Knowledge of one’s own behavioral state—whether one is walking, grooming, or resting—is critical for contextualizing sensory cues including interpreting visual motion and tracking odor sources. Additionally, awareness of one’s own posture is important to avoid initiating destabilizing or physically impossible actions. Ascending neurons (ANs), interneurons in the vertebrate spinal cord or insect ventral nerve cord (VNC) that project to the brain, may provide such high-fidelity behavioral state signals. However, little is known about what ANs encode and where they convey signals in any brain. To address this gap, we performed a large-scale functional screen of AN movement encoding, brain targeting, and motor system patterning in the adult fly, Drosophila melanogaster. Using a new library of AN sparse driver lines, we measured the functional properties of 247 genetically-identifiable ANs by performing two-photon microscopy recordings of neural activity in tethered, behaving flies. Quantitative, deep network-based neural and behavioral analyses revealed that ANs nearly exclusively encode high-level behaviors—primarily walking as well as resting and grooming—rather than low-level joint or limb movements. ANs that convey self-motion—resting, walking, and responses to gust-like puff stimuli—project to the brain’s anterior ventrolateral protocerebrum (AVLP), a multimodal, integrative sensory hub, while those that encode discrete actions—eye grooming, turning, and proboscis extension—project to the brain’s gnathal ganglion (GNG), a locus for action selection. The structure and polarity of AN projections within the VNC are predictive of their functional encoding and imply that ANs participate in motor computations while also relaying state signals to the brain. Illustrative of this are ANs that temporally integrate proboscis extensions over tens-of-seconds, likely through recurrent interconnectivity. Thus, in line with long-held theoretical predictions, ascending populations convey high-level behavioral state signals almost exclusively to brain regions implicated in sensory feature contextualization and action selection.

Mating induces pronounced changes in female reproductive behavior, typically including a dramatic reduction in sexual receptivity. In Drosophila, postmating behavioral changes are triggered by sex peptide (SP), a male seminal fluid peptide that acts via a receptor (SPR) expressed in sensory neurons (SPSNs) of the female reproductive tract. Here, we identify second-order neurons that mediate the behavioral changes induced by SP. These SAG neurons receive synaptic input from SPSNs in the abdominal ganglion and project to the dorsal protocerebrum. Silencing SAG neurons renders virgin females unreceptive, whereas activating them increases the receptivity of females that have already mated. Physiological experiments demonstrate that SP downregulates the excitability of the SPSNs, and hence their input onto SAG neurons. These data thus provide a physiological correlate of mating status in the female central nervous system and a key entry point into the brain circuits that control sexual receptivity.

In Caenorhabditis elegans, satiety quiescence mimics behavioral aspects of satiety and postprandial sleep in mammals. On the basis of calcium-imaging, genetics, and behavioral studies, here we report that a pair of amphid neurons, ASI, is activated by nutrition and regulates worms’ behavioral states specifically promoting satiety quiescence; ASI inhibits the switch from quiescence to dwelling (a browsing state) and accelerates the switch from dwelling to quiescence. The canonical TGFβ pathway, whose ligand is released from ASI, regulates satiety quiescence. The mutants of a ligand, a receptor and SMADs in the TGFβ pathway all eat more and show less quiescence than wild-type. The TGFβ receptor in downstream neurons RIM and RIC is sufficient for worms to exhibit satiety quiescence, suggesting neuronal connection from ASI to RIM and RIC is essential for feeding regulation through the TGFβ pathway. ASI also regulates satiety quiescence partly through cGMP signaling; restoring cGMP signaling in ASI rescues the satiety quiescence defect of cGMP signaling mutants. From these results, we propose that TGFβ and cGMP pathways in ASI connect nutritional status to promotion of satiety quiescence, a sleep-like behavioral state.

The way the hippocampus processes information and encodes memories in the form of "cell assemblies" is likely determined in part by how its circuits are wired up during development. In this issue, Xu et al. now provide new insight into how neurons arising from a single common precursor migrate to their final destination and form functionally synchronous ensembles.

