Biology subjects

Hamling, K. R.

Publications and source records attributed to Hamling, K. R..

11 recordsLinked to original sources

bioRxiv2026

Transcriptional profiling of extraocular motor neurons reveals sim1a as a candidate strabismus-related gene

Strabismus, or misalignment of the eyes, is a heritable disorder frequently associated with vision loss and decreased quality of life. Incomitant strabismus, where the degree of misalignment differs based on gaze angle, can arise from mutations in genes that regulate the development of extraocular motor neurons. To date, few such genes have been identified. The extraocular motor system is highly conserved across vertebrates, suggesting a comparative transcriptomic discovery approach would be fruitful. Using bulk and single-cell sequencing in a small accessible vertebrate, the larval zebrafish, we identified genes expressed in subpopulations of extraocular motor neurons in cranial nuclei nIII/nIV. We next assessed extraocular motor neuron number and vestibulo-ocular reflex performance after CRISPR/Cas9-mediated mutagenesis of three genes with suggestive expression patterns: sim1a, nav2a, one-cut1, and one known to disrupt nIII/nIV motor neuron specification: phox2a. Loss of sim1a impaired the vestibulo-ocular reflex without change to nIII/nIV motor neuron number. Our data suggest that constitutive disruptions to sim1a can impair nIII/nIV-dependent eye movements. More broadly, our work illuminates considerable transcriptomic diversity among extraocular motor neuron subpopulations, and establishes a pipeline to identify genes relevant to ocular motor disease etiology.

neuroscience↗

bioRxiv2025

Activation of cannabinoid receptor CB1 leads to aberrant myelination in development

The endocannabinoid system (ECS) has a widespread role in the development and function of the central nervous system (CNS). Cannabinoid receptors like CB1 and CB2 can be activated with exogenous cannabinoids most popularly known as tetrahydrocannabinol (THC) or cannabis and cannabidiol (CBD). The components of the ECS are expressed early in fetal development, and prenatal exposure to cannabis can lead to structural changes in white matter. White matter is composed of neuronal axons ensheathed in myelin, a lipid-rich insulation that facilitates saltatory conduction and maintains axon integrity. In the CNS, myelin is made by specialized glial cells called oligodendrocytes (OLs), which in addition to neurons also express components of the ECS. However, while several studies have focused on how the ECS regulates neuronal development, there is a limited understanding of its impact on OL development or myelin formation. Therefore, our current study set out to understand how pharmacological activation of the ECS alters OL differentiation and myelin formation in vivo. We administered WIN 55,212-2 (WIN 55), a CB1 and CB2 agonist, to larval zebrafish and longitudinally analyzed OL development and myelination in vivo. Interestingly, we observed an increase in non-axonal ensheathments in the spinal cord, which appeared to be surrounding neuronal cell bodies. These non-axonal ensheathments were dependent on CB1, as the addition of WIN 55 in a global CB1 mutant prevented this phenotype. Furthermore, this ectopic cell body ensheathment occurred independently from normal myelination processes, as individual OLs did not exhibit changes in the number of myelin sheaths, sheath length, or total myelin output. This study shows that activation of CB receptors in vivo leads to increased non-axonal ensheathment without significantly changing OL differentiation or normal myelin formation. Future studies can further investigate the pathways that drive this phenotype to better understand how exogenous cannabinoid activation can regulate the precision of oligodendrocyte ensheathment.

neuroscience↗

bioRxiv2024

A brainstem circuit for gravity-guided vertical navigation

The sensation of gravity anchors our perception of the environment and is crucial for navigation. However, the neural circuits that transform gravity into commands for navigation are undefined. We first determined that larval zebrafish (Danio rerio) navigate vertically by maintaining a consistent heading across a series of upward climb or downward dive bouts. Gravity-blind mutant fish swim with more variable heading and excessive veering, leading to inefficient vertical navigation. After targeted photoablation of ascending vestibular neurons and spinal projecting midbrain neurons, but not vestibulospinal neurons, vertical navigation was impaired. These data define a sensorimotor circuit that uses evolutionarily-conserved brainstem architecture to transform gravitational signals into persistent heading for vertical navigation. The work lays a foundation to understand how vestibular inputs allow animals to move efficiently through their environment.

