Adult Drosophila Brain Research Articles

Synaptic connectome of a neurosecretory network in the Drosophila brain.

Hormones mediate inter-organ signaling which is crucial in orchestrating diverse behaviors and physiological processes including sleep and activity, feeding, growth, metabolism and reproduction. The pars intercerebralis and pars lateralis in insects represent major hubs which contain neurosecretory cells (NSC) that produce various hormones. To obtain insight into how hormonal signaling is regulated, we have characterized the synaptic connectome of NSC in the adult Drosophila brain. Identification of neurons providing inputs to multiple NSC subtypes implicates diuretic hormone 44-expressing NSC as a major coordinator of physiology and behavior. Surprisingly, despite most NSC having dendrites in the subesophageal zone (primary taste processing center), gustatory inputs to NSC are largely indirect. We also deciphered pathways via which diverse olfactory inputs are relayed to NSC. Further, our analyses revealed substantial inputs from descending neurons to NSC, suggesting that descending neurons regulate both endocrine and motor output to synchronize physiological changes with appropriate behaviors. In contrast to NSC inputs, synaptic output from NSC is sparse and mostly mediated by corazonin NSC. Therefore, we additionally determine putative paracrine interconnectivity between NSC subtypes and hormonal pathways from NSC to peripheral tissues by analyzing single-cell transcriptomic datasets. Our comprehensive characterization of the Drosophila neurosecretory network connectome provides a platform to understand complex hormonal networks and how they orchestrate animal behaviors and physiology.

A LAT1-Like Amino Acid Transporter Regulates Neuronal Activity in the Drosophila Mushroom Bodies.

The proper functioning of neural circuits that integrate sensory signals is essential for individual adaptation to an ever-changing environment. Many molecules can modulate neuronal activity, including neurotransmitters, receptors, and even amino acids. Here, we ask whether amino acid transporters expressed by neurons can influence neuronal activity. We found that minidiscs (mnd), which encodes a light chain of a heterodimeric amino acid transporter, is expressed in different cell types of the adult Drosophila brain: in mushroom body neurons (MBs) and in glial cells. Using live calcium imaging, we found that MND expressed in α/β MB neurons is essential for sensitivity to the L-amino acids: Leu, Ile, Asp, Glu, Lys, Thr, and Arg. We found that the Target Of Rapamycin (TOR) pathway but not the Glutamate Dehydrogenase (GDH) pathway is involved in the Leucine-dependent response of α/β MB neurons. This study strongly supports the key role of MND in regulating MB activity in response to amino acids.

Synapse-Specific Trapping of SNARE Machinery Proteins in the Anesthetized Drosophila Brain.

General anesthetics disrupt brain network dynamics through multiple pathways, in part through postsynaptic potentiation of inhibitory ion channels as well as presynaptic inhibition of neuroexocytosis. Common clinical general anesthetic drugs, such as propofol and isoflurane, have been shown to interact and interfere with core components of the exocytic release machinery to cause impaired neurotransmitter release. Recent studies however suggest that these drugs do not affect all synapse subtypes equally. We investigated the role of the presynaptic release machinery in multiple neurotransmitter systems under isoflurane general anesthesia in the adult female Drosophila brain using live-cell super-resolution microscopy and optogenetic readouts of exocytosis and neural excitability. We activated neurotransmitter-specific mushroom body output neurons and imaged presynaptic function under isoflurane anesthesia. We found that isoflurane impaired synaptic release and presynaptic protein dynamics in excitatory cholinergic synapses. In contrast, isoflurane had little to no effect on inhibitory GABAergic or glutamatergic synapses. These results present a distinct inhibitory mechanism for general anesthesia, whereby neuroexocytosis is selectively impaired at excitatory synapses, while inhibitory synapses remain functional. This suggests a presynaptic inhibitory mechanism that complements the other inhibitory effects of these drugs.

The spatial and temporal structure of neural activity across the fly brain

What are the spatial and temporal scales of brainwide neuronal activity? We used swept, confocally-aligned planar excitation (SCAPE) microscopy to image all cells in a large volume of the brain of adult Drosophila with high spatiotemporal resolution while flies engaged in a variety of spontaneous behaviors. This revealed neural representations of behavior on multiple spatial and temporal scales. The activity of most neurons correlated (or anticorrelated) with running and flailing over timescales that ranged from seconds to a minute. Grooming elicited a weaker global response. Significant residual activity not directly correlated with behavior was high dimensional and reflected the activity of small clusters of spatially organized neurons that may correspond to genetically defined cell types. These clusters participate in the global dynamics, indicating that neural activity reflects a combination of local and broadly distributed components. This suggests that microcircuits with highly specified functions are provided with knowledge of the larger context in which they operate.

