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.

Reduced activity of vertically acting motoneurons during convergence.

Previous studies have revealed unexpected relationships between the firing rates of horizontally acting motoneurons and vergence. During a vergence task, for example, antidromically identified abducens internuclear neurons show a negative correlation between vergence angle and firing rate, which is the opposite of the modulation displayed by the medial rectus motoneurons to which they project. For a given horizontal eye position, medial rectus motoneurons discharge at a higher rate if the eyes are converged than if the same eye position is reached during a task that requires version; paradoxically, however, the horizontal rectus eye muscles show corelaxation during vergence. These complex and unexpected relationships inspired the present author to investigate whether the tonic firing rates of vertically acting motoneurons in oculomotor nucleus are correlated with vergence angle. Monkeys were trained to fixate a single, randomly selected, visual target among an array of 60 red plus-shaped LEDs, arranged at 12 different distances in three-dimensional space. The targets were arranged to permit dissociation of vertical eye position and vergence angle. Here I report, for the first time, that most vertically acting motoneurons in oculomotor nucleus show a significant negative correlation between tonic firing rate and vergence angle. This suggests the possibility that there may be a general corelaxation of extraocular muscles during vergence.NEW & NOTEWORTHY An array of 60 plus-shaped LEDs, positioned at various locations in three-dimensional space, was used to elicit conjugate and disjunctive saccades while single neurons in oculomotor nucleus were recorded from rhesus monkeys. This study demonstrates that most vertically acting motoneurons in oculomotor nucleus discharge at a lower rate when the eyes are converged.

Functional Organization of Vestibulo-Ocular Responses in Abducens Motoneurons.

Vestibulo-ocular reflexes (VORs) are the dominating contributors to gaze stabilization in all vertebrates. During horizontal head movements, abducens motoneurons form the final element of the reflex arc that integrates visuovestibular inputs into temporally precise motor commands for the lateral rectus eye muscle. Here, we studied a possible differentiation of abducens motoneurons into subtypes by evaluating their morphology, discharge properties, and synaptic pharmacology in semi-intact in vitro preparations of larval Xenopus laevis Extracellular nerve recordings during sinusoidal head motion revealed a continuum of resting rates and activation thresholds during vestibular stimulation. Differences in the sensitivity to changing stimulus frequencies and velocities allowed subdividing abducens motoneurons into two subgroups, one encoding the frequency and velocity of head motion (Group I), and the other precisely encoding angular velocity independent of stimulus frequency (Group II). Computational modeling indicated that Group II motoneurons are the major contributor to actual eye movements over the tested stimulus range. The segregation into two functional subgroups coincides with a differential activation of glutamate receptor subtypes. Vestibular excitatory inputs in Group I motoneurons are mediated predominantly by NMDA receptors and to a lesser extent by AMPA receptors, whereas an AMPA receptor-mediated excitation prevails in Group II motoneurons. Furthermore, glycinergic ipsilateral vestibular inhibitory inputs are activated during the horizontal VOR, whereas the tonic GABAergic inhibition is presumably of extravestibular origin. These findings support the presence of physiologically and pharmacologically distinct functional subgroups of extraocular motoneurons that act in concert to mediate the large dynamic range of extraocular motor commands during gaze stabilization.SIGNIFICANCE STATEMENT Outward-directed gaze-stabilizing eye movements are commanded by abducens motoneurons that combine different sensory inputs including signals from the vestibular system about ongoing head movements (vestibulo-ocular reflex). Using an amphibian model, this study investigates whether different types of abducens motoneurons exist that become active during different types of eye movements. The outcome of this study demonstrates the presence of specific motoneuronal populations with pharmacological profiles that match their response dynamics. The evolutionary conservation of the vestibulo-ocular circuitry makes it likely that a similar motoneuronal organization is also implemented in other vertebrates. Accordingly, the physiological and pharmacological understanding of specific motoneuronal contributions to eye movements might help in designing drug therapies for human eye movement dysfunctions such as abducens nerve palsy.

Usefulness of intraoperative electromyographic monitoring of oculomotor and abducens nerves during skull base surgery.

