María A. Davis-López de Carrizosa

Complementary Actions of BDNF and Neurotrophin-3 on the Firing Patterns and Synaptic Composition of Motoneurons

Neurotrophins, as target-derived factors, are essential for neuronal survival during development, but during adulthood, their scope of actions widens to become also mediators of synaptic and morphological plasticity. Target disconnection by axotomy produces an initial synaptic stripping ensued by synaptic rearrangement upon target reinnervation. Using abducens motoneurons of the oculomotor system as a model for axotomy, we report that trophic support by brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) or a mixture of both, delivered to the stump of severed axons, results in either the prevention of synaptic stripping when administered immediately after lesion or in a promotion of reinnervation of afferents to abducens motoneurons once synaptic stripping had occurred, in concert with the recovery of synaptic potentials evoked from the vestibular nerve. Synaptotrophic effects, however, were larger when both neurotrophins were applied together. The axotomy-induced reduction in firing sensitivities related to eye movements were also restored to normal values when BDNF and NT-3 were administered, but discharge characteristics recovered in a complementary manner when only one neurotrophin was used. This is the first report to show selective retrograde trophic dependence of circuit-driven firing properties in vivo indicating that NT-3 restored the phasic firing, whereas BDNF supported the tonic firing of motoneurons during eye movement performance. Therefore, our data report a link between the synaptotrophic actions of neurotrophins, retrogradely delivered, and the alterations of neuronal firing patterns during motor behaviors. These trophic actions could be responsible, in part, for synaptic rearrangements that alter circuit stability and synaptic balance during plastic events of the brain.

Nerve growth factor regulates the firing patterns and synaptic composition of motoneurons.

Target-derived neurotrophins exert powerful synaptotrophic actions in the adult brain and are involved in the regulation of different forms of synaptic plasticity. Target disconnection produces a profound synaptic stripping due to the lack of trophic support. Consequently, target reinnervation leads to synaptic remodeling and restoration of cellular functions. Extraocular motoneurons are unique in that they normally express the TrkA neurotrophin receptor in the adult, a feature not seen in other cranial or spinal motoneurons, except after lesions such as axotomy or in neurodegenerative diseases like amyotrophic lateral sclerosis. We investigated the effects of nerve growth factor (NGF) by retrogradely delivering this neurotrophin to abducens motoneurons of adult cats. Axotomy reduced the density of somatic boutons and the overall tonic and phasic firing modulation. Treatment with NGF restored synaptic inputs and firing modulation in axotomized motoneurons. When K252a, a selective inhibitor of tyrosine kinase activity, was applied to specifically test TrkA effects, the NGF-mediated restoration of synapses and firing-related parameters was abolished. Discharge variability and recruitment threshold were, however, increased by NGF compared with control or axotomized motoneurons. Interestingly, these parameters returned to normal following application of REX, an antibody raised against neurotrophin receptor p75 (p75NTR). In conclusion, NGF, acting retrogradely through TrkA receptors, supports afferent boutons and regulates the burst and tonic signals correlated with eye movements. On the other hand, p75NTR activation regulates recruitment threshold, which impacts on firing regularity. To our knowledge, this is the first report showing powerful synaptotrophic effects of NGF on motoneurons in vivo.

Dual Encoding of Muscle Tension and Eye Position by Abducens Motoneurons

Extraocular muscle tension associated with spontaneous eye movements has a pulse-slide-step profile similar to that of motoneuron firing rate. Existing models only relate motoneuron firing to eye position, velocity and acceleration. We measured and quantitatively compared lateral rectus muscle force and eye position with the firing of abducens motoneurons in the cat to determine fundamental encoding correlations. During fixations (step), muscle force increased exponentially with eccentric eye position, consistent with a model of estimate ensemble motor innervation based on neuronal sensitivities and recruitment order. Moreover, firing rate in all motoneurons tested was better related to eye position than to muscle tension during fixations. In contrast, during the postsaccadic slide phase, the time constant of firing rate decay was closely related to that of muscle force decay, suggesting that all motoneurons encode muscle tension as well. Discharge characteristics of abducens motoneurons formed overlapping clusters of phasic and tonic motoneurons, thus, tonic units recruited earlier and had a larger slide signal. We conclude that the slide signal is a discharge characteristic of the motoneuron that controls muscle tension during the postsaccadic phase and that motoneurons are specialized for both tension and position-related properties. The organization of signal content in the pool of abducens motoneurons from the very phasic to the very tonic units is possibly a result of the differential trophic background received from distinct types of muscle fibers.