Neural connectivity molecules best identify the heterogeneous clock and dopaminergic cell types in the Drosophila adult brain.

Our recent single-cell sequencing of most adult Drosophila circadian neurons indicated notable and unexpected heterogeneity. To address whether other populations are similar, we sequenced a large subset of adult brain dopaminergic neurons. Their gene expression heterogeneity is similar to that of clock neurons, i.e., both populations have two to three cells per neuron group. There was also unexpected cell-specific expression of neuron communication molecule messenger RNAs: G protein-coupled receptor or cell surface molecule (CSM) transcripts alone can define adult brain dopaminergic and circadian neuron cell type. Moreover, the adult expression of the CSM DIP-beta in a small group of clock neurons is important for sleep. We suggest that the common features of circadian and dopaminergic neurons are general, essential for neuronal identity and connectivity of the adult brain, and that these features underlie the complex behavioral repertoire of Drosophila.

Regulation and modulation of biogenic amine neurotransmission in Drosophila and Caenorhabditis elegans.

Neurotransmitters are crucial for the relay of signals between neurons and their target. Monoamine neurotransmitters dopamine (DA), serotonin (5-HT), and histamine are found in both invertebrates and mammals and are known to control key physiological aspects in health and disease. Others, such as octopamine (OA) and tyramine (TA), are abundant in invertebrates. TA is expressed in both Caenorhabditis elegans and Drosophila melanogaster and plays important roles in the regulation of essential life functions in each organism. OA and TA are thought to act as the mammalian homologs of epinephrine and norepinephrine respectively, and when triggered, they act in response to the various stressors in the fight-or-flight response. 5-HT regulates a wide range of behaviors in C. elegans including egg-laying, male mating, locomotion, and pharyngeal pumping. 5-HT acts predominantly through its receptors, of which various classes have been described in both flies and worms. The adult brain of Drosophila is composed of approximately 80 serotonergic neurons, which are involved in modulation of circadian rhythm, feeding, aggression, and long-term memory formation. DA is a major monoamine neurotransmitter that mediates a variety of critical organismal functions and is essential for synaptic transmission in invertebrates as it is in mammals, in which it is also a precursor for the synthesis of adrenaline and noradrenaline. In C. elegans and Drosophila as in mammals, DA receptors play critical roles and are generally grouped into two classes, D1-like and D2-like based on their predicted coupling to downstream G proteins. Drosophila uses histamine as a neurotransmitter in photoreceptors as well as a small number of neurons in the CNS. C. elegans does not use histamine as a neurotransmitter. Here, we review the comprehensive set of known amine neurotransmitters found in invertebrates, and discuss their biological and modulatory functions using the vast literature on both Drosophila and C. elegans. We also suggest the potential interactions between aminergic neurotransmitters systems in the modulation of neurophysiological activity and behavior.

Parkin Knockdown Modulates Dopamine Release in the Central Complex, but Not the Mushroom Body Heel, of Aging Drosophila.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons leading to reduced locomotion. Mutations of parkin gene in Drosophila produce the same phenotypes as vertebrate models, but the effect of parkin knockdown on dopamine release is not known. Here, we report age-dependent, spatial variation of dopamine release in the brain of parkin-RNAi adult Drosophila. Dopamine was repetitively stimulated by local application of acetylcholine and quantified by fast-scan cyclic voltammetry in the central complex or mushroom body heel. In the central complex, the main area controlling locomotor function, dopamine release is maintained for repeated stimulations in aged control flies, but lower concentrations of dopamine are released in the central complex of aged parkin-RNAi flies. In the mushroom body heel, the dopamine release decrease in older parkin-RNAi flies is similar to controls. There is not significant dopaminergic neuronal loss even in older parkin knockdown flies, which indicates that the changes in stimulated dopamine release are due to alterations of neuronal function. In young parkin-RNAi flies, locomotion is inhibited by 30%, while in older parkin-RNAi flies it is inhibited by 85%. Overall, stimulated dopamine release is modulated by parkin in an age and brain region dependent manner. Correlating the functional state of the dopaminergic system with behavioral phenotypes provides unique insights into the PD mechanism. Drosophila can be used to study dopamine functionality in PD, elucidate how genetics influence dopamine, and test potential therapies to maintain dopamine release.