Neural circuits connecting the cerebral cortex, the basal ganglia and the thalamus are fundamental networks for sensorimotor processing and their dysfunction has been consistently implicated in neuropsychiatric disorders1-9. These recursive, loop circuits have been investigated in animal models and by clinical neuroimaging, however, direct functional access to developing human neurons forming these networks has been limited. Here, we use human pluripotent stem cells to reconstruct an in vitro cortico-striatal-thalamic-cortical circuit by creating a four-part loop assembloid. More specifically, we generate regionalized neural organoids that resemble the key elements of the cortico-striatal-thalamic-cortical circuit, and functionally integrate them into loop assembloids using custom 3D-printed biocompatible wells. Volumetric and mesoscale calcium imaging, as well as extracellular recordings from individual parts of these assembloids reveal the emergence of synchronized patterns of neuronal activity. In addition, a multi–step rabies retrograde tracing approach demonstrate the formation of neuronal connectivity across the network in loop assembloids. Lastly, we apply this system to study heterozygous loss of ASH1L gene associated with autism spectrum disorder and Tourette syndrome and discover aberrant synchronized activity in disease model assembloids. Taken together, this human multi-cellular platform will facilitate functional investigations of the cortico-striatal-thalamic-cortical circuit in the context of early human development and in disease conditions.

Recent insights into genome organization have emphasized the importance of A/B chromatin compartments. While our previous research showed that Brd2 depletion weakens compartment boundaries and promotes A/B mixing 1, Hinojosa-Gonzalez et al.2 were unable to replicate the findings. In response, we revisited our Micro-C data and successfully replicated the original results using the default parameters in the cooltools software package. We show that, after correcting inconsistencies with the selection and phasing of the compartment profiles, the decrease in B compartment strength persists but the change in compartment identity is to a much lesser extent than originally reported. To further assess the regulatory role of Brd2, we used saddle plots to determine the strength of compartmentalization and observed a consistent decrease of compartment strength especially at B compartments upon Brd2 depletion. This study highlights the importance of selecting appropriate parameters and analytical tools for compartment analysis and carefully interpreting the results.

Astrocytes are predominant glial cells that tile the central nervous system and participate in well-established functional and morphological interactions with neurons, blood vessels, and other glia. These ubiquitous cells display rich intracellular Ca signaling, which has now been studied for over 30 years. In this review, we provide a summary and perspective of recent progress concerning the study of astrocyte intracellular Ca signaling as well as discussion of its potential functions. Progress has occurred in the areas of imaging, silencing, activating, and analyzing astrocyte Ca signals. These insights have collectively permitted exploration of the relationships of astrocyte Ca signals to neural circuit function and behavior in a variety of species. We summarize these aspects along with a framework for mechanistically interpreting behavioral studies to identify directly causal effects. We finish by providing a perspective on new avenues of research concerning astrocyte Ca signaling.

Many brain functions depend on the ability of neural networks to temporally integrate transient inputs to produce sustained discharges. This can occur through cell-autonomous mechanisms in individual neurons and through reverberating activity in recurrently connected neural networks. We report a third mechanism involving temporal integration of neural activity by a network of astrocytes. Previously, we showed that some types of interneurons can generate long-lasting trains of action potentials (barrage firing) following repeated depolarizing stimuli. Here we show that calcium signaling in an astrocytic network correlates with barrage firing; that active depolarization of astrocyte networks by chemical or optogenetic stimulation enhances; and that chelating internal calcium, inhibiting release from internal stores, or inhibiting GABA transporters or metabotropic glutamate receptors inhibits barrage firing. Thus, networks of astrocytes influence the spatiotemporal dynamics of neural networks by directly integrating neural activity and driving barrages of action potentials in some populations of inhibitory interneurons.

Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron-glia interactions. Here, we show the first electrophysiological recordings from Drosophila astrocytes and characterize their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current-voltage relationship, low membrane resistance, high capacitance, and dye-coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic pre-motor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via Eaat1, and blocking Eaat1 extended glutamatergic interneuron-evoked inhibitory post-synaptic currents in motor neurons. The neuronal synapses were always located within a micron of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters. This article is protected by copyright. All rights reserved.

Clathrin/AP2-coated vesicles are the principal endocytic carriers originating at the plasma membrane. In experiments reported here, we have used spinning disk confocal and lattice light sheet microscopy to study the assembly dynamics of coated pits on the dorsal and ventral membranes of migrating U373 glioblastoma cells stably expressing AP2-EGFP and on lateral protrusions from immobile SUM159 breast carcinoma cells, gene edited to express AP2-EGFP. On U373 cells, coated pits initiated on the dorsal membrane at the front of the lamellipodium, as well as at the approximate boundary between the lamellipodium and lamella, and continued to grow as they were swept back toward the cell body; coated pits were absent from the corresponding ventral membrane. We observed a similar dorsal/ventral asymmetry on membrane protrusions from SUM159 cells. Stationary-coated pits formed and budded on the remainder of the dorsal and ventral surfaces of both types of cells. These observations support a previously proposed model that invokes net membrane deposition at the leading edge due to an imbalance between the endocytic and exocytic membrane flow at the front of a migrating cell.