The mating decisions of Drosophila melanogaster females are primarily revealed through either of two discrete actions: opening of the vaginal plates to allow copulation, or extrusion of the ovipositor to reject the male. Both actions are triggered by the male courtship song, and both are dependent upon the female's mating status. Virgin females are more likely to open their vaginal plates in response to song; mated females are more likely to extrude their ovipositor. Here, we examine the neural cause and behavioral consequence of ovipositor extrusion. We show that the DNp13 descending neurons act as command-type neurons for ovipositor extrusion, and that ovipositor extrusion is an effective deterrent only when performed by females that have previously mated. The DNp13 neurons respond to male song via direct synaptic input from the pC2l auditory neurons. Mating status does not modulate the song responses of DNp13 neurons, but rather how effectively they can engage the motor circuits for ovipositor extrusion. We present evidence that mating status information is mediated by ppk sensory neurons in the uterus, which are activated upon ovulation. Vaginal plate opening and ovipositor extrusion are thus controlled by anatomically and functionally distinct circuits, highlighting the diversity of neural decision-making circuits even in the context of closely related behaviors with shared exteroceptive and interoceptive inputs.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly . Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly's brain.

Understanding information processing in the brain requires monitoring neuronal activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope empowered by all-optical laser scanning, we imaged neuronal activity in vivo at up to 3,000 frames per second and submicrometer spatial resolution. This imaging method enabled monitoring of both supra- and subthreshold electrical activity down to 345 μm below the brain surface in head-fixed awake mice.

Imaging neurons and neural circuits over large volumes at high speed and subcellular resolution is a difficult task. Incorporating a Bessel focus module into a two-photon fluorescence mesoscope, we achieved rapid volumetric imaging of neural activity over the mesoscale with synaptic resolution. We applied the technology to calcium imaging of entire dendritic spans of neurons as well as neural ensembles within multiple cortical regions over two hemispheres of the awake mouse brain.

Mating and egg laying are tightly cooordinated events in the reproductive life of all oviparous females. Oviposition is typically rare in virgin females but is initiated after copulation. Here we identify the neural circuitry that links egg laying to mating status in Drosophila melanogaster. Activation of female-specific oviposition descending neurons (oviDNs) is necessary and sufficient for egg laying, and is equally potent in virgin and mated females. After mating, sex peptide-a protein from the male seminal fluid-triggers many behavioural and physiological changes in the female, including the onset of egg laying. Sex peptide is detected by sensory neurons in the uterus, and silences these neurons and their postsynaptic ascending neurons in the abdominal ganglion. We show that these abdominal ganglion neurons directly activate the female-specific pC1 neurons. GABAergic (γ-aminobutyric-acid-releasing) oviposition inhibitory neurons (oviINs) mediate feed-forward inhibition from pC1 neurons to both oviDNs and their major excitatory input, the oviposition excitatory neurons (oviENs). By attenuating the abdominal ganglion inputs to pC1 neurons and oviINs, sex peptide disinhibits oviDNs to enable egg laying after mating. This circuitry thus coordinates the two key events in female reproduction: mating and egg laying.

The ability to measure synaptic connectivity and properties is essential for understanding neuronal circuits. However, existing methods that allow such measurements at cellular resolution are laborious and technically demanding. Here, we describe a system that allows such measurements in a high-throughput way by combining two-photon optogenetics and volumetric Ca2+ imaging with whole-cell recording. We reveal a circuit motif for generating fast undulatory locomotion in zebrafish.

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

The motor cortex controls skilled arm movement by sending temporal patterns of activity to lower motor centres. Local cortical dynamics are thought to shape these patterns throughout movement execution. External inputs have been implicated in setting the initial state of the motor cortex, but they may also have a pattern-generating role. Here we dissect the contribution of local dynamics and inputs to cortical pattern generation during a prehension task in mice. Perturbing cortex to an aberrant state prevented movement initiation, but after the perturbation was released, cortex either bypassed the normal initial state and immediately generated the pattern that controls reaching or failed to generate this pattern. The difference in these two outcomes was probably a result of external inputs. We directly investigated the role of inputs by inactivating the thalamus; this perturbed cortical activity and disrupted limb kinematics at any stage of the movement. Activation of thalamocortical axon terminals at different frequencies disrupted cortical activity and arm movement in a graded manner. Simultaneous recordings revealed that both thalamic activity and the current state of cortex predicted changes in cortical activity. Thus, the pattern generator for dexterous arm movement is distributed across multiple, strongly interacting brain regions.