Cell‐specific responses to Tau pathology in the aging brain

AbstractBackgroundGene expression profiling has emerged as a promising unbiased approach to characterize putative mechanisms of Alzheimer’s disease (AD). We previously highlighted transcriptional signatures that are driven by Tau pathology or aging in a longitudinal Drosophila model of tauopathy and human RNA‐sequencing data. However, cellular heterogeneity confounds the interpretation of bulk‐tissue gene expression. Defining cell types and characterizing cell‐specific processes that precipitate neurodegeneration is crucial for elucidating AD pathogenesis.MethodWe use a highly tractable, longitudinal, Drosophila model of tauopathy and single cell transcriptomics (scRNA‐seq) to explore cell‐specific contributions to tau‐mediated gene expression while accounting for age‐related changes. Brains of adult Drosophila pan‐neuronally expressing a mutant variant of human Tau (R406W) and respective controls were dissociated for droplet‐based scRNA‐seq at 1‐, 10‐, and 20‐ days post‐eclosion. We computed differentially expressed genes (DEGs) for each cell subpopulation and profiled gene regulatory networks (regulons), highlighting cell‐specific Tau‐ and aging‐related changes. Tau‐induced changes were compared with previously published bulk RNA‐sequencing data and gene co‐expression networks. To address cross‐species conservation of single cell transcriptional signatures, we leverage published human single nuclei RNA‐sequencing (snRNA‐seq) data.ResultWe identified 96 cell subpopulations clustered by transcriptomic similarities in the adult Drosophila brain and annotated 59 with cell identities. We mapped half of all DEGs identified in our published bulk‐tissue RNA‐sequencing to cell subpopulations. We characterized 183 regulons in our dataset and demonstrated network‐level expression changes driven by Tau. Most Tau‐induced gene expression changes were observed in glutamatergic and cholinergic excitatory neurons while fewer changes were seen in GABAergic inhibitory neurons. Likewise, we observed Tau‐induced reductions in cholinergic and glutamatergic neuron numbers. Correlation of cluster‐specific transcriptional profiles between our data and mammalian snRNA‐seq demonstrate conservation of gene expression among inferred cell identities. Strikingly, we found abundant innate immune gene expression signatures among neuronal subpopulations, and pinpointed cell autonomous Nuclear Factor Kappa B (NFkB) signaling as a modifier in Tau‐induced neurodegeneration.ConclusionOur results comprise a powerful single cell transcriptomic resource for studying tau‐mediated disruption of gene expression and dynamic age‐dependent changes at cellular resolution in a tractable model system.

Mettl3-dependent m6A modification attenuates the brain stress response in Drosophila

N6-methyladenosine (m6A), the most prevalent internal modification on eukaryotic mRNA, plays an essential role in various stress responses. The brain is uniquely vulnerable to cellular stress, thus defining how m6A sculpts the brain’s susceptibility may provide insight to brain aging and disease-related stress. Here we investigate the impact of m6A mRNA methylation in the adult Drosophila brain with stress. We show that m6A is enriched in the adult brain and increases with heat stress. Through m6A-immunoprecipitation sequencing, we show 5′UTR Mettl3-dependent m6A is enriched in transcripts of neuronal processes and signaling pathways that increase upon stress. Mettl3 knockdown results in increased levels of m6A targets and confers resilience to stress. We find loss of Mettl3 results in decreased levels of nuclear m6A reader Ythdc1, and knockdown of Ythdc1 also leads to stress resilience. Overall, our data suggest that m6A modification in Drosophila dampens the brain’s biological response to stress.

TauLUM, an in vivo Drosophila sensor of tau multimerization, identifies neuroprotective interventions in tauopathy

SummaryTau protein aggregates are a defining neuropathological feature of “tauopathies,” a group of neurodegenerative disorders that include Alzheimer’s disease. In the current study, we develop a Drosophila split-luciferase-based sensor of tau-tau interaction. This model, which we term “tauLUM,” allows investigators to quantify tau multimerization at individual time points or longitudinally in adult, living animals housed in a 96-well plate. TauLUM causes cell death in the adult Drosophila brain and responds to both pharmacological and genetic interventions. We find that transgenic expression of an anti-tau intrabody or pharmacological inhibition of HSP90 reduces tau multimerization and cell death in tauLUM flies, establishing the suitability of this system for future drug and genetic modifier screening. Overall, our studies position tauLUM as a powerful in vivo discovery platform that leverages the advantages of the Drosophila model organism to better understand tau multimerization.

