UNLABELLED: Neurons respond to specific features of sensory stimuli. In the visual system, for example, some neurons respond to motion of small but not large objects, whereas other neurons prefer motion of the entire visual field. Separate neurons respond equally to local and global motion but selectively to additional features of visual stimuli. How and where does response selectivity emerge? Here, we show that wide-field (WF) cells in retino-recipient layers of the mouse superior colliculus (SC) respond selectively to small moving objects. Moreover, we identify two mechanisms that contribute to this selectivity. First, we show that input restricted to a small portion of the broad dendritic arbor of WF cells is sufficient to trigger dendritic spikes that reliably propagate to the soma/axon. In vivo whole-cell recordings reveal that nearly every action potential evoked by visual stimuli has characteristics of spikes initiated in dendrites. Second, inhibitory input from a different class of SC neuron, horizontal cells, constrains the range of stimuli to which WF cells respond. Horizontal cells respond preferentially to the sudden appearance or rapid movement of large stimuli. Optogenetic reduction of their activity reduces movement selectivity and broadens size tuning in WF cells by increasing the relative strength of responses to stimuli that appear suddenly or cover a large region of space. Therefore, strongly propagating dendritic spikes enable small stimuli to drive spike output in WF cells and local inhibition helps restrict responses to stimuli that are both small and moving.

SIGNIFICANCE STATEMENT: How do neurons respond selectively to some sensory stimuli but not others? In the visual system, a particularly relevant stimulus feature is object motion, which often reveals other animals. Here, we show how specific cells in the superior colliculus, one synapse downstream of the retina, respond selectively to object motion. These wide-field (WF) cells respond strongly to small objects that move slowly anywhere through a large region of space, but not to stationary objects or full-field motion. Action potential initiation in dendrites enables small stimuli to trigger visual responses and inhibitory input from cells that prefer large, suddenly appearing, or quickly moving stimuli restricts responses of WF cells to objects that are small and moving.

Understanding how the brain operates requires understanding how large sets of neurons function together. Modern recording technology makes it possible to simultaneously record the activity of hundreds of neurons, and technological developments will soon allow recording of thousands or tens of thousands. As with all experimental techniques, these methods are subject to confounds that complicate the interpretation of such recordings, and could lead to erroneous scientific conclusions. Here we discuss methods for assessing and improving the quality of data from these techniques and outline likely future directions in this field.

Functional imaging in behaving animals is essential to understanding brain function. However, artifacts resulting from animal motion, including locomotion, can severely corrupt functional measurements. To dampen tissue motion, we designed a new optical window with minimal optical aberrations. Using the newly developed high-speed continuous volumetric imaging system based on an optical phase-locked ultrasound lens, we quantified motion of the cerebral cortex and hippocampal surface during two-photon functional imaging in behaving mice. We find that the out-of-plane motion is generally greater than the axial dimension of the point-spread-function during mouse locomotion, which indicates that high-speed continuous volumetric imaging is necessary to minimize motion artifacts.

Neural circuitry has evolved to form distributed networks that act dynamically across large volumes. Conventional microscopy collects data from individual planes and cannot sample circuitry across large volumes at the temporal resolution relevant to neural circuit function and behaviors. Here we review emerging technologies for rapid volume imaging of neural circuitry. We focus on two critical challenges: the inertia of optical systems, which limits image speed, and aberrations, which restrict the image volume. Optical sampling time must be long enough to ensure high-fidelity measurements, but optimized sampling strategies and point-spread function engineering can facilitate rapid volume imaging of neural activity within this constraint. We also discuss new computational strategies for processing and analyzing volume imaging data of increasing size and complexity. Together, optical and computational advances are providing a broader view of neural circuit dynamics and helping elucidate how brain regions work in concert to support behavior.

Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm(2)) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view.

This chapter reviews the application of new genetically encoded tools in feeding circuits that regulate appetite. Rapid activation and inhibition of agouti related peptide (AgRP) neurons conclusively established a causal role for rapid control of food intake. Chemogenetic activation of AgRP neurons using hM3Dq avoids the invasive protocols required for ChR2 activation. ChR2 distributes into axons, and selective optogenetic activation of AgRP neuron axon projection fields in distinct brain areas was used to examine their individual contribution to feeding behavior. Some of the brain areas targeted by AgRP neuron axon projections have been examined further for cell type specific control of appetite. Rodents with bed nucleus of stria terminalis (BNST) lesions show hyperphagia and obesity, indicating that reduced BNST output promotes feeding. pro-opiomelanocortin (POMC) neurons regulate feeding over longer timescales. parabrachial nucleus (PBN) neurons have a powerful inhibitory role on food intake, but their inhibition does not strongly elevate food intake.

Membrane remodeling is an essential part for transfer of components to and from the cell surface and membrane-bound organelles, and for changes in cell shape, particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging, mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and non-invasive illumination of the newly developed lattice light sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and showed that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and remained relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and in multicellular assemblies.

The nociceptive flexor withdrawal reflex has an august place in the history of neuroscience. In this issue of Neuron, Hilde et al. (2016) advance our understanding of this reflex by characterizing the molecular identity and circuit connectivity of component interneurons. They assess how a DNA-binding factor Satb2 controls cell position, molecular identity, pre-and postsynaptic targeting, and function of a population of inhibitory sensory relay interneurons that serve to integrate both proprioceptive and nociceptive afferent information.

The nociceptive flexor withdrawal reflex has an august place in the history of neuroscience. In this issue of Neuron, Hilde et al. (2016) advance our understanding of this reflex by characterizing the molecular identity and circuit connectivity of component interneurons. They assess how a DNA-binding factor Satb2 controls cell position, molecular identity, pre-and postsynaptic targeting, and function of a population of inhibitory sensory relay interneurons that serve to integrate both proprioceptive and nociceptive afferent information.

Animal species display enormous variation for innate behaviours, but little is known about how this diversity arose. Here, using an unbiased genetic approach, we map a courtship song difference between wild isolates of Drosophila simulans and Drosophila mauritiana to a 966 base pair region within the slowpoke (slo) locus, which encodes a calcium-activated potassium channel. Using the reciprocal hemizygosity test, we confirm that slo is the causal locus and resolve the causal mutation to the evolutionarily recent insertion of a retroelement in a slo intron within D. simulans. Targeted deletion of this retroelement reverts the song phenotype and alters slo splicing. Like many ion channel genes, slo is expressed widely in the nervous system and influences a variety of behaviours; slo-null males sing little song with severely disrupted features. By contrast, the natural variant of slo alters a specific component of courtship song, illustrating that regulatory evolution of a highly pleiotropic ion channel gene can cause modular changes in behaviour.

Animals collect sensory information from the world and make adaptive choices about how to respond to it. Here, we reveal a network motif in the brain for one of the most fundamental behavioral choices made by bilaterally symmetric animals: whether to respond to a sensory stimulus by moving to the left or to the right. We define network connectivity in the hindbrain important for the lateralized escape behavior of zebrafish and then test the role of neurons by using laser ablations and behavioral studies. Key inhibitory neurons in the circuit lie in a column of morphologically similar cells that is one of a series of such columns that form a developmental and functional ground plan for building hindbrain networks. Repetition within the columns of the network motif we defined may therefore lie at the foundation of other lateralized behavioral choices.