Kathleen E. Cullen’s research while affiliated with Johns Hopkins University and other places

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here, we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

The vestibular system is vital for maintaining stable vision during daily activities. When peripheral vestibular input is lost, patients initially experience impaired gaze stability due to reduced effectiveness of the vestibular-ocular-reflex pathway. To aid rehabilitation, patients are often prescribed gaze-stabilization exercises during which they make self-initiated active head movements. Analyzing statistical pattern of sequences of stereotyped behaviors to characterize degrees of randomness or repeatability has proven to be a powerful approach for diagnosing disease states, yet this approach has not been applied to patients with vestibular loss. Accordingly, here we investigated whether the patterning of head movements is altered in vestibular-loss patients by using trial-based analysis and sample-entropy measurement. The subjects completed gaze-stability exercises in both the yaw and pitch directions. In trial-based analysis, we calculated the trial-to-trial variability of head movement duration and peak velocity for each individual head movement. Our results showed that healthy individuals exhibited a temporally repetitive (correlated) structure in peak velocity, especially for head movements in the pitch direction, which was absent in most patients. In the sample entropy analysis, which measures the irregularity or randomness of a time series, our results revealed that head-movement generation was more regular in vestibular-loss patients compared to healthy controls. Together, these analyses suggest that vestibular-loss patients display less flexibility in the patterning of their head motions. Our results provide the first experimental evidence that temporal head stability is a valuable metric for distinguishing individuals with impaired vestibular function from healthy ones.

The vestibular system is integral to behavior; the loss of peripheral vestibular function leads to disabling consequences, such as blurred vision, dizziness, and unstable posture, severely limiting activities of daily living. Fortunately, the vestibular system’s well-defined peripheral structure and well-understood encoding strategies offer unique opportunities for developing sensory prostheses to restore vestibular function. While these devices show promising results in both animal models and implanted patients, substantial room for improvement remains. Research from an engineering perspective has largely focused on optimizing stimulation protocol to improve outcomes. However, this approach has often been pursued in isolation from research in neuroscience that has enriched our understanding of neural responses at the synaptic, cellular, and circuit levels. Accordingly, this review bridges the domains of neuroscience and engineering to consider recent progress and challenges in vestibular prosthesis development. We advocate for interdisciplinary approaches that leverage studies of neural circuits at the population level, especially in light of recent advancement in large-scale recording technology, to identify impediments still to overcome and to develop more naturalistic stimulation strategies. Fully integrating neuroscience and engineering in the context of prosthesis development will help advance the field forward and ultimately improve patient outcomes.

Swim tests are highly effective for identifying vestibular deficits in rodents by offering significant vestibular motor challenges with reduced proprioceptive input, unlike rotarod and balance beam tests. Traditional swim tests rely on subjective assessments, limiting objective quantification and reproducibility. We present a novel instrumented swim test using a miniature motion sensor with a 3D accelerometer and 3D gyroscope affixed to the rodent’s head. This setup robustly quantifies six-dimensional motion—three translational and three rotational axes—during swimming with high temporal resolution. We demonstrate the test’s capabilities by comparing head movements of Gpr156-/- mutant mice, which have impaired otolith organ development, to their heterozygous littermates. Our results show axis-specific differences in head movement probability distribution functions and dynamics that identify mice with the Gpr156 mutation. Axis-specific power spectrum analyses reveal selective movement alterations within distinct frequency ranges. Additionally, our spherical visualization and 3D analysis quantifies swimming performance based on head vector distance from upright. We use this analysis to generate a single classifier metric—a weighted average of an animal’s head deviation from upright during swimming. This metric effectively distinguishes animals with vestibular dysfunction from those with normal vestibular function. Overall, this instrumented swim test provides quantitative metrics for assessing performance and identifying subtle, axis- and frequency-specific deficits not captured by existing systems. This novel quantitative approach can enhance understanding of rodent sensorimotor function including enabling more selective and reproducible studies of vestibular-motor deficits.

Otolith organs in the inner ear and neuromasts in the fish lateral-line harbor two populations of hair cells oriented to detect stimuli in opposing directions. The underlying mechanism is highly conserved: the transcription factor EMX2 is regionally expressed in just one hair cell population and acts through the receptor GPR156 to reverse cell orientation relative to the other population. In mouse and zebrafish, loss of Emx2 results in sensory organs that harbor only one hair cell orientation and are not innervated properly. In zebrafish, Emx2 also confers hair cells with reduced mechanosensory properties. Here, we leverage mouse and zebrafish models lacking GPR156 to determine how detecting stimuli of opposing directions serves vestibular function, and whether GPR156 has other roles besides orienting hair cells. We find that otolith organs in Gpr156 mouse mutants have normal zonal organization and normal type I-II hair cell distribution and mechano-electrical transduction properties. In contrast, gpr156 zebrafish mutants lack the smaller mechanically evoked signals that characterize Emx2-positive hair cells. Loss of GPR156 does not affect orientation-selectivity of afferents in mouse utricle or zebrafish neuromasts. Consistent with normal otolith organ anatomy and afferent selectivity, Gpr156 mutant mice do not show overt vestibular dysfunction. Instead, performance on two tests that engage otolith organs is significantly altered – swimming and off-vertical-axis rotation. We conclude that GPR156 relays hair cell orientation and transduction information downstream of EMX2, but not selectivity for direction-specific afferents. These results clarify how molecular mechanisms that confer bi-directionality to sensory organs contribute to function, from single hair cell physiology to animal behavior.

