Cerebellar contributions to a brainwide network for flexible behavior in mice.

The cerebellum regulates nonmotor behavior, but the routes of influence are not well characterized. Here we report a necessary role for the posterior cerebellum in guiding a reversal learning task through a network of diencephalic and neocortical structures, and in flexibility of free behavior. After chemogenetic inhibition of lobule VI vermis or hemispheric crus I Purkinje cells, mice could learn a water Y-maze but were impaired in ability to reverse their initial choice. To map targets of perturbation, we imaged c-Fos activation in cleared whole brains using light-sheet microscopy. Reversal learning activated diencephalic and associative neocortical regions. Distinctive subsets of structures were altered by perturbation of lobule VI (including thalamus and habenula) and crus I (including hypothalamus and prelimbic/orbital cortex), and both perturbations influenced anterior cingulate and infralimbic cortex. To identify functional networks, we used correlated variation in c-Fos activation within each group. Lobule VI inactivation weakened within-thalamus correlations, while crus I inactivation divided neocortical activity into sensorimotor and associative subnetworks. In both groups, high-throughput automated analysis of whole-body movement revealed deficiencies in across-day behavioral habituation to an open-field environment. Taken together, these experiments reveal brainwide systems for cerebellar influence that affect multiple flexible responses.

Automated high-throughput mouse transsynaptic viral tracing using iDISCO+ tissue clearing, light-sheet microscopy, and BrainPipe.

Transsynaptic viral tracing requires tissue sectioning, manual cell counting, and anatomical assignment, all of which are time intensive. We describe a protocol for BrainPipe, a scalable software for automated anatomical alignment and object counting in light-sheet microscopy volumes. BrainPipe can be generalized to new counting tasks by using a new atlas and training a neural network for object detection. Combining viral tracing, iDISCO+ tissue clearing, and BrainPipe facilitates mapping of cerebellar connectivity to the rest of the murine brain. For complete details on the use and execution of this protocol, please refer to Pisano et al. (2021).

Machine vision methods for analyzing social interactions.

Recent developments in machine vision methods for automatic, quantitative analysis of social behavior have immensely improved both the scale and level of resolution with which we can dissect interactions between members of the same species. In this paper, we review these methods, with a particular focus on how biologists can apply them to their own work. We discuss several components of machine vision-based analyses: methods to record high-quality video for automated analyses, video-based tracking algorithms for estimating the positions of interacting animals, and machine learning methods for recognizing patterns of interactions. These methods are extremely general in their applicability, and we review a subset of successful applications of them to biological questions in several model systems with very different types of social behaviors.

Evidence for an audience effect in mice: male social partners alter the male vocal response to female cues.

Mice (Mus musculus) form large and dynamic social groups and emit ultrasonic vocalizations in a variety of social contexts. Surprisingly, these vocalizations have been studied almost exclusively in the context of cues from only one social partner, despite the observation that in many social species the presence of additional listeners changes the structure of communication signals. Here, we show that male vocal behavior elicited by female odor is affected by the presence of a male audience - with changes in vocalization count, acoustic structure and syllable complexity. We further show that single sensory cues are not sufficient to elicit this audience effect, indicating that multiple cues may be necessary for an audience to be apparent. Together, these experiments reveal that some features of mouse vocal behavior are only expressed in more complex social situations, and introduce a powerful new assay for measuring detection of the presence of social partners in mice.

The contribution of ultrasonic vocalizations to mouse courtship.

Vocalizations transmit information to social partners, and mice use these signals to exchange information during courtship. Ultrasonic vocalizations from adult males are tightly associated with their interactions with females, and vary as a function of male quality. Work in the last decade has established that the spectrotemporal features of male vocalizations are not learned, but that female attention toward specific vocal features is modified by social experience. Additionally, progress has been made on elucidating how mouse vocalizations are encoded in the auditory system, and on the olfactory circuits that trigger their production. Together these findings provide us with important insights into how vocal communication can contribute to social interactions.

Phase shifts in binaural stimuli provide directional cues for sound localisation in the field cricket Gryllus bimaculatus.

The cricket's auditory system is a highly directional pressure difference receiver whose function is hypothesised to depend on phase relationships between the sound waves propagating through the auditory trachea that connects the left and right hearing organs. We tested this hypothesis by measuring the effect of experimentally constructed phase shifts in acoustic stimuli on phonotactic behavior of Gryllus bimaculatus, the oscillatory response patterns of the tympanic membrane, and the activity of the auditory afferents. The same artificial calling song was played simultaneously at the left and right sides of the cricket, but one sound pattern was shifted in phase by 90 deg (carrier frequencies between 3.6 and 5.4 kHz). All three levels of auditory processing are sensitive to experimentally induced acoustic phase shifts, and the response characteristics are dependent on the carrier frequency of the sound stimulus. At lower frequencies, crickets steered away from the sound leading in phase, while tympanic membrane vibrations and auditory afferent responses were smaller when the ipsilateral sound was leading. In contrast, opposite responses were observed at higher frequencies in all three levels of auditory processing. Minimal responses occurred near the carrier frequency of the cricket's calling song, suggesting a stability at this frequency. Our results indicate that crickets may use directional cues arising from phase shifts in acoustic signals for sound localisation, and that the response properties of pressure difference receivers may be analysed with phase-shifted sound stimuli to further our understanding of how insect auditory systems are adapted for directional processing.