Relative contribution of lateral vestibular neuron and abducens internuclear neuron inputs to the discharge activity of medial rectus motoneurons.

Medial rectus motoneurons mediate nasally directed horizontal eye movements. These motoneurons receive two major excitatory inputs, from the abducens internuclear neurons (ABD Ints) and neurons of the lateral vestibular nucleus whose axons course through the ascending tract of Deiters (ATD). In the present work, we have recorded in the alert chronic cat preparation the discharge activity of these two premotor neurons simultaneously with eye movements, to discern their relative contribution to the firing pattern of medial rectus motoneurons. ABD Int discharge was accurately correlated with eye movements, displaying high sensitivities to eye position and eye velocity. ATD neurons also discharged in relation to spontaneous and vestibular eye movements but showed significantly lower eye position and eye velocity sensitivities. Outstandingly, ATD neurons presented a significantly lower eye position threshold for recruitment compared to both ABD Ints and medial rectus motoneurons. Therefore, ATD neurons exhibited eye position and velocity signals during spontaneous and vestibular eye movements, which were of lower magnitude than those of ABD Ints, but due to their low recruitment threshold, they could play a significant role in facilitating ABD Int signal transmission onto medial rectus motoneurons.

BDNF Influence on Adult Terminal Axon Sprouting after Partial Deafferentation.

BDNF is a neurotrophin family member implicated in many different neuronal functions, from neuronal survival during development to synaptic plasticity associated with processes of learning and memory. Its presence in the oculomotor system has previously been demonstrated, as it regulates afferent composition of extraocular motoneurons and their firing pattern. Moreover, BDNF expression increases after extraocular motoneuron partial deafferentation, in parallel with terminal axon sprouting from the remaining axons. To elucidate whether BDNF could play an active role in this process, we performed partial deafferentation of the medial rectus motoneurons through transection of one of the two main afferents, that is, the ascending tract of Deiters, and injected BDNF into the motoneuron target muscle, the medial rectus. Furthermore, to check whether BDNF could stimulate axon sprouting without lesions, we performed the same experiment without any lesions. Axon terminal sprouting was assessed by calretinin immunostaining, which specifically labels the remaining afferent system on medial rectus motoneurons, the abducens internuclear neurons. The results presented herein show that exogenous BDNF stimulated terminal axon growth, allowing the total recovery of synaptic coverage around the motoneuron somata. Moreover, calretinin staining in the neuropil exceeded that present in the control situation. Thus, BDNF could also stimulate axonal sprouting in the neuropil of intact animals. These results point to an active role of BDNF in plastic adaptations that take place after partial deafferentation.

Role of vascular endothelial growth factor as a critical neurotrophic factor for the survival and physiology of motoneurons.

Vascular endothelial growth factor (VEGF) was discovered by its angiogenic activity. However, during evolution, it appeared earlier as a neurotrophic factor required for the development of the nervous system in invertebrates lacking a circulatory system. We aimed at reviewing recent evidence indicating that VEGF has neuroprotective effects in neurons exposed to a variety of insults. Of particular interest is the link established between VEGF and motoneurons, especially after the design of the VEGFδ/δ mutant mice. These mice are characterized by low levels of VEGF and develop muscle weakness and motoneuron degeneration resembling amyotrophic lateral sclerosis. The administration of VEGF through several routes to animal models of amyotrophic lateral sclerosis delays motor impairment and motoneuron degeneration and increases life expectancy. There are new recent advances in the role of VEGF in the physiology of motoneurons. Our experimental aims use the extraocular (abducens) motoneurons lesioned by axotomy as a model for studying VEGF actions. Axotomized abducens motoneurons exhibit severe alterations in their discharge activity and a loss of synaptic boutons. The exogenous administration of VEGF to axotomized abducens motoneurons, either from the transected nerve or intraventricularly, fully restores the synaptic and discharge properties of abducens motoneurons, despite being axotomized. In addition, when an anti-VEGF neutralizing antibody is delivered from the muscle to intact, uninjured abducens motoneurons, these cells display alterations in their discharge pattern and a loss of synaptic boutons that resemble the state of axotomy. All these data indicate that VEGF is an essential neurotrophic factor for motoneurons.