Otolith organs in the inner ear and neuromasts in the fish lateral-line harbor two populations of hair cells oriented to detect stimuli in opposing directions. The underlying mechanism is highly conserved: the transcription factor EMX2 is regionally expressed in just one hair cell population and acts through the receptor GPR156 to reverse cell orientation relative to the other population. In mouse and zebrafish, loss of Emx2 results in sensory organs that harbor only one hair cell orientation and are not innervated properly. In zebrafish, Emx2 also confers hair cells with reduced mechanosensory properties. Here, we leverage mouse and zebrafish models lacking GPR156 to determine how detecting stimuli of opposing directions serves vestibular function, and whether GPR156 has other roles besides orienting hair cells. We find that otolith organs in Gpr156 mouse mutants have normal zonal organization and normal type I-II hair cell distribution and mechano-electrical transduction properties. In contrast, gpr156 zebrafish mutants lack the smaller mechanically-evoked signals that characterize Emx2-positive hair cells. Loss of GPR156 does not affect orientation-selectivity of afferents in mouse utricle or zebrafish neuromasts. Consistent with normal otolith organ anatomy and afferent selectivity, Gpr156 mutant mice do not show overt vestibular dysfunction. Instead, performance on two tests that engage otolith organs is significantly altered – swimming and off-vertical-axis rotation. We conclude that GPR156 relays hair cell orientation and transduction information downstream of EMX2, but not selectivity for direction-specific afferents. These results clarify how molecular mechanisms that confer bi-directionality to sensory organs contribute to function, from single hair cell physiology to animal behavior.

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

Quantitatively relating behavior to underlying biology is crucial in life science. Although progress in keypoint tracking tools has reduced barriers to recording postural data, identifying specific behaviors from this data remains challenging. Manual behavior coding is labor-intensive and inconsistent, while automatic methods struggle to explicitly define complex behaviors, even when they seem obvious to the human eye. Here, we demonstrate an effective technique for detecting circling in mice, a form of locomotion characterized by stereotyped spinning. Despite circling's extensive history as a behavioral marker, there currently exists no standard automated detection method. We developed a circling detection technique using simple postprocessing of keypoint data obtained from videos of freely-exploring (Cib2−/−;Cib3−/−) mutant mice, a strain previously found to exhibit circling behavior. Our technique achieves statistical parity with independent human observers in matching occurrence times based on human consensus, and it accurately distinguishes between videos of wild type mice and mutants. Our pipeline provides a convenient, noninvasive, quantitative tool for analyzing circling mouse models without the need for software engineering experience. Additionally, as the concepts underlying our approach are agnostic to the behavior being analyzed, and indeed to the modality of the recorded data, our results support the feasibility of algorithmically detecting specific research-relevant behaviors using readily-interpretable parameters tuned on the basis of human consensus.

Introduction: Aging is associated with loss of balance, with falls being one of the leading causes of death among the elderly in the USA. Gaze stabilization exercises (GSE) improve balance control in vestibular populations and could be useful to prevent falls in healthy individuals. However, the extent to which aging affects head kinematics in GSE is unknown. Methods: Forty-eight younger (n = 25, 24 ± 6 years, 60% female) and older (n = 23, 66 ± 5 years, 56% female) adults completed six 30-s GSE. Participants were asked to maintain gaze fixation on a stationary target while continuously performing head movements in pitch (e.g., vertical) and yaw (e.g., horizontal) directions. The visual target was placed on the wall 1 m or 2 m away or handheld at arm's length. Head kinematics were recorded with an inertial measurement unit placed on the back of the participants' head. Results: Older adults took significantly more time (e.g., delay) to complete cycles of head rotation in both pitch and yaw compared to younger participants across all GSE. Such delay was further increased during yaw head rotation while fixating gaze of the 1 m target. The average peak velocity (APV) and average angular displacement (AAD), however, were equivalent between groups in all GSE. Conclusion: Aging leads to the maintenance of head rotation APV and AAD at the expense of delayed cycles of head rotation, suggesting an age-dependent prioritization strategy (e.g., adapt duration first, range second) during continuous head movements. The distance of the visual target and head movement direction influenced elderly performance and should be considered when prescribing GSE to older populations.