BACKGROUND: Intraoperative neurophysiologic monitoring of the extraocular cranial nerve (EOCN) is not commonly performed because of technical difficulty and risk, reliability of the result and predictability of the postoperative function of the EOCN. METHODS: We performed oculomotor nerve (CN III) and abducens nerve (CN VI) intraoperative monitoring in patients with skull base surgery by recording the spontaneous muscle activity (SMA) and compound muscle action potential (CMAP). Two types of needle electrodes of different length were percutaneously inserted into the extraocular muscles with the free-hand technique. We studied the relationships between the SMA and CMAP and postoperative function of CN III and CN VI. RESULTS: A total of 23 patients were included. Nineteen oculomotor nerves and 22 abducens nerves were monitored during surgery, respectively. Neurotonic discharge had a positive predictive value of less than 50% and negative predictive value of more than 80% for postoperative CN III and CN VI dysfunction. The latency of patients with postoperative CN III dysfunction was 2.79 ± 0.13 ms, longer than that with intact CN III function (1.73 ± 0.11 ms). One patient had transient CN VI dysfunction, whose CMAP latency (2.54 ms) was longer than that of intact CN VI function (2.11 ± 0.38 ms). There was no statistically significant difference between patients with paresis and with intact function. CONCLUSIONS: The method of intraoperative monitoring of EOCNs described here is safe and useful to record responses of SMA and CMAP. Neurotonic discharge seems to have limited value in predicting the postoperative function of CN III and CN VI. The onset latency of CMAP longer than 2.5 ms after tumor removal is probably relevant to postoperative CN III and CN VI dysfunction. However, a definite quantitative relationship has not been found between the amplitude and stimulation intensity of CMAP and the postoperative outcome of CN III and CN VI.

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.

Mechanics of mouse ocular motor plant quantified by optogenetic techniques.

Rigorous descriptions of ocular motor mechanics are often needed for models of ocular motor circuits. The mouse has become an important tool for ocular motor studies, yet most mechanical data come from larger species. Recordings of mouse abducens neurons indicate the mouse mechanics share basic viscoelastic properties with larger species but have considerably longer time constants. Time constants can also be extracted from the rate at which the eye re-centers when released from an eccentric position. The displacement can be accomplished by electrically stimulating ocular motor nuclei, but electrical stimulation may also activate nearby ocular motor circuitry. We achieved specific activation of abducens motoneurons through photostimulation in transgenic mice expressing channelrhodopsin in cholinergic neurons. Histology confirmed strong channelrhodopsin expression in the abducens nucleus with relatively little expression in nearby ocular motor structures. Stimulation was delivered as 20- to 1,000-ms pulses and 40-Hz trains. Relaxations were modeled best by a two-element viscoelastic system. Time constants were sensitive to stimulus duration. Analysis of isometric relaxation of isolated mouse extraocular muscles suggest the dependence is attributable to noninstantaneous decay of active forces in non-twitch fibers following stimulus offset. Time constants were several times longer than those obtained in primates, confirming that the mouse ocular motor mechanics are relatively sluggish. Finally, we explored the effects of 0.1- to 20-Hz sinusoidal photostimuli and demonstrated their potential usefulness in characterizing ocular motor mechanics, although this application will require further data on the temporal relationship between photostimulation and neuronal firing in extraocular motoneurons.

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.

Reaching the limit of the oculomotor plant: 3D kinematics after abducens nerve stimulation during the torsional vestibulo-ocular reflex.

Accumulating evidence shows that the oculomotor plant is capable of implementing aspects of three-dimensional kinematics such as Listing's law and the half-angle rule. But these studies have only examined the eye under static conditions or with movements that normally obey these rules (e.g., saccades and pursuit). Here we test the capability of the oculomotor plant to rearrange itself as necessary for non-half-angle behavior. Three monkeys (Macaca mulatta) fixated five vertically displaced targets along the midsagittal plane while sitting on a motion platform that rotated sinusoidally about the naso-occipital axis. This activated the torsional, rotational vestibulo-ocular reflex, which exhibits a zero-angle or negative-angle rule (depending on the visual stimulus). On random sinusoidal cycles, we stimulated the abducens nerve and observed the resultant eye movements. If the plant has rearranged itself to implement this non-half-angle behavior, then stimulation should reveal this behavior. On the other hand, if the plant is only capable of half-angle behavior, then stimulation should reveal a half-angle rule. We find the latter to be true and therefore additional neural signals are likely necessary to implement non-half-angle behavior.

