Flying insects exhibit remarkable navigational abilities controlled by their compact nervous systems. Optic flow, the pattern of changes in the visual scene induced by locomotion, is a crucial sensory cue for robust self-motion estimation, especially during rapid flight. Neurons that respond to specific, large-field optic flow patterns have been studied for decades, primarily in large flies, such as houseflies, blowflies, and hover flies. The best-known optic-flow sensitive neurons are the large tangential cells of the dipteran lobula plate, whose visual-motion responses, and to a lesser extent, their morphology, have been explored using single-neuron neurophysiology. Most of these studies have focused on the large, Horizontal and Vertical System neurons, yet the lobula plate houses a much larger set of 'optic-flow' sensitive neurons, many of which have been challenging to unambiguously identify or to reliably target for functional studies. Here we report the comprehensive reconstruction and identification of the Lobula Plate Tangential Neurons in an Electron Microscopy (EM) volume of a whole Drosophila brain. This catalog of 58 LPT neurons (per brain hemisphere) contains many neurons that are described here for the first time and provides a basis for systematic investigation of the circuitry linking self-motion to locomotion control. Leveraging computational anatomy methods, we estimated the visual motion receptive fields of these neurons and compared their tuning to the visual consequence of body rotations and translational movements. We also matched these neurons, in most cases on a one-for-one basis, to stochastically labeled cells in genetic driver lines, to the mirror-symmetric neurons in the same EM brain volume, and to neurons in an additional EM data set. Using cell matches across data sets, we analyzed the integration of optic flow patterns by neurons downstream of the LPTs and find that most central brain neurons establish sharper selectivity for global optic flow patterns than their input neurons. Furthermore, we found that self-motion information extracted from optic flow is processed in distinct regions of the central brain, pointing to diverse foci for the generation of visual behaviors.

Many neurons in the central nervous system produce a single primary cilium that serves as a specialized signaling organelle. Several neuromodulatory G-protein-coupled receptors (GPCRs) localize to primary cilia in neurons, although it is not understood how GPCR signaling from the cilium impacts circuit function and behavior. We find that the vertebrate ancient long opsin A (VALopA), a G-coupled GPCR extraretinal opsin, targets to cilia of zebrafish spinal neurons. In the developing 1-d-old zebrafish, brief light activation of VALopA in neurons of the central pattern generator circuit for locomotion leads to sustained inhibition of coiling, the earliest form of locomotion. We find that a related extraretinal opsin, VALopB, is also G-coupled, but is not targeted to cilia. Light-induced activation of VALopB also suppresses coiling, but with faster kinetics. We identify the ciliary targeting domains of VALopA. Retargeting of both opsins shows that the locomotory response is prolonged and amplified when signaling occurs in the cilium. We propose that ciliary localization provides a mechanism for enhancing GPCR signaling in central neurons.

The interplay between two major forebrain structures - cortex and subcortical striatum - is critical for flexible, goal-directed action. Traditionally, it has been proposed that striatum is critical for selecting what type of action is initiated while the primary motor cortex is involved in the online control of movement execution. Recent data indicates that striatum may also be critical for specifying movement execution. These alternatives have been difficult to reconcile because when comparing very distinct actions, as in the vast majority of work to date, they make essentially indistinguishable predictions. Here, we develop quantitative models to reveal a somewhat paradoxical insight: only comparing neural activity during similar actions makes strongly distinguishing predictions. We thus developed a novel reach-to-pull task in which mice reliably selected between two similar, but distinct reach targets and pull forces. Simultaneous cortical and subcortical recordings were uniquely consistent with a model in which cortex and striatum jointly specify flexible parameters of action during movement execution.

Sparse coding can improve discrimination of sensory stimuli by reducing overlap between their representations. Two factors, however, can offset sparse coding's benefits: similar sensory stimuli have significant overlap and responses vary across trials. To elucidate the effects of these 2 factors, we analyzed odor responses in the fly and mouse olfactory regions implicated in learning and discrimination-the mushroom body (MB) and the piriform cortex (PCx). We found that neuronal responses fall along a continuum from extremely reliable across trials to extremely variable or stochastic. Computationally, we show that the observed variability arises from noise within central circuits rather than sensory noise. We propose this coding scheme to be advantageous for coarse- and fine-odor discrimination. More reliable cells enable quick discrimination between dissimilar odors. For similar odors, however, these cells overlap and do not provide distinguishing information. By contrast, more unreliable cells are decorrelated for similar odors, providing distinguishing information, though these benefits only accrue with extended training with more trials. Overall, we have uncovered a conserved, stochastic coding scheme in vertebrates and invertebrates, and we identify a candidate mechanism, based on variability in a winner-take-all (WTA) inhibitory circuit, that improves discrimination with training.

