SUMMARY Different individuals exhibit distinct behaviors, but studying the neuronal basis of individuality is a daunting challenge. Here, we considered this question in the vomeronasal organ, a pheromone-detecting epithelium containing hundreds of distinct neuronal types. Using light-sheet microscopy, we characterized in each animal the abundance of 17 physiologically-defined types, altogether recording from a half-million sensory neurons. Inter-animal differences were much larger than predicted by chance, and different physiological cell types showed distinct patterns of variability. One neuronal type was present in males and nearly absent in females. Surprisingly, this apparent sexual dimorphism was generated by plasticity, as exposure to female scents or single ligands led to both the elimination of this cell type and alterations in olfactory behavior. That an all-or-none apparent sex difference in neuronal types is controlled by experience—even in a sensory system devoted to “innate” behaviors—highlights the extraordinary role of “nurture” in neural individuality.

SUMMARY In social interactions among mammals, individuals are recognized by olfactory cues, but identifying the key signals among thousands of compounds remains a major challenge. To address this need, we developed a new technique, Component-Activity Matching (CAM), to select candidate ligands that “explain” patterns of bioactivity across diverse complex mixtures. Using mouse urine from eight different sexes and strains, we identified 23 components to explain firing rates in seven of eight functional classes of vomeronasal sensory neurons. Focusing on a class of neurons selective for females, we identified a novel family of vomeronasal ligands, steroid carboxylic acids. These ligands accounted for much of the neuronal activity of urine from some female strains, were necessary for normal levels of male investigatory behavior of female scents and sufficed to trigger mounting behavior. CAM represents the first step towards an exhaustive characterization of the molecular cues for natural behavior in a mammalian olfactory system.

Highlights d Demonstration of head-mounted microendoscopic calcium imaging in behaving macaques d Surgical protocols developed for preparing the animal for calcium imaging d Proof-of-concept plug-and-play calcium imaging with stable recording capability d Imaging in the dorsal premotor cortex exhibited dynamics selective to direction of reach

HighlightsFirst demonstration of head-mounted microendoscopic calcium imaging in behaving macaque.Surgical protocols developed for preparing the animal for calcium imaging, including virus injections to express GCaMP and chronic implantation of a GRIN lens to enable optical access to gyral cortex.Proof of concept plug-and-play calcium imaging in behaving macaques with months long stable recording capability allowing populations of individual neurons to be tracked longitudinally.Bilateral calcium imaging from dorsal premotor cortex exhibited dynamics selective to the animal's direction of reach and allowed decoding of the animal's motor behavior Summary A major effort is now underway across the brain sciences to identify, characterize and manipulate mesoscale neural circuits in order to elucidate the mechanisms underlying sensory perception, cognition and behavior. Optical imaging technologies, in conjunction with genetically encoded sensors and actuators, serve as important tools toward these goals, allowing access to large-scale genetically defined neuronal populations. In particular, one-photon miniature microscopes, coupled with genetically encoded calcium indicators and microendoscopic gradient-refractive index (GRIN) lenses, enable unprecedented readout of neural circuit dynamics in cortical and deep subcortical brain regions during active behavior in rodents. This has already led to breakthrough discoveries across a wide array of rodent brain regions and behaviors. However, in order to study the neural circuit mechanisms underlying more complex and clinically relevant human behaviors and cognitive functions, it is crucial to translate this technology to non-human primates. Here, we describe the first successful application of this technology in the rhesus macaque. We identified a viral strategy for robust expression of GCaMP, optimized a surgical protocol for microendoscope GRIN lens insertion, and created a chronic cranial chamber and lens mounting system for imaging in gyral cortex. Using these methods, we demonstrate the ability to perform plug-and-play, head-mounted recordings of cellular-resolution calcium dynamics from over 100 genetically-targeted neurons simultaneously in dorsal premotor cortex while the macaque performs a naturalistic motor reach task with the head unrestrained and freely moving. The recorded population of neurons exhibited calcium dynamics selective to the direction of reach, which we show can be used to decode the animal's trial-by-trial motor behavior. Recordings were stable over several months, allowing us to longitudinally track large populations of individual neurons and monitor their relationship to motor behavior over time. Finally, we demonstrate the ability to conduct simultaneous, multi-site imaging in bilateral dorsal premotor cortices, offering an opportunity to study distributed networks underlying complex behavior and cognition. Together, this work establishes headmounted microendoscopic calcium imaging in macaque as a powerful new approach for studying the neural ci...

Imaging permits the visualization of neural activity from the whole-mount vomeronasal sensory epithelium with single-cell resolution. The preparation preserves an intact tissue environment, enabling the robust detection of cellular responses upon chemical stimulation and study of the precise 3D mapping of vomeronasal sensory neuron (VSN) functional types within the epithelium. Using objective-coupled planar illumination (OCPI) microscopy to perform fast volumetric imaging, we routinely record the responses of thousands of VSNs for hours from a single intact vomeronasal organ preparation. Here we document the preparation of the whole-mounted vomeronasal epithelium, multichannel stimulus delivery, and three-dimensional calcium imaging by OCPI microscopy.