neuroscience↗

bioRxiv2024

Motor neurons are dispensable for the assembly of a sensorimotor circuit for gaze stabilization

Sensorimotor reflex circuits engage distinct neuronal subtypes, defined by precise connectivity, to transform sensation into compensatory behavior. Whether and how motor neuron populations specify the subtype fate and/or sensory connectivity of their pre-motor partners remains controversial. Here, we discovered that motor neurons are dispensable for proper connectivity in the vestibular reflex circuit that stabilizes gaze. We first measured activity following vestibular sensation in premotor projection neurons after constitutive loss of their extraocular motor neuron partners. We observed normal responses and topography indicative of unchanged functional connectivity between sensory neurons and projection neurons. Next, we show that projection neurons remain anatomically and molecularly poised to connect appropriately with their down-stream partners. Lastly, we show that the transcriptional signatures that typify projection neurons develop independently of motor partners. Our findings comprehensively overturn a long-standing model: that connectivity in the circuit for gaze stabilization is retrogradely determined by motor partner-derived signals. By defining the contribution of motor neurons to specification of an archetypal sensorimotor circuit, our work speaks to comparable processes in the spinal cord and advances our understanding of principles of neural development.

neuroscience↗

bioRxiv2023

The vestibulospinal nucleus is a locus of balance development

Mature vertebrates maintain posture using vestibulospinal neurons that transform sensed instability into reflexive commands to spinal motor circuits. Postural stability improves across development. However, due to the complexity of terrestrial locomotion, vestibulospinal contributions to postural refinement in early life remain unexplored. Here we leveraged the relative simplicity of underwater locomotion to quantify the postural consequences of losing vestibulospinal neurons during development in larval zebrafish of undifferentiated sex. By comparing posture at two timepoints, we discovered that later lesions of vestibulospinal neurons led to greater instability. Analysis of thousands of individual swim bouts revealed that lesions disrupted movement timing and corrective reflexes without impacting swim kinematics, and that this effect was particularly strong in older larvae. Using a generative model of swimming, we showed how these disruptions could account for the increased postural variability at both timepoints. Finally, late lesions disrupted the fin/trunk coordination observed in older larvae, linking vestibulospinal neurons to postural control schemes used to navigate in depth. Since later lesions were considerably more disruptive to postural stability, we conclude that vestibulospinal contributions to balance increase as larvae mature. Vestibulospinal neurons are highly conserved across vertebrates; we therefore propose that they are a substrate for developmental improvements to postural control. SIGNIFICANCE STATEMENTMany animals experience balance improvements during early life. Mature vertebrates use vestibulospinal neurons to transform sensed instability into postural corrections. To understand if/how these neurons shape postural development, we ablated them at two developmentally important timepoints in larval zebrafish. Loss of vestibulospinal neurons disrupted specific stabilizing behaviors (swim timing, tilt correction, and fin/body coordination) more profoundly in older fish. We conclude that postural development happens in part by changes to vestibulospinal neurons -- a significant step towards understanding how developing brains gain the ability to balance.

neuroscience↗

bioRxiv2023

The nature and origin of synaptic inputs to vestibulospinal neurons in the larval zebrafish

