Efficient training approaches for optimizing behavioral performance and reducing head fixation time.

The use of head fixation has become routine in systems neuroscience. However, whether the behavior changes with head fixation, whether animals can learn aspects of a task while freely moving and transfer this knowledge to the head fixed condition, has not been examined in much detail. Here, we used a novel floating platform, the "Air-Track", which simulates free movement in a real-world environment to address the effect of head fixation and developed methods to accelerate training of behavioral tasks for head fixed mice. We trained mice in a Y maze two choice discrimination task. One group was trained while head fixed and compared to a separate group that was pre-trained while freely moving and then trained on the same task while head fixed. Pre-training significantly reduced the time needed to relearn the discrimination task while head fixed. Freely moving and head fixed mice displayed similar behavioral patterns, however, head fixation significantly slowed movement speed. The speed of movement in the head fixed mice depended on the weight of the platform. We conclude that home-cage pre-training improves learning performance of head fixed mice and that while head fixation obviously limits some aspects of movement, the patterns of behavior observed in head fixed and freely moving mice are similar.

Coordination between Eye Movement and Whisking in Head-Fixed Mice Navigating a Plus Maze.

Navigation through complex environments requires motor planning, motor preparation, and the coordination between multiple sensory-motor modalities. For example, the stepping motion when we walk is coordinated with motion of the torso, arms, head, and eyes. In rodents, movement of the animal through the environment is coordinated with whisking. Even head-fixed mice navigating a plus maze position their whiskers asymmetrically with the bilateral asymmetry signifying the upcoming turn direction. Here we report that, in addition to moving their whiskers, on every trial mice also move their eyes conjugately in the direction of the upcoming turn. Not only do mice move their eyes, but they coordinate saccadic eye movement with the asymmetric positioning of the whiskers. Our analysis shows that asymmetric positioning of whiskers predicted the turn direction that mice will make at an earlier stage than eye movement. Consistent with these results, our observations also revealed that whisker asymmetry increases before saccadic eye movement. Importantly, this work shows that when rodents plan for active behavior, their motor plans can involve both eye and whisker movement. We conclude that, when mice are engaged in and moving through complex real-world environments, their behavioral state can be read out in the movement of both their whiskers and eyes.

Real-Time Closed-Loop Feedback in Behavioral Time Scales Using DeepLabCut.

Computer vision approaches have made significant inroads into offline tracking of behavior and estimating animal poses. In particular, because of their versatility, deep-learning approaches have been gaining attention in behavioral tracking without any markers. Here, we developed an approach using DeepLabCut for real-time estimation of movement. We trained a deep-neural network (DNN) offline with high-speed video data of a mouse whisking, then transferred the trained network to work with the same mouse, whisking in real-time. With this approach, we tracked the tips of three whiskers in an arc and converted positions into a TTL output within behavioral time scales, i.e., 10.5 ms. With this approach, it is possible to trigger output based on movement of individual whiskers, or on the distance between adjacent whiskers. Flexible closed-loop systems like the one we have deployed here can complement optogenetic approaches and can be used to directly manipulate the relationship between movement and neural activity.

Whisking Asymmetry Signals Motor Preparation and the Behavioral State of Mice.

A central function of the brain is to plan, predict, and imagine the effect of movement in a dynamically changing environment. Here we show that in mice head-fixed in a plus-maze, floating on air, and trained to pick lanes based on visual stimuli, the asymmetric movement, and position of whiskers on the two sides of the face signals whether the animal is moving, turning, expecting reward, or licking. We show that (1) whisking asymmetry is coordinated with behavioral state, and that behavioral state can be decoded and predicted based on asymmetry, (2) even in the absence of tactile input, whisker positioning and asymmetry nevertheless relate to behavioral state, and (3) movement of the nose correlates with asymmetry, indicating that facial expression of the mouse is itself correlated with behavioral state. These results indicate that the movement of whiskers, a behavior that is not instructed or necessary in the task, can inform an observer about what a mouse is doing in the maze. Thus, the position of these mobile tactile sensors reflects a behavioral and movement-preparation state of the mouse.SIGNIFICANCE STATEMENT Behavior is a sequence of movements, where each movement can be related to or can trigger a set of other actions. Here we show that, in mice, the movement of whiskers (tactile sensors used to extract information about texture and location of objects) is coordinated with and predicts the behavioral state of mice: that is, what mice are doing, where they are in space, and where they are in the sequence of behaviors.

