Impact of Purkinje Cell Simple Spike Synchrony on Signal Transmission from Flocculus.

Purkinje cells (PCs) in the cerebellar flocculus carry rate-coded information that ultimately drives eye movement. Floccular PCs lying nearby each other exhibit partial synchrony of their simple spikes (SS). Elsewhere in the cerebellum, PC SS synchrony has been demonstrated to influence activity of the PCs' synaptic targets, and some suggest it constitutes another vector for information transfer. We investigated in the cerebellar flocculus the extent to which the rate code and PC synchrony interact. One motivation for the study was to explain the cerebellar deficits in ataxic mice like tottering; we speculated that PC synchrony has a positive effect on rate code transmission that is lost in the mutants. Working in transgenic mice whose PCs express channelrhodopsin, we exploited a property of optogenetics to control PC synchrony: pulsed photostimulation engenders stimulus-locked spiking, whereas continuous photostimulation engenders spiking whose timing is unconstrained. We photoactivated flocculus PCs using pulsed stimuli with sinusoidally varying timing vs. continuous stimuli with sinusoidally varying intensity. Recordings of PC pairs confirmed that pulsed stimuli engendered greater PC synchrony. We quantified the efficiency of transmission of the evoked PC firing rate modulation from the amplitudes of firing rate modulation and eye movement. Rate code transmission was slightly poorer in the conditions that generated greater PC synchrony, arguing against our motivating speculation regarding the origin of ataxia in tottering. Floccular optogenetic stimulation prominently augmented a 250-300 Hz local field potential oscillation, and we demonstrate relationships between the oscillation power and the evoked PC synchrony.

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.

Flocculus Purkinje cell signals in mouse Cacna1a calcium channel mutants of escalating severity: an investigation of the role of firing irregularity in ataxia.

Mutation of the Cacna1a gene for the P/Q (CaV2.1) calcium channel invariably leads to cerebellar dysfunction. The dysfunction has been attributed to disrupted rhythmicity of cerebellar Purkinje cells, but the hypothesis remains unproven. If irregular firing rates cause cerebellar dysfunction, then the irregularity and behavioral deficits should covary in a series of mutant strains of escalating severity. We compared firing irregularity in floccular and anterior vermis Purkinje cells in the mildly affected rocker and moderately affected tottering Cacna1a mutants and normal C57BL/6 mice. We also measured the amplitude and timing of modulations of floccular Purkinje cell firing rate during the horizontal vestibuloocular reflex (VOR, 0.25-1 Hz) and the horizontal and vertical optokinetic reflex (OKR, 0.125-1 Hz). We recorded Purkinje cells selective for rotational stimulation about the vertical axis (VAPCs) and a horizontal axis (HAPCs). Irregularity scaled with behavioral deficit severity in the flocculus but failed to do so in the vermis, challenging the irregularity hypothesis. Mutant VAPCs exhibited unusually strong modulation during VOR and OKR, the response augmentation scaling with phenotypic severity. HAPCs exhibited increased OKR modulation but in tottering only. The data contradict prior claims that modulation amplitude is unaffected in tottering but support the idea that attenuated compensatory eye movements in Cacna1a mutants arise from defective transfer of Purkinje cell signals to downstream circuitry, rather than attenuated synaptic transmission within the cerebellar cortex. Shifts in the relative sizes of the VAPC and HAPC populations raise the possibility that Cacna1a mutations influence the development of floccular zone architecture.

Dynamics of abducens nucleus neurons in the awake mouse.