Tau-Induced Elevation of the Activity-Regulated Cytoskeleton Associated Protein Arc1 Causally Mediates Neurodegeneration in the Adult Drosophila Brain

Alzheimer’s disease and other tauopathies are neurodegenerative disorders pathologically defined by aggregated forms of tau protein in the brain. While synaptic degradation is a well-established feature of tau-induced neurotoxicity, the underlying mechanisms of how pathogenic forms of tau drive synaptic dysfunction are incompletely understood. Synaptic function and subsequent memory consolidation are dependent upon synaptic plasticity, the ability of synapses to adjust their structure and strength in response to changes in activity. The activity regulated cytoskeleton associated protein ARC acts in the nucleus and at postsynaptic densities to regulate various forms of synaptic plasticity. ARC harbors a retrovirus-like Gag domain that facilitates ARC multimerization and capsid formation. Trans-synaptic transfer of RNA-containing ARC capsids is required for synaptic plasticity. While ARC is elevated in brains of patients with Alzheimer’s disease and genetic variants in ARC increase susceptibility to Alzheimer’s disease, mechanistic insight into the role of ARC in Alzheimer’s disease is lacking. Using a Drosophila model of tauopathy, we find that pathogenic tau significantly increases multimeric species of the protein encoded by the Drosophila homolog of ARC, Arc1, in the adult fly brain. We find that Arc1 is elevated within nuclei and the neuropil of tau transgenic Drosophila, but does not localize to synaptic vesicles or presynaptic terminals. Lastly, we find that genetic manipulation of Arc1 modifies tau-induced neurotoxicity, suggesting that tau-induced Arc1 elevation mediates neurodegeneration. Taken together, our results suggest that ARC elevation in human Alzheimer’s disease is a consequence of tau pathology and is a causal factor contributing to neuronal death.

Classification and genetic targeting of cell types in the primary taste and premotor center of the adult Drosophila brain.

Neural circuits carry out complex computations that allow animals to evaluate food, select mates, move toward attractive stimuli, and move away from threats. In insects, the subesophageal zone (SEZ) is a brain region that receives gustatory, pheromonal, and mechanosensory inputs and contributes to the control of diverse behaviors, including feeding, grooming, and locomotion. Despite its importance in sensorimotor transformations, the study of SEZ circuits has been hindered by limited knowledge of the underlying diversity of SEZ neurons. Here, we generate a collection of split-GAL4 lines that provides precise genetic targeting of 138 different SEZ cell types in adult Drosophila melanogaster, comprising approximately one third of all SEZ neurons. We characterize the single-cell anatomy of these neurons and find that they cluster by morphology into six supergroups that organize the SEZ into discrete anatomical domains. We find that the majority of local SEZ interneurons are not classically polarized, suggesting rich local processing, whereas SEZ projection neurons tend to be classically polarized, conveying information to a limited number of higher brain regions. This study provides insight into the anatomical organization of the SEZ and generates resources that will facilitate further study of SEZ neurons and their contributions to sensory processing and behavior.

NeuroRetriever: Automatic Neuron Segmentation for Connectome Assembly.

Segmenting individual neurons from a large number of noisy raw images is the first step in building a comprehensive map of neuron-to-neuron connections for predicting information flow in the brain. Thousands of fluorescence-labeled brain neurons have been imaged. However, mapping a complete connectome remains challenging because imaged neurons are often entangled and manual segmentation of a large population of single neurons is laborious and prone to bias. In this study, we report an automatic algorithm, NeuroRetriever, for unbiased large-scale segmentation of confocal fluorescence images of single neurons in the adult Drosophila brain. NeuroRetriever uses a high-dynamic-range thresholding method to segment three-dimensional morphology of single neurons based on branch-specific structural features. Applying NeuroRetriever to automatically segment single neurons in 22,037 raw brain images, we successfully retrieved 28,125 individual neurons validated by human segmentation. Thus, automated NeuroRetriever will greatly accelerate 3D reconstruction of the single neurons for constructing the complete connectomes.