VEGF and Neuronal Survival.

Vascular endothelial growth factor (VEGF) is well known for its angiogenic activity, but recent evidence has revealed a neuroprotective action of this factor on injured or diseased neurons. In the present review, we summarize the most relevant findings that have contributed to establish a link between VEGF deficiency and neuronal degeneration. At issue, 1) mutant mice with reduced levels of VEGF show adult-onset muscle weakness and motoneuron degeneration resembling amyotrophic lateral sclerosis (ALS), 2) administration of VEGF to different animal models of motoneuron degeneration improves motor performance and ameliorates motoneuronal degeneration, and 3) there is an association between low plasmatic levels of VEGF and human ALS. Altogether, the results presented in this review highlight VEGF as an essential motoneuron neurotrophic factor endowed with promising therapeutic potential for the treatment of motoneuron disorders.

VEGF is an essential retrograde trophic factor for motoneurons.

VEGF was initially discovered due to its angiogenic activity and therefore named "vascular endothelial growth factor." However, its more recently discovered neurotrophic activity may be evolutionarily more ancient. Our previous work showed that all the changes produced by axotomy on the firing activity and synaptic inputs of abducens motoneurons were completely restored after VEGF administration. Therefore, we hypothesized that the lack of VEGF delivered by retrograde transport from the periphery should also affect the physiology of otherwise intact abducens motoneurons. For VEGF retrograde blockade, we chronically applied a neutralizing VEGF antibody to the lateral rectus muscle. Recordings of extracellular single-unit activity and eye movements were made in alert cats before and after the application of the neutralizing antibody. Our data revealed that intact, noninjured abducens motoneurons retrogradely deprived of VEGF exhibited noticeable changes in their firing pattern. There is a general decrease in firing rate and a significant reduction in eye position and eye velocity sensitivity (i.e., a decrease in the tonic and phasic components of their discharge, respectively). Moreover, by means of confocal immunocytochemistry, motoneurons under VEGF blockade showed a marked reduction in the density of afferent synaptic terminals contacting with their cell bodies. Altogether, the present findings demonstrate that the lack of retrogradely delivered VEGF renders abducens motoneurons into an axotomy-like state. This indicates that VEGF is an essential retrograde factor for motoneuronal synaptic drive and discharge activity.

MIF versus SIF Motoneurons, What Are Their Respective Contribution in the Oculomotor Medial Rectus Pool?

Multiply-innervated muscle fibers (MIFs) are peculiar to the extraocular muscles as they are non-twitch but produce a slow build up in tension on repetitive stimulation. The motoneurons innervating MIFs establish en grappe terminals along the entire length of the fiber, instead of the typical en plaque terminals that singly-innervated muscle fibers (SIFs) motoneurons establish around the muscle belly. MIF motoneurons have been proposed to participate only in gaze holding and slow eye movements. We aimed to discern the function of MIF motoneurons by recording medial rectus motoneurons of the oculomotor nucleus. Single-unit recordings in awake cats demonstrated that electrophysiologically-identified medial rectus MIF motoneurons participated in different types of eye movements, including fixations, rapid eye movements or saccades, convergences, and the slow and fast phases of the vestibulo-ocular nystagmus, the same as SIF motoneurons did. However, MIF medial rectus motoneurons presented lower firing frequencies, were recruited earlier and showed lower eye position (EP) and eye velocity (EV) sensitivities than SIF motoneurons. MIF medial rectus motoneurons were also smaller, had longer antidromic latencies and a lower synaptic coverage than SIF motoneurons. Peristimulus time histograms (PSTHs) revealed that electrical stimulation to the myotendinous junction, where palisade endings are located, did not recurrently affect the firing probability of medial rectus motoneurons. Therefore, we conclude there is no division of labor between MIF and SIF motoneurons based on the type of eye movement they subserve.SIGNIFICANCE STATEMENT In addition to the common singly-innervated muscle fiber (SIF), extraocular muscles also contain multiply-innervated muscle fibers (MIFs), which are non-twitch and slow in contraction. MIF motoneurons have been proposed to participate only in gaze holding and slow eye movements. In the present work, by single-unit extracellular recordings in awake cats, we demonstrate, however, that both SIF and MIF motoneurons, electrophysiologically-identified, participate in the different types of eye movements. However, MIF motoneurons showed lower firing rates (FRs), recruitment thresholds, and eye-related sensitivities, and could thus contribute to the fine adjustment of eye movements. Electrical stimulation of the myotendinous junction activates antidromically MIF motoneurons but neither MIF nor SIF motoneurons receive a synaptic reafferentation that modifies their discharge probability.