The mechanics of the eyeball and orbital tissues (the "ocular motor plant") are a fundamental determinant of ocular motor signal processing. The mouse is used increasingly in ocular motor physiology, but little is known about its plant mechanics. One way to characterize the mechanics is to determine relationships between extraocular motoneuron firing and eye movement. We recorded abducens nucleus neurons in mice executing compensatory eye movements during 0.1- to 1.6-Hz oscillation in the light. We analyzed firing rates to extract eye position and eye velocity sensitivities, from which we determined time constants of a viscoelastic model of the plant. The majority of abducens neurons were already active with the eye in its central rest position, with only 6% recruited at more abducted positions. Firing rates exhibited largely linear relationships to eye movement, although there was a nonlinearity consisting of increasing modulation in proportion to eye movement as eye amplitudes became small (due to reduced stimulus amplitude or reduced alertness). Eye position and velocity sensitivities changed with stimulus frequency as expected for an ocular motor plant dominated by cascaded viscoelasticities. Transfer function poles lay at approximately 0.1 and 0.9 s. Compared with previously studied animal species, the mouse plant is stiffer than the rabbit but laxer than cat and rhesus. Differences between mouse and rabbit can be explained by scaling for eye size (allometry). Differences between the mouse and cat or rhesus can be explained by differing ocular motor repertoires of animals with and without a fovea or area centralis.

Crossover trial of gabapentin and memantine as treatment for acquired nystagmus.

We conducted a masked, crossover, therapeutic trial of gabapentin (1,200mg/day) versus memantine (40 mg/day) for acquired nystagmus in 10 patients (aged 28-61 years; 7 female; 3 multiple sclerosis [MS]; 6 post-stroke; 1 post-traumatic). Nystagmus was pendular in 6 patients (4 oculopalatal tremor; 2 MS) and jerk upbeat, hemi-seesaw, torsional, or upbeat-diagonal in each of the others. For the group, both drugs reduced median eye speed (p < 0.001), gabapentin by 32.8% and memantine by 27.8%, and improved visual acuity (p < 0.05). Each patient improved with 1 or both drugs. Side effects included unsteadiness with gabapentin and lethargy with memantine. Both drugs should be considered as treatment for acquired forms of nystagmus.

Probing the mechanism of saccade-associated head movements through observations of head movement propensity and cognition in the elderly.

Humans may accomplish gaze shifts by eye-only saccades or combined eye-head saccades. The mechanisms that determine whether the head moves remain poorly understood. Many observations can be explained if phylogenetically ancient circuits generate eye-head saccades by default and frontal cerebral structures interrupt this synergy when eye-only saccades are preferable. Saccade-associated head movements have been reported to increase in the elderly. To test the hypothesis of frontal inhibition of head movements, we investigated whether the increase is associated with a decline in frontal cognitive function. We measured head movement tendencies and cognition in volunteers aged 61-80. Measures of head movement tendency included the customary range of eye eccentricity, customary range of head eccentricity, range of target eccentricities evoking predominantly eye-only saccades, and two measures of head amplitude variation as a function of target eccentricity. Cognitive measures encompassed verbal fluency, verbal memory, non-verbal memory, and executive function. There was no correlation between cognition and any measure of head movement tendency. We combined these elderly data with measurements of head movements in a group aged 21-67 and found mildly reduced, not increased, head movement tendencies with age. However, when confronted with a task that could be accomplished without moving the head, young subjects were more likely to cease all head movements. While inconclusive regarding the hypothesis of inhibition of saccade-associated head movements by cerebral structures, the results indicate the need to distinguish between mechanisms that define head movement tendencies and mechanisms that adapt head motion to the geometry of a specific task.

Eye-head coupling tendencies in stationary and moving subjects.

Humans exhibit considerable individuality in their propensity to make head movements during horizontal saccades. These variations originate in multiple quantifiable characteristics, including individuals' preferred ranges of gaze, eye-in-head, and head-on-neck eccentricity. Such "eye-head tendencies" have been uniformly assessed in seated subjects. It is unknown whether they continue to influence behavior when subjects are in motion. Previous studies of eye-head coordination in subjects ambulating in laboratories would predict that wholly different eye-head tendencies become ascendant when subjects ambulate. We tested this prediction by recording eye and head positions in normal subjects in an outdoor environment as they spontaneously regarded their surroundings while seated, passively riding in a wheelchair, and ambulating. Individuals exhibited the usual subject-to-subject variations in the preferred ranges of eye, head, and gaze position, but their own behavior was similar across the different conditions. While ambulation did affect some of the measured eye-head tendencies, passively riding had similar effects, indicating that these effects relate more to motion through the environment than to the act of walking. In a surprising departure from studies of eye-head coordination in subjects ambulating in laboratory environments, neither head nor gaze was particularly strongly aligned with the direction of travel. Thus, the neural mechanisms of walking do not demand that specific gaze or head orientations be maintained continuously, at least not in the common situation of a non-challenging path that can be negotiated without much attention. In such situations eye and head control is flexible, and the eye-head tendencies manifesting when stationary can emerge.