Selective dendritic localization of mRNA in Drosophila mushroom body output neurons.

Memory-relevant neuronal plasticity is believed to require local translation of new proteins at synapses. Understanding this process requires the visualization of the relevant mRNAs within these neuronal compartments. Here, we used single-molecule fluorescence in situ hybridization to localize mRNAs at subcellular resolution in the adult Drosophila brain. mRNAs for subunits of nicotinic acetylcholine receptors and kinases could be detected within the dendrites of co-labeled mushroom body output neurons (MBONs) and their relative abundance showed cell specificity. Moreover, aversive olfactory learning produced a transient increase in the level of CaMKII mRNA within the dendritic compartments of the γ5β'2a MBONs. Localization of specific mRNAs in MBONs before and after learning represents a critical step towards deciphering the role of dendritic translation in the neuronal plasticity underlying behavioral change in Drosophila.

Behavioral Improvements and its Molecular Mechanism of Ilex kudingcha C.J. Tseng on Animal Model of Alzheimer’s Disease

Alzheimer's disease (AD) is a common chronic neurodegenerative disease with well-defined pathophysiological mechanisms. Ilex kudingcha (IK) C.J. Tseng is commonly known as bitter tea or “Khom” tea in Vietnam. The present study was conducted to investigate the anti-dementia effect of IK using olfactory bulbectomized (OBX) mice. OBX mice were daily treated with IK extract (540 mg/kg) or reference drug, tacrine (2.5 mg/kg) 1 week before and continuously for 3 days after the OBX surgery. The object recognition test, modified Y maze test and fear conditioning test were employed to analyze non-spatial short-term, spatial short-term and long-term memories of the mice respectively. Administration of IK extract and tacrine attenuated these OBX-induced cognitive deficits in mice. The effects of IK and tacrine on spatial short-term memory impairment were reversed by scopolamine, a muscarinic receptor antagonist. The amyloid-beta (Aβ) production in adult transgenic Drosophila brain flies was also investigated by using Western blotting with APP-HA antibody. These results indicated that IK extract improves short-term and long-term memory disturbances in OBX mice and that muscarinic receptor may play a role on these actions. In addition, our result also showed that IK extract reduces the expression of amyloid precursor protein (APP) in brain of AD model using Drosophila melanogaster.

Glial granules contain germline proteins in the Drosophila brain, which regulate brain transcriptome

Membraneless RNA-protein granules play important roles in many different cell types and organisms. In particular, granules found in germ cells have been used as a paradigm to study large and dynamic granules. These germ granules contain RNA and proteins required for germline development. Here, we unexpectedly identify large granules in specific subtypes of glial cells (“glial granules”) of the adult Drosophila brain which contain polypeptides with previously characterized roles in germ cells including scaffold Tudor, Vasa, Polar granule component and Piwi family proteins. Interestingly, our super-resolution microscopy analysis shows that in the glial granules, these proteins form distinct partially overlapping clusters. Furthermore, we show that glial granule scaffold protein Tudor functions in silencing of transposable elements and in small regulatory piRNA biogenesis. Remarkably, our data indicate that the adult brain contains a small population of cells, which express both neuroblast and germ cell proteins. These distinct cells are evolutionarily conserved and expand during aging suggesting the existence of age-dependent signaling. Our work uncovers previously unknown glial granules and indicates the involvement of their components in the regulation of brain transcriptome.

Connectomics Analysis Reveals First-, Second-, and Third-Order Thermosensory and Hygrosensory Neurons in the Adult Drosophila Brain

SummaryAnimals exhibit innate and learned preferences for temperature and humidity—conditions critical for their survival and reproduction. Leveraging a whole-brain electron microscopy volume, we studied the adult Drosophila melanogaster circuitry associated with antennal thermo- and hygrosensory neurons. We have identified two new target glomeruli in the antennal lobe, in addition to the five known ones, and the ventroposterior projection neurons (VP PNs) that relay thermo- and hygrosensory information to higher brain centers, including the mushroom body and lateral horn, seats of learned and innate behavior. We present the first connectome of a thermo- and hygrosensory neuropil, the lateral accessory calyx (lACA), by reconstructing neurons downstream of heating- and cooling-responsive VP PNs. A few mushroom body-intrinsic neurons solely receive thermosensory input from the lACA, while most receive additional olfactory and thermo- and/or hygrosensory PN inputs. Furthermore, several classes of lACA-associated neurons form a local network with outputs to other brain neuropils, suggesting that the lACA serves as a hub for thermo- and hygrosensory circuitry. For example, DN1a neurons link thermosensory PNs in the lACA to the circadian clock via the accessory medulla. Finally, we survey strongly connected downstream partners of VP PNs across the protocerebrum; these include a descending neuron targeted by dry-responsive VP PNs, meaning that just two synapses might separate hygrosensory inputs from motor circuits. These data provide a comprehensive first- and second-order layer analysis of Drosophila thermo- and hygrosensory systems and an initial survey of third-order neurons that could directly modulate behavior.