Axons Giving Rise to the Palisade Endings of Feline Extraocular Muscles Display Motor Features

Palisade endings are nerve specializations found in the extraocular muscles (EOMs) of mammals, including primates. They have long been postulated to be proprioceptors. It was recently demonstrated that palisade endings are cholinergic and that in monkeys they originate from the EOM motor nuclei. Nevertheless, there is considerable difference of opinion concerning the nature of palisade ending function. Palisade endings in EOMs were examined in cats to test whether they display motor or sensory characteristics. We injected an anterograde tracer into the oculomotor or abducens nuclei and combined tracer visualization with immunohistochemistry and α-bungarotoxin staining. Employing immunohistochemistry, we performed molecular analyses of palisade endings and trigeminal ganglia to determine whether cat palisade endings are a cholinergic trigeminal projection. We confirmed that palisade endings are cholinergic and showed, for the first time, that they, like extraocular motoneurons, are also immunoreactive for calcitonin gene-related peptide. Following tracer injection into the EOM nuclei, we observed tracer-positive palisade endings that exhibited choline acetyl transferase immunoreactivity. The tracer-positive nerve fibers supplying palisade endings also established motor terminals along the muscle fibers, as demonstrated by α-bungarotoxin. Neither the trigeminal ganglion nor the ophthalmic branch of the trigeminal nerve contained cholinergic elements. This study confirms that palisade endings originate in the EOM motor nuclei and further indicates that they are extensions of the axons supplying the muscle fiber related to the palisade. The present work excludes the possibility that they receive cholinergic trigeminal projections. These findings call into doubt the proposed proprioceptive function of palisade endings.

Palisade Endings Are a Constant Feature in the Extraocular Muscles of Frontal-Eyed, But Not Lateral-Eyed, Animals

Purpose To test whether palisade endings are a general feature of mammalian extraocular muscles (EOMs). Methods Thirteen species, some frontal-eyed (human, monkey, cat, and ferret), and others lateral-eyed (pig, sheep, calf, horse, rabbit, rat, mouse, gerbil, and guinea pig) were analyzed. Palisade endings were labeled by using different combinations of immunofluorescence techniques. Three-dimensional reconstructions of immunolabeled palisade endings were done. Results In all frontal-eyed species, palisade endings were a consistent feature in the rectus EOMs. Their total number was high and they exhibited an EOM-specific distribution. In particular, the number of palisade endings in the medial recti was significantly higher than in the other rectus muscles. In the lateral-eyed animals, palisade endings were infrequent and, when present, their total number was rather low. They were only found in ungulates (sheep, calf, pig, and horse) and in rabbit. In rodents (rat, guinea pig, mouse, and gerbil) palisade endings were found infrequently (e.g., rat) or were completely absent. Palisade endings in frontal-eyed species and in some lateral-eyed species (pig, sheep, calf, and horse) had a uniform morphology. They generally lacked α-bungarotoxin staining, with a few exceptions in primates. Palisade endings in other lateral-eyed species (rabbit and rat) exhibited a simplified morphology and bound α-bungarotoxin. Conclusions Palisade endings are not a universal feature of mammalian EOMs. So, if they are proprioceptors, not all species require them. Because in frontal-eyed species, the medial rectus muscle has the highest number of palisade endings, they likely play a special role in convergence.

Functional Diversity of Neurotrophin Actions on the Oculomotor System

Neurotrophins play a principal role in neuronal survival and differentiation during development, but also in the maintenance of appropriate adult neuronal circuits and phenotypes. In the oculomotor system, we have demonstrated that neurotrophins are key regulators of developing and adult neuronal properties, but with peculiarities depending on each neurotrophin. For instance, the administration of NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor) or NT-3 (neurotrophin-3) protects neonatal extraocular motoneurons from cell death after axotomy, but only NGF and BDNF prevent the downregulation in ChAT (choline acetyltransferase). In the adult, in vivo recordings of axotomized extraocular motoneurons have demonstrated that the delivery of NGF, BDNF or NT-3 recovers different components of the firing discharge activity of these cells, with some particularities in the case of NGF. All neurotrophins have also synaptotrophic activity, although to different degrees. Accordingly, neurotrophins can restore the axotomy-induced alterations acting selectively on different properties of the motoneuron. In this review, we summarize these evidences and discuss them in the context of other motor systems.