Mouse Extraocular Muscles and the Musculotopic Organization of Their Innervation.

The organization of extraocular muscles (EOMs) and their motor nuclei was investigated in the mouse due to the increased importance of this model for oculomotor research. Mice showed a standard EOM organization pattern, although their eyes are set at the side of the head. They do have more prominent oblique muscles, whose insertion points differ from those of frontal-eyed species. Retrograde tracers revealed that the motoneuron layout aligns with the general vertebrate plan with respect to nuclei and laterality. The mouse departed in some significant respects from previously studied species. First, more overlap between the distributions of muscle-specific motoneuronal pools was present in the oculomotor nucleus (III). Furthermore, motoneuron dendrites for each pool filled the entire III and extended beyond the edge of the abducens nucleus (VI). This suggests mouse extraocular motoneuron afferents must target specific pools based on features other than dendritic distribution and nuclear borders. Second, abducens internuclear neurons are located outside the VI. We concluded this because no unlabeled abducens internuclear neurons were observed following lateral rectus muscle injections and because retrograde tracer injections into the III labeled cells immediately ventral and ventrolateral to the VI, not within it. This may provide an anatomical substrate for differential input to motoneurons and internuclear neurons that allows rodents to move their eyes more independently. Finally, while soma size measurements suggested motoneuron subpopulations supplying multiply and singly innervated muscle fibers are present, markers for neurofilaments and perineuronal nets indicated overlap in the size distributions of the two populations. Anat Rec, 302:1865-1885, 2019. © 2019 American Association for Anatomy.

Eye orientation during static tilts and its relationship to spontaneous head pitch in the laboratory mouse.

Both eye position and head orientation are influenced by the macular (otolith) organs, via the tilt maculo-ocular reflex (tiltMOR) and the vestibulo-collic reflexes, respectively. The mechanisms that control head position also influence the rest position of the eye because head orientation influences eye position through the tiltMOR. Despite the increasing popularity of mice for studies of vestibular and ocular motor functions, relatively little is known in this species about tiltMOR, spontaneous orientation of the head, and their interrelationship. We used 2D video oculography to determine in C57BL/6 mice the absolute horizontal and vertical positions of the eyes over body orientations spanning 360 degrees about the pitch and roll axes. We also determined head pitch during ambulation in the same animals. Eye elevation varied approximately sinusoidally as functions of pitch or roll angle. Over the central +/-30 degrees of pitch, sensitivity and gain in the light were 31.7 degrees/g and 0.53, respectively. The corresponding values for roll were 31.5 degrees/g and 0.52. Absolute positions adopted in light and darkness differed only slightly. During ambulation, mice carried the lambda-bregma plane at a downward pitch of 29 degrees , corresponding to a horizontal eye position of 64 degrees and a vertical eye position of 22 degrees . The vertical position is near the center of the range of eye movements produced by the pitch tiltMOR. The results indicate that the tiltMOR is robust in this species and favor standardizing pitch orientation across laboratories. The robust tiltMOR also has significant methodological implications for the practice of pupil-tracking video oculography in this species.

Idiosyncratic variations in eye-head coupling observed in the laboratory also manifest during spontaneous behavior in a natural setting.