Focal epilepsy is associated with intermittent brief population discharges (interictal spikes), which resemble sentinel spikes that often occur at the onset of seizures. Why interictal spikes self-terminate whilst seizures persist and propagate is incompletely understood. We used fluorescent glutamate and GABA sensors in an awake rodent model of neocortical seizures to resolve the spatiotemporal evolution of both neurotransmitters in the extracellular space. Interictal spikes were accompanied by brief glutamate transients which were maximal at the initiation site and rapidly propagated centrifugally. GABA transients lasted longer than glutamate transients and were maximal ∼1.5 mm from the focus where they propagated centripetally. Prior to seizure initiation GABA transients were attenuated, whilst glutamate transients increased, consistent with a progressive failure of local inhibitory restraint. As seizures increased in frequency, there was a gradual increase in the spatial extent of spike-associated glutamate transients associated with interictal spikes. Neurotransmitter imaging thus reveals a progressive collapse of an annulus of feed-forward GABA release, allowing seizures to escape from local inhibitory restraint.

Our nervous system contains billions of neurons that form precise connections with each other through interactions between cell surface proteins (CSPs). In Drosophila, the Dpr and DIP immunoglobulin protein subfamilies form homophilic or heterophilic interactions to instruct synaptic connectivity, synaptic growth and cell survival. However, the upstream regulation and downstream signaling mechanisms of Dprs and DIPs are not clear. In the Drosophila larval neuromuscular system, DIP-α is expressed in the dorsal and ventral type-Is motor neurons (MNs). We conducted an F1 dominant modifier genetic screen to identify regulators of Dprs and DIPs. We found that the transcription factor, huckebein (hkb), genetically interacts with DIP-α and is important for target recognition specifically in the dorsal Is MN, but not the ventral Is MN. Loss of hkb led to complete removal of DIP-α expression. We then confirmed that this specificity is through the dorsal Is MN specific transcription factor, even-skipped (eve), which acts downstream of hkb. Genetic interaction between hkb and eve revealed that they act in the same pathway to regulate dorsal Is MN connectivity. Our study provides insight into the transcriptional regulation of DIP-α and suggests that distinct regulatory mechanisms exist for the same CSP in different neurons.

L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular L-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of L-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular L-lactate dynamics in mice.

The endoplasmic reticulum (ER) and the Golgi apparatus are the first sorting stations along the secretory pathway of mammalian cells and have a crucial role in protein quality control and cellular homeostasis. While machinery components mediating ER-to-Golgi transport have been mapped, it is unclear how exchange between the two closely juxtaposed organelles is coordinated in living cells. Here, using gene editing to tag machinery components, live-cell confocal and stimulated emission depletion (STED) super-resolution microscopy, we show that ER-to-Golgi transport occurs via a dynamic network of tubules positive for the small GTPase ARF4. swCOPI machinery is tightly associated to this network and moves with tubular-vesicular structures. Strikingly, the ARF4 network appears to be continuous with the ER and ARF4 tubules remodel around static ER exit sites (ERES) defined by COPII machinery. We were further able to dissect the steps of ER-to-Golgi transport with functional trafficking assays. A wave of cargo released from the ER percolates through peripheral and Golgi-tethered ARF4 structures before filling the cis-Golgi. Perturbation via acute degradation of ARF4 shows an active regulatory role for the GTPase and COPI in anterograde transport. Our data supports a model in which anterograde ER-to-Golgi transport occurs via an ARF4 tubular-vesicular network directly connecting the ER and Golgi-associated pre-cisternae.

As observed in human language learning and song learning in birds, the fruit fly Drosophila melanogaster changes its' auditory behaviors according to prior sound experiences. Female flies that have heard male courtship songs of the same species are less responsive to courtship songs of different species. This phenomenon, known as song preference learning in flies, requires GABAergic input to pC1 neurons in the central brain, with these neurons playing a key role in mating behavior by integrating multimodal sensory and internal information. The neural circuit basis of this GABAergic input, however, has not yet been identified. Here, we find that pCd-2 neurons, totaling four cells per hemibrain and expressing the sex-determination gene doublesex, provide the GABAergic input to pC1 neurons for song preference learning. First, RNAi-mediated knockdown of GABA production in pCd-2 neurons abolished song preference learning. Second, pCd-2 neurons directly, and in many cases mutually, connect with pC1 neurons, suggesting the existence of reciprocal circuits between pC1 and pCd-2 neurons. Finally, GABAergic and dopaminergic inputs to pCd-2 neurons are necessary for song preference learning. Together, this study suggests that reciprocal circuits between pC1 and pCd-2 neurons serve as a sensory and internal state-integrated hub, allowing flexible control over female copulation. Consequently, this provides a neural circuit model that underlies experience-dependent auditory plasticity.