The nucleus prepositus predominantly outputs eye movement-related information during passive and active self-motion.

Maintaining a constant representation of our heading as we move through the world requires the accurate estimate of spatial orientation. As one turns (or is turned) toward a new heading, signals from the semicircular canals are relayed through the vestibular system to higher-order centers that encode head direction. To date, there is no direct electrophysiological evidence confirming the first relay point of head-motion signals from the vestibular nuclei, but previous anatomical and lesion studies have identified the nucleus prepositus as a likely candidate. Whereas burst-tonic neurons encode only eye-movement signals during head-fixed eye motion and passive vestibular stimulation, these neurons have not been studied during self-generated movements. Here, we specifically address whether burst-tonic neurons encode head motion during active behaviors. Single-unit responses were recorded from the nucleus prepositus of rhesus monkeys and compared for head-restrained and active conditions with comparable eye velocities. We found that neurons consistently encoded eye position and velocity across conditions but did not exhibit significant sensitivity to head position or velocity. Additionally, response sensitivities varied as a function of eye velocity, similar to abducens motoneurons and consistent with their role in gaze control and stabilization. Thus our results demonstrate that the primate nucleus prepositus chiefly encodes eye movement even during active head-movement behaviors, a finding inconsistent with the proposal that this nucleus makes a direct contribution to head-direction cell tuning. Given its ascending projections, however, we speculate that this eye-movement information is integrated with other inputs in establishing higher-order spatial representations.

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.

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 (p75(NTR)). 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, p75(NTR) 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.

Intraoperative lateral rectus electromyographic recordings optimized by deep intraorbital needle electrodes.

OBJECTIVE: We demonstrate the advantages and safety of long, intraorbitally-placed needle electrodes, compared to standard-length subdermal electrodes, when recording lateral rectus electromyography (EMG) during intracranial surgeries. METHODS: Insulated 25 mm and uninsulated 13 mm needle electrodes, aimed at the lateral rectus muscle, were placed in parallel during 10 intracranial surgeries, examining spontaneous and stimulation-induced EMG activities. Postoperative complications in these patients were reviewed, alongside additional patients who underwent long electrode placement in the lateral rectus. RESULTS: In 40 stimulation-induced recordings from 10 patients, the 25 mm electrodes recorded 6- to 26-fold greater amplitude EMG waveforms than the 13 mm electrodes. The 13 mm electrodes detected greater unwanted volume conduction upon facial nerve stimulation, typically exceeding the amplitude of abducens nerve stimulation. Except for one case with lateral canthus ecchymosis, no clinical or radiographic complications occurred in 36 patients (41 lateral rectus muscles) following needle placement. CONCLUSIONS: Intramuscular recordings from long electrode in the lateral rectus offers more reliable EMG monitoring than 13 mm needles, with excellent discrimination between abducens and facial nerve stimulations, and without significant complications from needle placement. SIGNIFICANCE: Long intramuscular electrode within the orbit for lateral rectus EMG recording is practical and reliable for abducens nerve monitoring.

Stimulation of abducens nucleus supports classical conditioning of the nictitating membrane response.

The acquisition and terminal performance of a classical conditioning group compared with a control group indicated that extension of the nictitating membrane elicited by direct electrical stimulation of the abducens nucleus was successfully conditioned to a previously neutral stimulus. The conditioning so obtained was associative and not due to such nonassociative factors as sensitization, pseudo-conditioning, or alteration in base-rate responding.

Goldfish abducens motoneurons: physiological and anatomical specialization.

During natural movements, the motoneurons innervating a single muscle have different patterns of activity that are correlated with differences in synaptic input. The caudal abducens motoneurons fire phasically in synchronous bursts before rapid posterior eye movements; the rostral abducens motoneurons fire only tonically when the eye is fixed or moving slowly. This physiological difference is not related to motoneuron size. In this respect the abducens motoneurons violate the "size principle" that has been advanced for spinal motoneurons. The difference is probably related to the present finding that the caudal but not the rostral cells receive numerous electrical synapses that are known to have a role in synchronizing phasic activity.

