinated fibers were not observed in any of the nerves. The location of the extraocular motor nuclei was established by retrograde axonal transport of HRP or cobaltic-lysine complex. The oculomotor nucleus is situated ventral to the posterior pole of the magnocellular mesencephalic nucleus and the trochlear nucleus is found caudal and dorsal to this. The abducens nucleus is situated at the level of the octavolateral efferent nucleus and consists of a single group of cells on each side of the ventral tegmentum. The oculomotor nucleus of G. petersii shows a somatotopic organization. The superior rectus muscle receives a contralateral innervation whereas the inferior rectus and oblique muscles and the internal rectus muscles receive an ipsilateral innervation. The superior oblique muscle is innervated by contralateral trochlear motoneurons and the external rectus by ipsilateral abducens motoneurons. The majority of extraocular motoneurons have piriform perikarya and long beaded dendrites that extend laterally in the oculomotor and abducens nuclei and rostrally in the trochlear nucleus. The terminal dendritic portions of trochlear motoneurons widely overlap with oculomotor dendrites and perikarya. In all three nuclei the axon originates opposite to the main dendrite. Collaterals of the hairpin-bend abducens axons could be identified in a few cases. The oculomotor system of G. petersii appears basically similar to that of other teleosts; the differences observed concern mainly the structure of the abducens nucleus, the intracranial and intracephalic course of the trochlear nerve, and the relatively small number of axons in each nerve.

Transneuronal retinal input to the primate Edinger-Westphal nucleus.

The Edinger-Westphal nucleus of the oculomotor nuclear complex provides preganglionic parasympathetic innervation to the pupil. We labelled its retinal input by transneuronal autoradiography after an eye injection of [3H]proline in the macaque monkey. The primary retinal projection to the pretectum terminated in the ipsilateral and contralateral olivary nuclei. These nuclei were intensely labelled and sharply delimited, with a mean diameter of 590 microm and a rostrocaudal length of 2.52 mm. The caudal half of the olivary nucleus on each side broke into multiple clumps of label. Fragments of label also surrounded each olivary nucleus. The exact pattern of pretectal labelling varied considerably among animals and even from side to side in the same animal. In 5 of 6 monkeys, label from the olivary nucleus reached the Edinger-Westphal nucleus transneuronally. In transverse sections, the Edinger-Westphal label appeared as a circular patch located on either side of the midbrain ventral to the cerebral aqueduct in the central gray matter. It averaged 230 microm in diameter and 610 microm in length. In Nissl-stained sections, the autoradiographic label corresponded to a distinct nucleus comprised of neurons that were smaller than neurons in nearby somatic subdivisions of the oculomotor complex. The mean area of Edinger-Westphal neurons was 295 microm2. Transneuronal retinal input to the Edinger-Westphal nucleus mediating pupillary constriction terminates in a single, well-defined cell group in the midbrain.

Generation of the ocular motor nuclei and their cell types in the rabbit.

Autoradiography of 3H-thymidine incorporation was combined with horseradish peroxidase (HRP) transport to distinguish the birthdates of motoneurons and internuclear neurons of the abducens nucleus, and of specific motor pools within the oculomotor nucleus. Motoneurons were identified by their retrograde transport of HRP from the extraocular muscles. In other experiments, internuclear neurons of the abducens nucleus were identified by their retrograde transport of HRP from the oculomotor nucleus. We found that motoneurons and internuclear neurons are generated simultaneously in the abducens nucleus, and suggest that the differentiation of these two neuron types may be controlled by the local environment. The motor pools of the oculomotor nucleus are generated sequentially. This may reflect the mechanism whereby nuclei are constructed.

Alpha- and gamma-motoneurons in the adult human spinal cord and somatic cranial nerve nuclei: the significance of dendroachitectonics studied by the Golgi method.

A modified Golgi method (Vaisamruat and Hess, '53) was found to give satisfactory impregnation of cell bodies and dendrites, but not of dendritic spines and axons, in adult human material fixed by immersion in formalin. Examination of the motor columns in the spinal cord intumescences and of the third and twelfth cranial nerve nuclei revealed four neuron types, based on dendritic field size and dendritic branching pattern. Two of these were recognized as alpha-motoneurons; one of them was seen only in the medial motor column of the spinal ventral horn, while the other was observed in the cranial motor nuclei as well as the spinal lateral motor column. Differences in somadendritic dimensions in this neuron type were thought to reflect motor unit size, and thus terminal axon field dimensions. Of the two types of gamma-motoneurons recognized in the spinal cord and oculomotor nucleus, one was a miniature version of the commoner type of alpha-motoneuron. On this basis, it is proposed that it may give rise to fusimotor axons with plate endings. The second type of gamma-motoneuron does not resemble any of the other motoneuron types, and its axons may therefore be thought to terminate in trail endings.

Organization of the six motor nuclei innervating the ocular muscles in lamprey.

The topography of motoneurons supplying each of the six ocular muscles of the lamprey, Lampetra fluviatilis, was studied by selective application of HRP to the cut nerves of identified muscles. In addition, the distributions of motoneuron populations to both eyes were studied simultaneously with fluorescein and rhodamine coupled dextran-amines (FDA and RDA) applied to cut ocular muscle nerves of either side. The motoneuron pool of the caudal oblique muscle is represented bilaterally in the trochlear (N IV) motor nucleus. The dorsal rectus muscle is innervated from a contralateral group of oculomotor (N III) motoneurons and the remaining four muscles exclusively from the ipsilateral side (N III and N VI). The inferior and posterior rectus muscles are both innervated by the abducens nerve. In contrast to all jawed vertebrates, only three eye muscles (the dorsal rectus, rostral rectus, and rostral oblique) are innervated by the oculomotor nerve in lampreys (N III). Lampreys have a motor nucleus similar to the accessory abducens nucleus previously described only in tetrapods. They lack the muscle homologous to the nasal rectus muscle of elasmobranchs and the medial rectus muscle of osteognathostomes. The distribution of the dendrites of different groups of motoneurons was studied and is considered in relation to inputs from tectum and the different cranial nerves.