Complete synaptic wiring diagrams, or connectomes, of whole brains open new opportunities for studying the structure-function relationship of neural circuits. However, the large number of nodes and edges in the graphs makes the analysis challenging. Here, we present the Connectome Interpreter (https://github.com/YijieYin/connectome_interpreter), an open-source software toolkit for efficient graph traversal to find polysynaptic pathways, compute the effective connectivity and receptive fields for arbitrarily deep neurons, slice out subcircuits, and non-linear but differentiable circuit modeling, implemented using efficient approaches tailored to the high density and size of connectomes such as that of the fruit fly Drosophila melanogaster. Our approach delivers results orders of magnitude faster than conventional methods in consumer computer hardware. We demonstrate the capabilities of our toolkit with select applications, including quantifying the density of polysynaptic connections in the whole adult fruit fly brain, exploring the necessity for non-linearities in circuit modeling, and combining known function of neurons with the connectome to aid in formulating hypotheses of circuit function.

Brains contain networks of interconnected neurons, so knowing the network architecture is essential for understanding brain function. We therefore mapped the synaptic-resolution connectome of an insect brain (Drosophila larva) with rich behavior, including learning, value-computation, and action-selection, comprising 3,013 neurons and 544,000 synapses. We characterized neuron-types, hubs, feedforward and feedback pathways, and cross-hemisphere and brain-nerve cord interactions. We found pervasive multisensory and interhemispheric integration, highly recurrent architecture, abundant feedback from descending neurons, and multiple novel circuit motifs. The brain’s most recurrent circuits comprised the input and output neurons of the learning center. Some structural features, including multilayer shortcuts and nested recurrent loops, resembled powerful machine learning architectures. The identified brain architecture provides a basis for future experimental and theoretical studies of neural circuits.

Making inferences about the computations performed by neuronal circuits from synapse-level connectivity maps is an emerging opportunity in neuroscience. The mushroom body (MB) is well positioned for developing and testing such an approach due to its conserved neuronal architecture, recently completed dense connectome, and extensive prior experimental studies of its roles in learning, memory and activity regulation. Here we identify new components of the MB circuit in , including extensive visual input and MB output neurons (MBONs) with direct connections to descending neurons. We find unexpected structure in sensory inputs, in the transfer of information about different sensory modalities to MBONs, and in the modulation of that transfer by dopaminergic neurons (DANs). We provide insights into the circuitry used to integrate MB outputs, connectivity between the MB and the central complex and inputs to DANs, including feedback from MBONs. Our results provide a foundation for further theoretical and experimental work.

Gastropod mollusks are known for their large, individually identifiable neurons, which are amenable to long-term intracellular recordings that can be repeated from animal to animal. The constancy of individual neurons can help distinguish state-dependent or temporal variation within an individual from actual variability between individual animals. Investigations into the circuitry underlying rhythmic swimming movements of the gastropod species, and have uncovered intra- and inter-individual variability in synaptic connectivity and serotonergic neuromodulation. has a reliably evoked escape swim behavior that is produced by a central pattern generator (CPG) composed of a small number of identifiable neurons. There is apparent individual variability in some of the connections between neurons that is inconsequential for the production of the swim behavior under normal conditions, but determines whether that individual can swim following a neural lesion. Serotonergic neuromodulation of synaptic strength intrinsic to the CPG creates neural circuit plasticity within an individual and contributes to reorganization of the network during recovery from injury and during learning. In , variability over time in the modulatory actions of serotonin and in expression of serotonin receptor genes in an identified neuron directly reflects variation in swimming behavior. Tracking behavior and electrophysiology over hours to days was necessary to identify the functional consequences of these intra-individual, time-dependent variations. This work demonstrates the importance of unambiguous neuron identification, properly assessing the animal and network states, and tracking behavior and physiology over time to distinguish plasticity within the same animal at different times from variability across individual animals.

Astrocyte dysfunction has previously been linked to multiple neurodegenerative disorders including Parkinson's disease (PD). Among their many roles, astrocytes are mediators of the brain immune response, and astrocyte reactivity is a pathological feature of PD. They are also involved in the formation and maintenance of the blood-brain barrier (BBB), but barrier integrity is compromised in people with PD. This study focuses on an unexplored area of PD pathogenesis by characterizing the interplay between astrocytes, inflammation and BBB integrity, and by combining patient-derived induced pluripotent stem cells with microfluidic technologies to generate a 3D human BBB chip. Here we report that astrocytes derived from female donors harboring the PD-related LRRK2 G2019S mutation are pro-inflammatory and fail to support the formation of a functional capillary in vitro. We show that inhibition of MEK1/2 signaling attenuates the inflammatory profile of mutant astrocytes and rescues BBB formation, providing insights into mechanisms regulating barrier integrity in PD. Lastly, we confirm that vascular changes are also observed in the human postmortem substantia nigra of both males and females with PD.

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.