Multiple lineages enable robust development of the neuropil-glia architecture in adult Drosophila.

Neural remodeling is essential for the development of a functional nervous system and has been extensively studied in the metamorphosis of Drosophila Despite the crucial roles of glial cells in brain functions, including learning and behavior, little is known of how adult glial cells develop in the context of neural remodeling. Here, we show that the architecture of neuropil-glia in the adult Drosophila brain, which is composed of astrocyte-like glia (ALG) and ensheathing glia (EG), robustly develops from two different populations in the larva: the larval EG and glial cell missing-positive (gcm+ ) cells. Whereas gcm+ cells proliferate and generate adult ALG and EG, larval EG dedifferentiate, proliferate and redifferentiate into the same glial subtypes. Each glial lineage occupies a certain brain area complementary to the other, and together they form the adult neuropil-glia architecture. Both lineages require the FGF receptor Heartless to proliferate, and the homeoprotein Prospero to differentiate into ALG. Lineage-specific inhibition of gliogenesis revealed that each lineage compensates for deficiency in the proliferation of the other. Together, the lineages ensure the robust development of adult neuropil-glia, thereby ensuring a functional brain.

Dopamine regulation of reproduction in insects

Dopamine (DA) regulates reproductive behavior and promotes maturation of the reproductive system in insects. In Drosophila , male courtship behavior is regulated by the protocerebral posteriolateral dopaminergic cluster neuron 2ab (PPL2ab). The ability to synthesize DA in the PPL2ab affects male-male and male-female courtship behavior. The DA level in the brain of adult male Drosophila influences sexual orientation; although, male-male courtship is induced when the DA level is too high or too low. DA also regulates changes in reproductive behavior and post-copulation behavior. As a neurohormone, DA regulates taste and smell perception in insects. In the central nervous system, DA regulation of reproductive behavior involves the DA/MH (molting hormone) receptor (DopEcR) and synergy between DA and ecdysone, with the tritocerebrum P1 neurons playing an important role in the male courtship information loop. DA can promote development in the eggs and regulate the reproductive state in social insects. The promotional effect of DA on egg development involves developmental stage differences in DA receptor expression and interactions among DA, juvenile hormone (JH), MH, and octopamine (OA). For example, the expression level of dopamine D2-like receptor (DD2R) in the corpora allata of D. melanogaster young females is far lower than that in sexually mature females. Meanwhile, the expression level of dopamine D1-like receptor (DopR) in young females is much higher than that in mature females. By contrast, the expression of DopR and DD2R in the fat body is the opposite to that in the corpora allata. The interactions among DA, JH, MH, and OA depend on the expression of certain genes and regulation of metabolic enzyme activity. In addition, the interactions among different hormones also involve the insulin-like signaling (ILPs) pathway. As to reproductive state regulation in social insects, it has been found that the reproductive state of worker bees is regulated by queen mandibular pheromone (QMP), larvae pheromones, and the ovarian DA D2 receptor gene. Research on DA regulation of insect reproduction will provide us with references for the development of new drugs, and for research on mechanisms in human brain science and nervous system diseases.

Millisecond two-photon optical ribbon imaging for small-animal functional connectome study.

We developed a high-speed two-photon optical ribbon imaging system, which combines galvo-mirrors for an arbitrary curve scan on a lateral plane and a tunable acoustic gradient-index lens for a 100kHz-1MHz axial scan. The system provides micrometer/millisecond spatiotemporal resolutions, which enable continuous readout of functional dynamics from small and densely packed neurons in a living adult Drosophila brain. Compared to sparse sampling techniques, the ribbon imaging modality avoids motion artifacts. Combined with a Drosophila anatomical connectome database, which is the most complete among all model animals, this technique paves the way toward establishing whole-brain functional connectome.