The tendency to generate head movements during saccades varies from person to person. Head movement tendencies can be measured as subjects fixate sequences of illuminated targets, but the extent to which such measures reflect eye-head coupling during more natural behaviors is unknown. We quantified head movement tendencies in 20 normal subjects in a conventional laboratory experiment and in an outdoor setting in which the subjects directed their gaze spontaneously. In the laboratory, head movement tendencies during centrifugal saccades could be described by the eye-only range (EOR), customary ocular motor range (COMR), and the customary head orientation range (CHOR). An analogous EOR, COMR, and CHOR could be extracted from the centrifugal saccades executed in the outdoor setting. An additional six measures were introduced to describe the preferred ranges of eyes-in-head and head-on-torso manifest throughout the outdoor recording, i.e., not limited to the orientations following centrifugal saccades. These 12 measured variables could be distilled by factor analysis to one indoor and six outdoor factors. The factors reflect separable tendencies related to preferred ranges of visual search, head eccentricity, and eye eccentricity. Multiple correlations were found between the indoor and outdoor factors. The results demonstrate that there are multiple types of head movement tendencies, but some of these influence behavior across rather different experimental settings and tasks. Thus behavior in the two settings likely relies on common neural mechanisms, and the laboratory assays of head movement tendencies succeed in probing the mechanisms underlying eye-head coupling during more natural behaviors.

Functional and genomic changes in the mouse ocular motor system in response to light deprivation from birth.

Previous studies have suggested that abnormal visual experience early in life induces ocular motor abnormalities. The purpose of this study was to determine how visual deprivation alters the function and gene expression profile of the ocular motor system in mice. We measured the effect of dark rearing on eye movements, gene expression in the oculomotor nucleus, and contractility of isolated extraocular muscles. In vivo eye movement recordings showed decreased gains for optokinetic and vestibulo-ocular reflexes, confirming an effect of dark rearing on overall ocular motor function. Saccade peak velocities were preserved, however, arguing that the quantitative changes in these reflexes were not secondary to limitations in force generation. Using microarrays and quantitative PCR, we found that dark rearing shifted the oculomotor nucleus transcriptome to a state of delayed/arrested development. The expression of 132 genes was altered by dark rearing; these genes fit in various functional categories (signal transduction, transcription/translation control, metabolism, synaptic function, cytoskeleton), and some were known to be associated with neuronal development and plasticity. Extraocular muscle contractility was impaired by dark rearing to a greater extent than expected from the in vivo ocular motility studies: changes included decreased force and shortening speed and evidence of abnormal excitability. The results indicate that normal development of the mouse ocular motor system and its muscles requires visual experience. The transcriptional pattern of arrested development may indicate that vision is required to establish the adult pattern, but it also may represent the plastic response of oculomotor nuclei to abnormal extraocular muscles.

Eliminating the Ant1 isoform produces a mouse with CPEO pathology but normal ocular motility.

PURPOSE: The adenine nucleotide transporter 1 gene (ANT1) encodes an inner mitochondrial membrane protein that transports ATP into the cell. Mutations within ANT1 produce a syndrome of chronic progressive external ophthalmoplegia (CPEO) in humans. Ant1 knockout (Ant1-/-) mice develop cardiomyopathy and mitochondrial myopathy of limb muscles. Because the extraocular muscles (EOM) are preferentially affected in human CPEO, the objective of this study was to determine whether Ant1-/- mice also exhibit an EOM mitochondrial myopathy. METHODS: ANT isoform expression of isolated EOMs, EOM morphology and mitochondrial content, mitochondrial structure and function, ocular motility in intact mice, and contractile performance in isolated muscle preparations were examined. RESULTS: Ant1-/- EOMs had the typical appearance of mitochondrial myopathy, including increase in mitochondrial size, number, and oxidative phosphorylation (OXPHOS) staining. However, there were no measurable ocular motor abnormalities in intact Ant1-/- mice, and their isolated EOMs did not show evidence of increased fatigability. EOMs of wild-type mice exhibited higher levels of Ant2 mRNA compared with hindlimb muscle, which may compensate for the Ant1 loss in mutant mouse EOMs and account for the normal EOM function. CONCLUSIONS: The Ant1-/- mice provide a model in which to study CPEO pathology and compensatory mechanisms.

Characterization of vestibular dysfunction in the mouse model for Usher syndrome 1F.