Tonic inhibition and ponto-geniculo-occipital-related activities shape abducens motoneuron discharge during REM sleep.

Eye movements, ponto-geniculo-occipital (PGO) waves, muscular atonia and desynchronized cortical activity are the main characteristics of rapid eye movement (REM) sleep. Although eye movements designate this phase, little is known about the activity of the oculomotor system during REM sleep. In this work, we recorded binocular eye movements by the scleral search-coil technique and the activity of identified abducens (ABD) motoneurons along the sleep-wake cycle in behaving cats. The activity of ABD motoneurons during REM sleep was characterized by a tonic decrease of their mean firing rate throughout this period, and short bursts and pauses coinciding with the occurrence of PGO waves. We demonstrate that the decrease in the mean firing discharge was due to an active inhibition of ABD motoneurons, and that the occurrence of primary and secondary PGO waves induced a pattern of simultaneous but opposed phasic activation and inhibition on each ABD nucleus. With regard to eye movements, during REM sleep ABD motoneurons failed to codify eye position as during alertness, but continued to codify eye velocity. The pattern of tonic inhibition and the phasic activations and inhibitions shown by ABD motoneurons coincide with those reported in other non-oculomotor motoneurons, indicating that the oculomotor system - contrary to what has been accepted until now - is not different from other motor systems during REM sleep, and that all motor systems are receiving similar command signals during this period.

Electrophysiology of Extraocular Cranial Nerves: Oculomotor, Trochlear, and Abducens Nerve.

The utility of extraocular cranial nerve electrophysiologic recordings lies primarily in the operating room during skull base surgeries. Surgical manipulation during skull base surgeries poses a risk of injury to multiple cranial nerves, including those innervating extraocular muscles. Because tumors distort normal anatomic relationships, it becomes particularly challenging to identify cranial nerve structures. Studies have reported the benefits of using intraoperative spontaneous electromyographic recordings and compound muscle action potentials evoked by electrical stimulation in preventing postoperative neurologic deficits. Apart from surgical applications, electromyography of extraocular muscles has also been used to guide botulinum toxin injections in patients with strabismus and as an adjuvant diagnostic test in myasthenia gravis. In this article, we briefly review the rationale, current available techniques to monitor extraocular cranial nerves, technical difficulties, clinical and surgical applications, as well as future directions for research.

Three-dimensional kinematics at the level of the oculomotor plant.

Motor systems often require that superfluous degrees of freedom be constrained. For the oculomotor system, a redundancy in the degrees of freedom occurs during visually guided eye movements and is solved by implementing Listing's law and the half-angle rule, kinematic constraints that limit the range of eye positions and angular velocities used by the eyes. These constraints have been attributed either to neurally generated commands or to the physical mechanics of the eye and its surrounding muscles and tissues (i.e., the ocular plant). To directly test whether the ocular plant implements the half-angle rule, critical to the maintenance of Listing's law, we microstimulated the abducens nerve with the eye at different initial vertical eye positions. We report that the electrically evoked eye velocity exhibits the same eye position dependence as seen in visually guided smooth-pursuit eye movements. These results support an important role for the ocular plant in providing a solution to the degrees-of-freedom problem during eye movements.

Anatomic and physiological characteristics of the ferret lateral rectus muscle and abducens nucleus.

The ferret has become a popular model for physiological and neurodevelopmental research in the visual system. We believed it important, therefore, to study extraocular whole muscle as well as single motor unit physiology in the ferret. Using extracellular stimulation, 62 individual motor units in the ferret abducens nucleus were evaluated for their contractile characteristics. Of these motor units, 56 innervated the lateral rectus (LR) muscle alone, while 6 were split between the LR and retractor bulbi (RB) muscle slips. In addition to individual motor units, the whole LR muscle was evaluated for twitch, tetanic peak force, and fatigue. The abducens nucleus motor units showed a twitch contraction time of 15.4 ms, a mean twitch tension of 30.2 mg, and an average fusion frequency of 154 Hz. Single-unit fatigue index averaged 0.634. Whole muscle twitch contraction time was 16.7 ms with a mean twitch tension of 3.32 g. The average fatigue index of whole muscle was 0.408. The abducens nucleus was examined with horseradish peroxidase conjugated with the subunit B of cholera toxin histochemistry and found to contain an average of 183 motoneurons. Samples of LR were found to contain an average of 4,687 fibers, indicating an LR innervation ratio of 25.6:1. Compared with cat and squirrel monkeys, the ferret LR motor units contract more slowly yet more powerfully. The functional visual requirements of the ferret may explain these fundamental differences.