During the last larval molt in Manduca sexta, in response to an increasing, then decreasing ecdysteroid titer, a number of transcription factors such as E75B, MHR3, MHR4, and betaFTZ-F1 appear and disappear in the abdominal epidermis leading to dopa decarboxylase (DDC) expression. Messenger RNAs for both the 20E-induced transcription factors, MHR3 and E75B, are maximal near the peak of the ecdysteroid titer with MHR4 mRNA appearing as the titer declines followed by betaFTZ-F1 and DDC mRNAs. E75B and MHR4 mRNA were not expressed in Manduca GV1 cells, either during exposure to 20E or after its removal. When either MHR3 dsRNA was transfected or E75B was constitutively expressed in these cells, MHR4 mRNA appeared in response to 20E by 6h. E75B was found to form a heterodimer with MHR3 using the BacterioMatch II two-hybrid assay. We conclude that MHR3 apparently suppresses MHR4 expression in the presence of 20E; the appearance of E75B then removes MHR3 by dimerization, allowing MHR4 to be expressed. Because of significant basal activity of the ddc promoter in the GV1 cells, we could perform rescue experiments by adding various factors. Constitutive expression of either E75B or MHR4 in the cells suppressed the significant basal activity of the 3.2kb ddc promoter in the GV1 cells, but 20E had no effect on this activity. Thus, E75B and MHR4 are 20E-induced inhibitory factors that suppress ddc expression and therefore act as ecdysteroid-regulated timers to coordinate the onset of ddc expression at the end of the molt.

Steroid hormones play key roles in development, growth, and reproduction in various animal phyla [1]. The insect steroid hormone, ecdysteroid, coordinates growth and maturation, represented by molting and metamorphosis [2]. In Drosophila melanogaster, the prothoracicotropic hormone (PTTH)-producing neurons stimulate peak levels of ecdysteroid biosynthesis for maturation [3]. Additionally, recent studies on PTTH signaling indicated that basal levels of ecdysteroid negatively affect systemic growth prior to maturation [4-8]. However, it remains unclear how PTTH signaling is regulated for basal ecdysteroid biosynthesis. Here, we report that Corazonin (Crz)-producing neurons regulate basal ecdysteroid biosynthesis by affecting PTTH neurons. Crz belongs to gonadotropin-releasing hormone (GnRH) superfamily, implying an analogous role in growth and maturation [9]. Inhibition of Crz neuronal activity increased pupal size, whereas it hardly affected pupariation timing. This phenotype resulted from enhanced growth rate and a delay in ecdysteroid elevation during the mid-third instar larval (L3) stage. Interestingly, Crz receptor (CrzR) expression in PTTH neurons was higher during the mid- than the late-L3 stage. Silencing of CrzR in PTTH neurons increased pupal size, phenocopying the inhibition of Crz neuronal activity. When Crz neurons were optogenetically activated, a strong calcium response was observed in PTTH neurons during the mid-L3, but not the late-L3, stage. Furthermore, we found that octopamine neurons contact Crz neurons in the subesophageal zone (SEZ), transmitting signals for systemic growth. Together, our results suggest that the Crz-PTTH neuronal axis modulates ecdysteroid biosynthesis in response to octopamine, uncovering a regulatory neuroendocrine system in the developmental transition from growth to maturation.

The density and distribution of regulatory information in non-coding DNA of eukaryotic genomes is largely unknown. Evolutionary analyses have estimated that ∼60% of nucleotides in intergenic regions of the D. melanogaster genome is functionally relevant. This estimate is difficult to reconcile with the commonly accepted idea that enhancers are compact regulatory elements that generally encompass less than 1 kilobase of DNA. Here, we approached this issue through a functional dissection of the regulatory region of the gene shavenbaby (svb). Most of the ∼90 kilobases of this large regulatory region is highly conserved in the genus Drosophila, though characterized enhancers occupy a small fraction of this region. By analyzing the regulation of svb in different contexts of Drosophila development, we found that the regulatory architecture that drives svb expression in the abdominal pupal epidermis is organized in a dramatically different way than the information that drives svb expression in the embryonic epidermis. While in the embryonic epidermis svb is activated by compact and dispersed enhancers, svb expression in the pupal epidermis is driven by large regions with enhancer activity, which occupy a great portion of the svb cis-regulatory DNA. We observed that other developmental genes also display a dense distribution of putative regulatory elements in their regulatory regions. Furthermore, we found that a large percentage of conserved non-coding DNA of the Drosophila genome is contained within putative regulatory DNA. These results suggest that part of the evolutionary constraint on non-coding DNA of Drosophila is explained by the density of regulatory information.

The saga of fluorescence recovery after photobleaching (FRAP) illustrates how disparate technical developments impact science. Starting with the classic 1976 Axelrod et al. work in Biophysical Journal, FRAP (originally fluorescence photobleaching recovery) opened the door to extraction of quantitative information from photobleaching experiments, laying the experimental and theoretical groundwork for quantifying both the mobility and the mobile fraction of a labeled population of proteins. Over the ensuing years, FRAP's reach dramatically expanded, with new developments in GFP technology and turn-key confocal microscopy, which enabled measurement of protein diffusion and binding/dissociation rates in virtually every compartment within the cell. The FRAP technique and data catalyzed an exchange of ideas between biophysicists studying membrane dynamics, cell biologists focused on intracellular dynamics, and systems biologists modeling the dynamics of cell activity. The outcome transformed the field of cellular biology, leading to a fundamental rethinking of long-held theories of cellular dynamism. Here, we review the pivotal FRAP studies that made these developments and conceptual changes possible, which gave rise to current models of complex cell dynamics.