The deaf-circling Ames waltzer (av) mouse harbors a mutation in the protocadherin 15 (Pcdh15) gene and is a model for inner ear defects associated with Usher syndrome type 1F. Earlier studies showed altered cochlear hair cell morphology in young av mice. In contrast, no structural abnormality consistent with significant vestibular dysfunction in young av mice was observed. Light and scanning electron microscopic studies showed that vestibular hair cells from presumptive null alleles Pcdh15(av-Tg) and Pcdh15(av-3J) are morphologically similar to vestibular sensory cells from control littermates, suggesting that the observed phenotype in these alleles might be a result of a central, rather than peripheral, defect. In the present study, a combination of physiologic and anatomic methods was used to more thoroughly investigate the source of vestibular dysfunction in Ames waltzer mice. Analysis of vestibular evoked potentials and angular vestibulo-ocular reflexes revealed a lack of physiologic response to linear and angular acceleratory stimuli in Pcdh15 mutant mice. Optokinetic reflex function was diminished but still present in the mutant animals, suggesting that the defect is primarily peripheral in nature. These findings indicate that the mutation in Pcdh15 results in either a functional abnormality in the vestibular receptor organs or that the defects are limited to the vestibular nerve. AM1-43 dye uptake has been shown to correlate with normal transduction function in hair cells. Dye uptake was found to be dramatically reduced in Pcdh15 mutants compared to control littermates, suggesting that the mutation affects hair cell function, although structural abnormalities consistent with significant vestibular dysfunction are not apparent by light and scanning electron microscopy in the vestibular neuroepithelia of young animals.

Neural integrator function in murine CACNA1A mutants.

Time constants of gaze holding are shortened in rocker and tottering mice, two strains whose cerebellar dysfunction stems from genetic alterations of the P/Q calcium channel. The finding suggests that in mice as in primates, the cerebellum contributes to the function of the neural integrator. Studying CACNA1A mutants may elucidate how cerebellar signals support gaze holding.

Internuclear ophthalmoparesis in episodic ataxia type 2.

Two patients sharing a novel mutation of the CACNA1A gene for P/Q calcium channels showed significant slowing of adducting saccades compared with normal subjects or patients with cerebellar disease. Internuclear ophthalmoparesis (INO) was clinically evident in one. While these findings might be specific to this mutation, INO in our patients with episodic ataxia type 2 suggested involvement outside the cerebellum, either in the brain-stem internuclear pathway or at the neuromuscular junction.

Abnormal eye movements predict disability in MS: two-year follow-up.

We conducted a two-year follow-up study of 40 patients with MS in whom we had reported that abnormal eye movements (AEM) were associated with greater general disability. AEM patients (17/40) remained significantly (p < .001) more disabled (median EDSS of 7.0) than those with normal eye movements (median EDSS of 5.0). AEM and great disability were associated with abnormal MRI signals in brainstem or cerebellum, where disease may involve control circuits for eye movements as well as descending motor pathways.

Amplitudes of head movements during putative eye-only saccades.

The mechanisms allowing humans and other primates to dissociate head and eye movements during saccades are poorly understood. A more precise knowledge of head movement behavior during apparent eye-only saccades may provide insight into those mechanisms. We studied the distributions of head amplitude in normal humans. In half of the subjects, these distributions indicated the presence of a population of minor ("residual") head movements during eye-only saccades, distinct from the continuum of head movements generated during frank eye-head saccades. Like full-sized head movements, the residual movements grew in proportion to target eccentricity, indicating their drive is derived from the premotor command for the saccade. Furthermore, their amplitudes related most strongly to the head amplitudes obtained when subjects produced full-sized head movements and were reduced when subjects were instructed to perform exclusively eye-only saccades. Both observations suggest that the drive for residual head movements originates downstream of the point in which the head movement command diverges from the generalized gaze shift command. The results are consistent with a model of head control in which a neural gate prevents the common gaze shift command from reaching the head premotor circuitry whenever an eye-only saccade is desired. However, the gate is either imperfect or the multiple pathways that relay gaze shift signals to the head motor circuitry allow for the gate to be circumvented. The results underscore the need for physiological studies to probe neuronal activity related to neck activation during eye-only saccades.