Fast, Flexible Closed-Loop Feedback: Tracking Movement in "Real-Millisecond-Time".

One of the principal functions of the brain is to control movement and rapidly adapt behavior to a changing external environment. Over the last decades our ability to monitor activity in the brain, manipulate it while also manipulating the environment the animal moves through, has been tackled with increasing sophistication. However, our ability to track the movement of the animal in real time has not kept pace. Here, we use a dynamic vision sensor (DVS) based event-driven neuromorphic camera system to implement real-time, low-latency tracking of a single whisker that mice can move at ∼25 Hz. The customized DVS system described here converts whisker motion into a series of events that can be used to estimate the position of the whisker and to trigger a position-based output interactively within 2 ms. This neuromorphic chip-based closed-loop system provides feedback rapidly and flexibly. With this system, it becomes possible to use the movement of whiskers or in principal, movement of any part of the body to reward, punish, in a rapidly reconfigurable way. These methods can be used to manipulate behavior, and the neural circuits that help animals adapt to changing values of a sequence of motor actions.

Classic Cadherins Mediate Selective Intracortical Circuit Formation in the Mouse Neocortex.

Understanding the molecular mechanisms underlying the formation of selective intracortical circuitry is one of the important questions in neuroscience research. "Barrel nets" are recently identified intracortical axonal trajectories derived from layer 2/3 neurons in layer 4 of the primary somatosensory (barrel) cortex. Axons of layer 2/3 neurons are preferentially distributed in the septal regions of layer 4 of the barrel cortex, where they show whisker-related patterns. Because cadherins have been viewed as potential candidates that mediate the formation of selective neuronal circuits, here we examined the role of cadherins in the formation of barrel nets. We disrupted the function of cadherins by expressing dominant-negative cadherin (dn-cadherin) using in utero electroporation and found that barrel nets were severely disrupted. Confocal microscopic analysis revealed that expression of dn-cadherin reduced the density of axons in septal regions in layer 4 of the barrel cortex. We also found that cadherins were important for the formation, rather than the maintenance, of barrel nets. Our results uncover an important role of cadherins in the formation of local intracortical circuitry in the neocortex.

Neuronal circuits with whisker-related patterns.

Neuronal circuits with whisker-related patterns, such as those observed in the rodent somatosensory barrel cortex, have been widely used as a model system for investigating the anatomical organization, development and physiological roles of functional neuronal circuits. Whisker-related patterns exist not only in the barrel cortex but also in subcortical structures along the trigeminal neuraxis from the brainstem to the cortex. Here, we briefly summarize the organization, formation, and function of each neuronal circuit with whisker-related patterns, including the novel axonal trajectories that we recently found with the aid of in utero electroporation. We also discuss their biological implications as model systems for the studies of functional neuronal circuits.

Whisker-related axonal patterns and plasticity of layer 2/3 neurons in the mouse barrel cortex.

Elucidating neuronal circuits and their plasticity in the cerebral cortex is one of the important questions in neuroscience research. Here we report novel axonal trajectories and their plasticity in the mouse somatosensory barrel cortex. We selectively visualized layer 2/3 neurons using in utero electroporation and examined the axonal trajectories of layer 2/3 neurons. We found that the axons of layer 2/3 neurons preferentially run in the septal regions of layer 4 and named this axonal pattern "barrel nets." The intensity of green fluorescent protein in the septal regions was markedly higher compared with that in barrel hollows. Focal in utero electroporation revealed that the axons in barrel nets were indeed derived from layer 2/3 neurons in the barrel cortex. During development, barrel nets became visible at postnatal day 10, which was well after the initial appearance of barrels. When whisker follicles were cauterized within 3 d after birth, the whisker-related pattern of barrel nets was altered, suggesting that cauterization of whisker follicles results in developmental plasticity of barrel nets. Our results uncover the novel axonal trajectories of layer 2/3 neurons with whisker-related patterns and their developmental plasticity in the mouse somatosensory cortex. Barrel nets should be useful for investigating the pattern formation and axonal reorganization of intracortical neuronal circuits.