A chronically implantable device for the controlled delivery of substances, and stimulation and recording of activity in severed nerves.

We describe the use of an implantable device for peripheral nerves that allows chronic simultaneous delivery of small volumes of solution, recording of both field and multiunit potentials, and electrical stimulation. This custom-made multifunctional device was attached to the cut end of the abducens (VIth) nerve for stimulation, recording and injection purposes. Our device consists of a polyethylene chamber with two electrodes that can be used for stimulation and recording and two Teflon tubes that serve as inlet and outlet for administering chemicals to the nerve fitted inside. Since the device is implanted in a retro-orbital position, we herein will refer to it as an intraorbitary device (IOD). The applicability of the IOD is demonstrated with an electrophysiological and anatomical account of the properties of the abducens nerve. Furthermore, it is shown that certain neuronal discharge properties can be inferred from the nerve recordings. The IOD can also be efficiently used for the delivery of small volume of pharmacological substances or conventional retrograde markers.

Neural Progenitor Cell Implants in the Lesioned Medial Longitudinal Fascicle of Adult Cats Regulate Synaptic Composition and Firing Properties of Abducens Internuclear Neurons

Transplants of neural progenitor cells (NPCs) into the injured CNS have been proposed as a powerful tool for brain repair, but, to date, few studies on the physiological response of host neurons have been reported. Therefore, we explored the effects of NPC implants on the discharge characteristics and synaptology of axotomized abducens internuclear neurons, which mediate gaze conjugacy for horizontal eye movements. NPCs were isolated from the subventricular zone of neonatal cats and implanted at the site of transection in the medial longitudinal fascicle of adult cats. Abducens internuclear neurons of host animals showed a complete restoration of axotomy-induced alterations in eye position sensitivity, but eye velocity sensitivity was only partially regained. Analysis of the inhibitory and excitatory components of the discharge revealed a normal re-establishment of inhibitory inputs, but only partial re-establishment of excitatory inputs. Moreover, their inhibitory terminal coverage was similar to that in controls, indicating that there was ultimately no loss of inhibitory synaptic inputs. Somatic coverage by synaptophysin-positive contacts, however, showed intermediate values between control animals and animals that had undergone axotomy, likely due to partial loss of excitatory inputs. We also demonstrated that severed axons synaptically contacted NPCs, most of which were VEGF immunopositive, and that abducens internuclear neurons expressed the VEGF receptor Flk1. Together, our results suggest that VEGF neurotrophic support might underlie the increased inhibitory-to-excitatory balance observed in the postimplant cells. The noteworthy improvement of firing properties of injured neurons following NPC implants indicates that these cells might provide a promising therapeutic strategy after neuronal lesions.

Palisade endings of extraocular muscles develop postnatally following different time courses

Purpose To analyze in a frontal-eyed mammal (cat) the postnatal development of palisade endings in extraocular muscles (EOMs) and to compare the spatiotemporal and quantitative patterns of palisade endings among individual rectus muscles. Methods Cats of different ages ranging from birth to adult stage were studied. EOM whole-mount preparations were fluorescently labeled using six combinations of triple staining and analyzed in the confocal laser scanning microscope. Results Palisade endings developed postnatally and passed in each rectus muscle through the same, three developmental steps but in a heterochronic sequence and to a different final density per muscle. Specifically, palisade ending development was first completed in the medial rectus and later in the inferior, lateral, and superior rectus. The highest density of palisade endings was observed in the medial rectus and the lowest in the lateral rectus whereas values for the inferior and superior rectus were in between. Palisade endings expressed high levels of growth associated protein 43 during development and were supplied by axons that established motor terminals. Conclusions Cats open their eyes 7 to 10 days after birth and later develop a complex three-dimensional visuomotor climbing and jumping behavior depending on accurate binocular vision and fine tuning of the ocular movements. Our findings indicate that palisade ending development correlates with important landmarks in visuomotor behavior and provide support for our previous notion that palisade endings play an important role for convergence eye movements in frontal-eyed species.

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.