Calcium channelopathy mutants and their role in ocular motor research.

Thanks to technical advances in eye movement recording, the mouse is destined to become increasingly important in ocular motor research. An advantage of this species is the wide range of existing mutant strains and techniques to generate new mutations affecting specific cell types. Mutations of ion channels may be used to modulate the intrinsic properties of neurons, and this approach may generate insight into the degree to which neuronal computations depend upon those intrinsic properties as opposed to the properties of circuits of neurons. Dendritic calcium currents carried by P-type voltage-activated calcium channels have been widely postulated to perform important computational functions in cerebellar Purkinje cells. Mutations of this channel lead to human diseases, and several ataxic strains of mice are now known to harbor mutations of this calcium channel. Murine P-channel mutants such as rocker are ataxic, but have normal or near-normal numbers of cerebellar Purkinje cells and thus offer the opportunity to study the effects of biophysical perturbations as opposed to outright cell destruction or inactivation. Initial studies of rocker mice reveal an array of ocular motor abnormalities, including static hyperdeviation of the eyes and an attenuation of vestibulo-ocular reflex gains at high stimulus frequencies. The pattern of gain and phase abnormalities is entirely different in lurcher, an ataxic mutant in which Purkinje cells degenerate. The ocular motor abnormalities of rocker progress with animal age, underscoring the importance of careful attention to animal age when performing ocular motor studies in this short-lived species.

Using eye movements to assess brain function in mice.

Examining eye movements is an important part of the neurological evaluation of humans; the distribution of the neural circuits that control these movements is such that they are disrupted--often in highly characteristic fashions--by many disease processes. Technical advances have made it possible to measure accurately the eye movements of mice, so it is now possible to use the detective power of eye movement recording to characterize neurological dysfunction in genetically altered strains. Here we introduce analytical tools used in ocular motor research and demonstrate their ability to reveal disorders of the visual pathways, inner ear, and cerebellum.

4-aminopyridine does not enhance flocculus function in tottering, a mouse model of vestibulocerebellar dysfunction and ataxia.

The potassium channel antagonist 4-aminopyridine (4-AP) improves a variety of motor abnormalities associated with disorders of the cerebellum. The most rigorous quantitative data relate to 4-AP's ability to improve eye movement deficits in humans referable to dysfunction of the cerebellar flocculus. Largely based on work in the ataxic mouse mutant tottering (which carries a mutation of the Cacna1a gene of the P/Q voltage-activated calcium channel), 4-AP is hypothesized to function by enhancing excitability or rhythmicity of floccular Purkinje cells. We tested this hypothesis by determining whether systemic or intrafloccular administration of 4-AP would ameliorate the eye movement deficits in tottering that are attributable to flocculus dysfunction, including the reductions in amplitude of the yaw-axis vestibulo-ocular reflex (VOR) and vision-enhanced vestibulo-ocular reflex (VVOR), and the optokinetic reflex (OKR) about yaw and roll axes. Because tottering's deficits increase with age, both young and elderly mutants were tested to detect any age-dependent 4-AP effects. 4-AP failed to improve VOR, VVOR, and OKR gains during sinusoidal stimuli, although it may have reduced the tendency of the mutants' responses to VOR and VVOR to decline over the course of a one-hour recording session. For constant-velocity optokinetic stimuli, 4-AP generated some enhancement of yaw OKR and upward-directed roll OKR, but the effects were also seen in normal C57BL/6 controls, and thus do not represent a specific reversal of the electrophysiological consequences of the tottering mutation. Data support a possible extra-floccular locus for the effects of 4-AP on habituation and roll OKR. Unilateral intrafloccular 4-AP injections did not affect ocular motility, except to generate mild eye elevations, consistent with reduced floccular output. Because 4-AP did not produce the effects expected if it normalized outputs of floccular Purkinje cells, there is a need for further studies to elucidate the drug's mechanism of action on cerebellar motor dysfunction.