Vestibulospinal neurons integrate sensed imbalance to regulate postural reflexes. As an evolutionarily-conserved neural population, understanding their synaptic and circuit-level properties can offer insight into vertebrate antigravity reflexes. Motivated by recent work, we set out to verify and extend the characterization of vestibulospinal neurons in the larval zebrafish. Using current clamp recordings together with stimulation, we observed that larval zebrafish vestibulospinal neurons are silent at rest, yet capable of sustained spiking following depolarization. Neurons responded systematically to a vestibular stimulus (translation in the dark); responses were abolished after chronic or acute loss of the utricular otolith. Voltage clamp recordings at rest revealed strong excitatory inputs with a characteristic multimodal distribution of amplitudes, as well as strong inhibitory inputs. Excitatory inputs within a particular mode (amplitude range) routinely violated refractory period criteria and exhibited complex sensory tuning, suggesting a non-unitary origin. Next, using a unilateral loss-of-function approach, we characterized the source of vestibular inputs to vestibulospinal neurons from each ear. We observed systematic loss of high-amplitude excitatory inputs after utricular lesions ipsilateral, but not contralateral to the recorded vestibulospinal neuron. In contrast, while some neurons had decreased inhibitory inputs after either ipsilateral or contralateral lesions, there were no systematic changes across the population of recorded neurons. We conclude that imbalance sensed by the utricular otolith shapes the responses of larval zebrafish vestibulospinal neurons through both excitatory and inhibitory inputs. Our findings expand our understanding of how a vertebrate model, the larval zebrafish, might use vestibulospinal input to stabilize posture. More broadly, when compared to recordings in other vertebrates, our data speak to conserved origins of vestibulospinal synaptic input.

neuroscience↗

bioRxiv2023

Scalable Apparatus to Measure Posture and Locomotion (SAMPL): a high-throughput solution to study unconstrained vertical behavior in small animals

Balance and movement are impaired in a wide variety of neurological disorders. Recent advances in behavioral monitoring provide unprecedented access to posture and loco-motor kinematics, but without the throughput and scalability necessary to screen candidate genes / potential therapeutics. We present a powerful solution: a Scalable Apparatus to Measure Posture and Locomotion (SAMPL). SAMPL includes extensible imaging hardware and low-cost open-source acquisition software with real-time processing. We first demonstrate that SAMPLs hardware and acquisition software can acquire data from D. melanogaster, C.elegans, and D. rerio as they move vertically. Next, we leverage SAMPLs throughput to rapidly (two weeks) gather a new zebrafish dataset. We use SAMPLs analysis and visualization tools to replicate and extend our current understanding of how zebrafish balance as they navigate through a vertical environment. Next, we discover (1) that key kinematic parameters vary systematically with genetic background, and (2) that such background variation is small relative to the changes that accompany early development. Finally, we simulate SAMPLs ability to resolve differences in posture or vertical navigation as a function of effect size and data gathered - key data for screens. Taken together, our apparatus, data, and analysis provide a powerful solution for laboratories using small animals to investigate balance and locomotor disorders at scale. More broadly, SAMPL is both an adaptable resource for laboratories looking process video-graphic measures of behavior in real-time, and an exemplar of how to scale hardware to enable the throughput necessary for screening.

neuroscience↗

bioRxiv2022

Determinants of Motor Neuron Functional Subtypes Important for Locomotor Speed

Locomotion requires precise control of the strength and speed of muscle contraction and is achieved by recruiting functionally-distinct subtypes of motor neurons (MNs). MNs are essential to movement and differentially susceptible in disease, but little is known about how MNs acquire functional subtype-specific features during development. Using single-cell RNA profiling in embryonic and larval zebrafish, we identify novel and conserved molecular signatures for MN functional subtypes, and identify genes expressed in both early post-mitotic and mature MNs. Assessing MN development in genetic mutants, we define a molecular program essential for MN functional subtype specification. Two evolutionarily-conserved transcription factors, Prdm16 and Mecom, are both functional subtype-specific determinants integral for fast MN development. Loss of prdm16 or mecom causes fast MNs to develop transcriptional profiles and innervation similar to slow MNs. These results reveal the molecular diversity of vertebrate axial MNs and demonstrate that functional subtypes are specified through intrinsic transcriptional codes.

neuroscience↗

bioRxiv2022

Neuronal birthdate reveals topography in a vestibular brainstem circuit for gaze stabilization