Subunit-specific synaptic delivery of AMPA receptors by auxiliary chaperone proteins TARPγ8 and GSG1L in classical conditioning.

AMPA receptor (AMPAR) trafficking has emerged as a fundamental concept for understanding mechanisms of learning and memory as well as many neurological disorders. Classical conditioning is a simple and highly conserved form of associative learning. Our studies use an ex vivo brainstem preparation in which to study cellular mechanisms underlying learning during a neural correlate of eyeblink conditioning. Two stages of AMPAR synaptic delivery underlie conditioning utilizing sequential trafficking of GluA1-containing AMPARs early in conditioning followed by replacement with GluA4 subunits later. Subunit-selective trafficking of AMPARs is poorly understood. Here, we focused on identification of auxiliary chaperone proteins that traffic AMPARs. The results show that auxiliary proteins TARPγ8 and GSG1L are colocalized with AMPARs on abducens motor neurons that generate the conditioning. Significantly, TARPγ8 was observed to chaperone GluA1-containing AMPARs during synaptic delivery early in conditioning while GSG1L chaperones GluA4 subunits later in conditioning. Interestingly, TARPγ8 remains at the membrane surface as GluA1 subunits are withdrawn and associates with GluA4 when they are delivered to synapses. These data indicate that GluA1- and GluA4-containing AMPARs are selectively chaperoned by TARPγ8 and GSG1L, respectively. Therefore, sequential subunit-selective trafficking of AMPARs during conditioning is achieved through the timing of their interactions with specific auxiliary proteins.

Identification of motoneurons supplying multiply- or singly-innervated extraocular muscle fibers in the rat.

In mammals, the extraocular muscle fibers can be categorized in singly-innervated and multiply-innervated muscle fibers. In the monkey oculomotor, trochlear and abducens nucleus the motoneurons of multiply-innervated muscle fibers lie separated from those innervating singly-innervated muscle fibers and show different histochemical properties. In order to discover, if this organization is a general feature of the oculomotor system, we investigated the location of singly-innervated muscle fiber and multiply-innervated muscle fiber motoneurons in the rat using combined tract-tracing and immunohistochemical techniques. The singly-innervated muscle fiber and multiply-innervated muscle fiber motoneurons of the medial and lateral rectus muscle were identified by retrograde tracer injections into the muscle belly or the distal myotendinous junction. The belly injections labeled the medial rectus muscle subgroup of the oculomotor nucleus or the greatest part of abducens nucleus, including some cells outside the medial border of abducens nucleus. In contrast, the distal injections labeled only a subset of the medial rectus muscle motoneurons and exclusively cells outside the medial border of abducens nucleus. The tracer detection was combined with immunolabeling using antibodies for perineuronal nets (chondroitin sulfate proteoglycan) and non-phosphorylated neurofilaments. In monkeys both antibodies permit a distinction between singly-innervated muscle fiber and multiply-innervated muscle fiber motoneurons. The experiments revealed that neurons labeled from a distal injection lack both markers and are assumed to represent multiply-innervated muscle fiber motoneurons, whereas those labeled from a belly injection are chondroitin sulfate proteoglycan- and non-phosphorylated neurofilament-immunopositive and assumed to represent singly-innervated muscle fiber motoneurons. The overall identification of multiply-innervated muscle fiber and singly-innervated muscle fiber motoneurons within the rat oculomotor nucleus, trochlear nucleus, and abducens nucleus revealed that the smaller multiply-innervated muscle fiber motoneurons tend to lie separate from the larger diameter singly-innervated muscle fiber motoneurons. Our data provide evidence that rat extraocular muscles are innervated by two sets of motoneurons that differ in their molecular, morphological, and anatomical properties.