Early development of respiratory motor circuits in larval zebrafish (Danio rerio).

Rhythm-generating circuits in the vertebrate hindbrain form synaptic connections with cranial and spinal motor neurons, to generate coordinated, patterned respiratory behaviors. Zebrafish provide a uniquely tractable model system to investigate the earliest stages in respiratory motor circuit development in vivo. In larval zebrafish, respiratory behaviors are carried out by muscles innervated by cranial motor neurons-including the facial branchiomotor neurons (FBMNs), which innervate muscles that move the jaw, buccal cavity, and operculum. However, it is unclear when FBMNs first receive functional synaptic input from respiratory pattern-generating neurons, and how the functional output of the respiratory motor circuit changes across larval development. In the current study, we used behavior and calcium imaging to determine how early FBMNs receive functional synaptic inputs from respiratory pattern-generating networks in larval zebrafish. Zebrafish exhibited patterned operculum movements by 3 days postfertilization (dpf), though this behavior became more consistent at 4 and 5 dpf. Also by 3dpf, FBMNs fell into two distinct categories ("rhythmic" and "nonrhythmic"), based on patterns of neural activity. These two neuron categories were arranged differently along the dorsoventral axis, demonstrating that FBMNs have already established dorsoventral topography by 3 dpf. Finally, operculum movements were coordinated with pectoral fin movements at 3 dpf, indicating that the operculum behavioral pattern was driven by synaptic input. Taken together, this evidence suggests that FBMNs begin to receive initial synaptic input from a functional respiratory central pattern generator at or prior to 3 dpf. Future studies will use this model to study mechanisms of normal and abnormal respiratory circuit development.

Key Features of Structural and Functional Organization of Zebrafish Facial Motor Neurons Are Resilient to Disruption of Neuronal Migration.

The location of neurons early in development can be critical for their ability to differentiate and receive normal synaptic inputs. Indeed, disruptions in neuronal positioning lead to a variety of neurological disorders. Neurons have, however, shifted their positions across phylogeny, suggesting that changes in location do not always spell functional disaster. To investigate the functional consequences of abnormal positioning, we leveraged previously reported genetic perturbations to disrupt normal neuronal migration-and thus positioning-in a population of cranial motor neurons, the facial branchiomotor neurons (FBMNs). We used a combination of topographical, morphological, physiological, and behavioral analyses to determine whether key functional features of FBMNs were still established in migration mutants, in spite of a dramatic rostrocaudal repositioning of these neurons in hindbrain. We discovered that FBMNs seem remarkably resilient to a disruption in positioning, suggesting that they may not rely heavily on rostrocaudal positioning to guide their functional development. Thus, the role of positioning may vary across the developing nervous system, with some populations-like facial motor neurons-exhibiting greater resilience to abnormal positioning that permits them to shift location as a part of evolutionary change.

TRP channel mediated neuronal activation and ablation in freely behaving zebrafish.

The zebrafish (Danio rerio) is a useful vertebrate model system in which to study neural circuits and behavior, but tools to modulate neurons in freely behaving animals are limited. As poikilotherms that live in water, zebrafish are amenable to thermal and pharmacological perturbations. We exploit these properties by using transient receptor potential (TRP) channels to activate or ablate specific neuronal populations using the chemical and thermal agonists of heterologously expressed TRPV1, TRPM8 and TRPA1.

Behavioral state modulates the activity of brainstem sensorimotor neurons.

Sensorimotor processing must be modulated according to the animal's behavioral state. A previous study demonstrated that motion responses were strongly state dependent in birds. Vestibular eye and head responses were significantly larger and more compensatory during simulated flight, and a flight-specific vestibular tail response was also characterized. In the current study, we investigated the neural substrates for these state-dependent vestibular behaviors by recording extracellularly from neurons in the vestibular nuclear complex and comparing their spontaneous activity and sensory responses during default and simulated flight states. We show that motion-sensitive neurons in the lateral vestibular nucleus are state dependent. Some neurons increased their spontaneous firing rates during flight, though their increased excitability was not reflected in higher sensory gains. However, other neurons exhibited state-dependent gating of sensory inputs, responding to rotational stimuli only during flight. These results demonstrate that vestibular processing in the brainstem is state dependent and lay the foundation for future studies to investigate the synaptic mechanisms responsible for these modifications.

State-dependent sensorimotor processing: gaze and posture stability during simulated flight in birds.

Vestibular responses play an important role in maintaining gaze and posture stability during rotational motion. Previous studies suggest that these responses are state dependent, their expression varying with the environmental and locomotor conditions of the animal. In this study, we simulated an ethologically relevant state in the laboratory to study state-dependent vestibular responses in birds. We used frontal airflow to simulate gliding flight and measured pigeons' eye, head, and tail responses to rotational motion in darkness, under both head-fixed and head-free conditions. We show that both eye and head response gains are significantly higher during flight, thus enhancing gaze and head-in-space stability. We also characterize state-specific tail responses to pitch and roll rotation that would help to maintain body-in-space orientation during flight. These results demonstrate that vestibular sensorimotor processing is not fixed but depends instead on the animal's behavioral state.

Canal and otolith contributions to compensatory tilt responses in pigeons.

Gaze-stabilizing eye and head responses compensate more effectively for low-frequency rotational motion when such motion stimulates the otolith organs, as during earth-horizontal axis rotations. However, the nature of the otolith signal responsible for this improvement in performance has not been previously determined. In this study, we used combinations of earth-horizontal axis rotational and translational motion to manipulate the magnitude of net linear acceleration experienced by pigeons, under both head-fixed and head-free conditions. We show that phase enhancement of eye and head responses to low-frequency rotational motion was causally related to the magnitude of dynamic net linear acceleration and not the gravitational acceleration component. We also show that canal-driven and otolith-driven eye responses were both spatially and temporally appropriate to combine linearly, and that a simple linear model combining canal- and otolith-driven components predicted eye responses to complex motion that were consistent with our experimental observations. However, the same model did not predict the observed head responses, which were spatially but not temporally appropriate to combine according to the same linear scheme. These results suggest that distinct vestibular processing substrates exist for eye and head responses in pigeons and that these are likely different from the vestibular processing substrates observed in primates.