Across the nervous system, neurons with similar attributes are topographically organized. This topography reflects developmental pressures. Oddly, vestibular (balance) nuclei are thought to be disorganized. By measuring activity in birthdated neurons, we revealed a functional map within the central vestibular projection nucleus that stabilizes gaze in the larval zebrafish. We first discovered that both somatic position and stimulus selectivity follow projection neuron birthdate. Next, with electron microscopy and loss-of-function assays, we found that patterns of peripheral innervation to projection neurons were similarly organized by birthdate. Lastly, birthdate revealed spatial patterns of axonal arborization and synapse formation to projection neuron outputs. Collectively, we find that development reveals previously hidden organization to the input, processing, and output layers of a highly-conserved vertebrate sensorimotor circuit. The spatial and temporal attributes we uncover constrain the developmental mechanisms that may specify the fate, function, and organization of vestibulo-ocular reflex neurons. More broadly, our data suggest that, like invertebrates, temporal mechanisms may assemble vertebrate sensorimotor architecture.

neuroscience↗

bioRxiv2022

Tilt In Place Microscopy (TIPM): a simple, low-cost solution to image neural responses to body rotations

Animals use information about gravity and other destabilizing forces to balance and navigate through their environment. Measuring how brains respond to these forces requires considerable technical knowledge and/or financial resources. We present a simple alternative: Tilt In Place Microscopy (TIPM). TIPM is a low-cost and non-invasive way to measure neural activity following rapid changes in body orientation. Here we used TIPM to study vestibulospinal neurons in larval zebrafish during and immediately after roll tilts. Vestibulospinal neurons responded with reliable increases in activity that varied as a function of ipsilateral tilt amplitude. TIPM differentiated tonic (i.e. sustained tilt) from phasic responses, revealing coarse topography of stimulus sensitivity in the lateral vestibular nucleus. Neuronal variability across repeated sessions was minor relative to trial-to-trial variability, allowing us to use TIPM for longitudinal studies of the same neurons across two developmental timepoints. There, we observed global increases in response strength, and systematic changes in the neural representation of stimulus direction. Our data extend classical characterization of the body tilt representation by vestibulospinal neurons and establish TIPMs utility to study the neural basis of balance, especially in developing animals. Significance StatementVestibular sensation influences everything from navigation to interoception. Here we detail a straight-forward, validated and nearly-universal approach to image how the nervous system senses and responds to body tilts. We use our new method to replicate and expand upon past findings of tilt sensing by a conserved population of spinal-projecting vestibular neurons. The simplicity and broad compatibility of our approach will democratize the study of the brains response to destabilization, particularly across development.

neuroscience↗

bioRxiv2021

Synaptic Encoding of Vestibular Sensation Regulates Movement Timing and Coordination

Vertebrate vestibular circuits use sensory signals derived from the inner ear to guide both corrective and volitional movements. A major challenge in the neuroscience of balance is to link the synaptic and cellular substrates that encode body tilts to specific behaviors that stabilize posture and enable efficient locomotion. Here we address this problem by measuring the development, synaptic architecture, and behavioral contributions of vestibulospinal neurons in the larval zebrafish. First, we find that vestibulospinal neurons are born and are functionally mature before larvae swim freely, allowing them to act as a substrate for postural regulation. Next, we map the synaptic inputs to vestibulospinal neurons that allow them to encode posture. Further, we find that this synaptic architecture allows them to respond to linear acceleration in a directionally-tuned and utricle-dependent manner; they are thus poised to guide corrective movements. After loss of vestibulospinal neurons, larvae adopted eccentric postures with disrupted movement timing and weaker corrective kinematics. We used a generative model of swimming to demonstrate that together these disruptions can account for the increased postural variability. Finally, we observed that lesions disrupt vestibular-dependent coordination between the fins and trunk during vertical swimming, linking vestibulospinal neurons to navigation. We conclude that vestibulospinal neurons turn synaptic representations of body tilt into defined corrective behaviors and coordinated movements. As the need for stable locomotion is common and the vestibulospinal circuit is highly conserved our findings reveal general mechanisms for neuronal control of balance.

neuroscience↗