John S. Stahl

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.

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.

Mutation of the Cacna1a gene for the P/Q (Ca_V2.1) 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.

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.

Downbeat nystagmus (DBN) is a common eye movement complication of cerebellar disease. Use of mice to study pathophysiology of vestibulocerebellar disease is increasing, but it is unclear if mice can be used to study DBN; it has not been reported in this species. We determined whether DBN occurs in the ataxic mutant tottering, which carries a mutation in the Cacna1a gene for P/Q calcium channels. Spontaneous DBN occurred only rarely, and its magnitude did not exhibit the relationship to head tilt seen in human patients. DBN during yaw rotation was more common and shares some properties with the tilt-independent, gaze-independent component of human DBN, but differs in its dependence on vision. Hyperactivity of otolith circuits responding to pitch tilts is hypothesized to contribute to the gaze-independent component of human DBN. Mutants exhibited hyperactivity of the tilt maculo-ocular reflex (tiltMOR) in pitch. The hyperactivity may serve as a surrogate for DBN in mouse studies. TiltMOR hyperactivity correlates with hyperdeviation of the eyes and upward deviation of the head during ambulation; these may be alternative surrogates. Muscimol inactivation of the cerebellar flocculus suggests a floccular role in the tiltMOR hyperactivity and provides insight into the rarity of frank DBN in ataxic mice.

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.

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.