Translation information processing is regulated by protein kinase C-dependent mechanism in Purkinje cells in murine posterior vermis.

The cerebellar posterior vermis generates an estimation of our motion (translation) and orientation (tilt) in space using cues originating from semicircular canals and otolith organs. Theoretical work has laid out the basic computations necessary for this signal transformation, but details on the cellular loci and mechanisms responsible are lacking. Using a multicomponent modeling approach, we show that canal and otolith information are spatially and temporally matched in mouse posterior vermis Purkinje cells and that Purkinje cell responses combine translation and tilt information. Purkinje cell-specific inhibition of protein kinase C decreased and phase-shifted the translation component of Purkinje cell responses, but did not affect the tilt component. Our findings suggest that translation and tilt signals reach Purkinje cells via separate information pathways and that protein kinase C-dependent mechanisms regulate translation information processing in cerebellar cortex output neurons.

Short-term plasticity after partial deafferentation in the oculomotor system.

Medial rectus motoneurons are innervated by two main pontine inputs. The specific function of each of these two inputs remains to be fully understood. Indeed, selective partial deafferentation of medial rectus motoneurons, performed by the lesion of either the vestibular or the abducens input, initially induces similar changes in motoneuronal discharge. However, at longer time periods, the responses to both lesions are dissimilar. Alterations on eye movements and motoneuronal discharge induced by vestibular input transection recover completely 2 months post-lesion, whereas changes induced by abducens internuclear lesion are more drastic and permanent. Functional recovery could be due to some kind of plastic process, such as reactive synaptogenesis, developed by the remaining intact input, which would occupy the vacant synaptic spaces left after lesion. Herein, by means of confocal microscopy, immunocytochemistry and retrograde labeling, we attempt to elucidate the possible plastic processes that take place after partial deafferentation of medial rectus motoneuron. 48 h post-injury, both vestibular and abducens internuclear lesions produced a reduced synaptic coverage on these motoneurons. However, 96 h after vestibular lesion, there was a partial recovery in the number of synaptic contacts. This suggests that there was reactive synaptogenesis. This recovery was preceded by an increase in somatic neurotrophin content, suggesting a role of these molecules in presynaptic axonal sprouting. The rise in synaptic coverage might be due to terminal sprouting performed by the remaining main input, i.e., abducens internuclear neurons. The present results may improve the understanding of this apparently redundant input system.

Effects of Selective Deafferentation on the Discharge Characteristics of Medial Rectus Motoneurons.

Medial rectus motoneurons receive two main pontine inputs: abducens internuclear neurons, whose axons course through the medial longitudinal fasciculus (MLF), and neurons in the lateral vestibular nucleus, whose axons project through the ascending tract of Deiters (ATD). Abducens internuclear neurons are responsible for conjugate gaze in the horizontal plane, whereas ATD neurons provide medial rectus motoneurons with a vestibular input comprising mainly head velocity. To reveal the relative contribution of each input to the oculomotor physiology, single-unit recordings from medial rectus motoneurons were obtained in the control situation and after selective deafferentation from cats with unilateral transection of either the MLF or the ATD. Both MLF and ATD transection produced similar short-term alterations in medial rectus motoneuron firing pattern, which were more drastic in MLF of animals. However, long-term recordings revealed important differences between the two types of lesion. Thus, while the effects of the MLF section were permanent, 2 months after ATD lesioning all motoneuronal firing parameters were similar to the control. These findings indicated a more relevant role of the MLF pathway in driving motoneuronal firing and evidenced compensatory mechanisms following the ATD lesion. Confocal immunocytochemistry revealed that MLF transection produced also a higher loss of synaptic boutons, mainly at the dendritic level. Moreover, 2 months after ATD transection, we observed an increase in synaptic coverage around motoneuron cell bodies compared with short-term data, which is indicative of a synaptogenic compensatory mechanism of the abducens internuclear pathway that could lead to the observed firing and morphological recovery.SIGNIFICANCE STATEMENT Eye movements rely on multiple neuronal circuits for appropriate performance. The abducens internuclear pathway through the medial longitudinal fascicle (MLF) and the vestibular neurons through the ascending tract of Deiters (ATD) are a dual system that supports the firing of medial rectus motoneurons. We report the effect of sectioning the MLF or the ATD pathway on the firing of medial rectus motoneurons, as well as the plastic mechanisms by which one input compensates for the lack of the other. This work shows that while the effects of MLF transection are permanent, the ATD section produces transitory effects. A mechanism based on axonal sprouting and occupancy of the vacant synaptic space due to deafferentation is the base for the mechanism of compensation on the medial rectus motoneuron.

Extraocular Motor System Exhibits a Higher Expression of Neurotrophins When Compared with Other Brainstem Motor Systems.

Extraocular motoneurons resist degeneration in diseases such as amyotrophic lateral sclerosis. The main objective of the present work was to characterize the presence of neurotrophins in extraocular motoneurons and muscles of the adult rat. We also compared these results with those obtained from other cranial motor systems, such as facial and hypoglossal, which indeed suffer neurodegeneration. Immunocytochemical analysis was used to describe the expression of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in oculomotor, trochlear, abducens, facial, and hypoglossal nuclei of adult rats, and Western blots were used to describe the presence of neurotrophins in extraocular, facial (buccinator), and tongue muscles, which are innervated by the above-mentioned motoneurons. In brainstem samples, brain-derived neurotrophic factor was present both in extraocular and facial motoneuron somata, and to a lesser degree, in hypoglossal motoneurons. Neurotrophin-3 was present in extraocular motor nuclei, while facial and hypoglossal motoneurons were almost devoid of this protein. Finally, nerve growth factor was not present in the soma of any group of motoneurons, although it was present in dendrites of motoneurons located in the neuropil. Neuropil optical density levels were higher in extraocular motoneuron nuclei when compared with facial and hypoglossal nuclei. Neurotrophins could be originated in target muscles, since Western blot analyses revealed the presence of the three molecules in all sampled muscles, to a larger extent in extraocular muscles when compared with facial and tongue muscles. We suggest that the different neurotrophin availability could be related to the particular resistance of extraocular motoneurons to neurodegeneration.

Extraocular motoneurons of the adult rat show higher levels of vascular endothelial growth factor and its receptor Flk-1 than other cranial motoneurons.

Recent studies show a relationship between the deficit of vascular endothelial growth factor (VEGF) and motoneuronal degeneration, such as that occurring in amyotrophic lateral sclerosis (ALS). VEGF delivery protects motoneurons from cell death and delayed neurodegeneration in animal models of ALS. Strikingly, extraocular motoneurons show lesser vulnerability to neurodegeneration in ALS compared to other cranial or spinal motoneurons. Therefore, the present study investigates possible differences in VEGF and its main receptor VEGFR-2 or Flk-1 between extraocular and non-extraocular brainstem motoneurons. We performed immunohistochemistry and Western blot to determine the presence of VEGF and Flk-1 in rat motoneurons located in the three extraocular motor nuclei (abducens, trochlear and oculomotor) and to compare it to that observed in two other brainstem nuclei (hypoglossal and facial) that are vulnerable to degeneration. Extraocular motoneurons presented higher amounts of VEGF and its receptor Flk-1 than other brainstem motoneurons, and thus these molecules could be participating in their higher resistance to neurodegeneration. In conclusion, we hypothesize that differences in VEGF availability and signaling could be a contributing factor to the different susceptibility of extraocular motoneurons, when compared with other motoneurons, in neurodegenerative diseases.