Longitudinal brain imaging is essential for understanding neural mechanisms. Here, we present a saline-free, chronic preparation for repeated neural recording in adult Drosophila over multiple days. We describe steps for mounting flies, performing manual surgery on the head cuticle without external saline, and resealing the opening to create a transparent optical window. We demonstrate the utility of this approach by tracking single-neuron spiking and neuronal calcium dynamics over 7-10 days. This protocol is potentially applicable to other insect species.

The capacity to engineer organisms with multiple transgenic components is crucial to synthetic biology and basic biology research. For the former field, transgenic organisms allow the creation of novel biological functions; for the latter, such organisms provide potent means of dissecting complex biological pathways. However, the size limitations of a single transgenesis event and challenges associated with the assembly of multiple DNA fragments hinder the efficient integration of multiple transgenes. To overcome these hurdles, here we introduce a building block for synthetic design termed an integrated genetic array (IGA), which incorporates all genetic components into a single locus to prevent their separation during genetic manipulations. Since the natural recombination rate for genes located in the same locus is near zero, to construct IGAs we developed the Super Recombinator (SuRe) system, which uses CRISPR/Cas9, alone or in combination with site-specific serine recombinases, for in vivo transgene recombination at a single genomic locus. SuRe effectively doubles the number of elements assembled in each recombination round, exponentially accelerating IGA construction. By preventing the separation of transgenic elements, SuRe greatly reduces screening burdens, as validated here through studies of Drosophila melanogaster and Caenorhabditis elegans . To optimize SuRe, we compared CRISPR/Cas9-induced homology-directed recombination to site-specific recombination using various serine recombinases. Optimized versions of SuRe achieved efficiency and fidelity values near their theoretical maxima and allowed the generation of recombinant products up to 4.2 Mbp in size in Drosophila . Using SuRe, we created fruit flies with 12 transgenic elements for fluorescence voltage imaging of neural activity in precisely defined cell-types. Mathematical modeling of the scalability of SuRe to large transgene assemblies showed that integration times and gene assembly workloads respectively scale logarithmically and linearly with the number of transgenes, both major improvements over conventional approaches. Overall, SuRe enables the efficient integration of multiple genes at individual loci, up to the chromosomal scale. ### Competing Interest Statement The authors of this manuscript (J Luo, MJ Schnitzer, C Huang) are also inventors on the related patent application "Genetic tools for recombining transgenes at the same locus" (US Patent App. 18/248,978, 2023).

In dynamic environments, animals make behavioural decisions on the basis of the innate valences of sensory cues and information learnt about these cues across multiple timescales1-3. However, it remains unclear how the innate valence of a sensory stimulus affects the acquisition of learnt valence information and subsequent memory dynamics. Here we show that in the Drosophila brain, interconnected short- and long-term memory units of the mushroom body jointly regulate memory through dopamine signals that encode innate and learnt sensory valences. By performing time-lapse in vivo voltage-imaging studies of neural spiking in more than 500 flies undergoing olfactory associative conditioning, we found that protocerebral posterior lateral 1 dopamine neurons (PPL1-DANs)4 heterogeneously and bidirectionally encode innate and learnt valences of punishment, reward and odour cues. During learning, these valence signals regulate memory storage and extinction in mushroom body output neurons (MBONs)5. During initial conditioning bouts, PPL1-γ1pedc and PPL1-γ2α'1 neurons control short-term memory formation, which weakens inhibitory feedback from MBON-γ1pedc>α/β to PPL1-α'2α2 and PPL1-α3. During further conditioning, this diminished feedback allows these two PPL1-DANs to encode the net innate plus learnt valence of the conditioned odour cue, which gates long-term memory formation. A computational model constrained by the fly connectome6,7 and our spiking data explains how dopamine signals mediate the circuit interactions between short- and long-term memory traces, yielding predictions that our experiments confirmed. Overall, the mushroom body achieves flexible learning through the integration of innate and learnt valences in parallel learning units sharing feedback interconnections. This hybrid physiological-anatomical mechanism may be a general means by which dopamine regulates memory dynamics in other species and brain structures, including the vertebrate basal ganglia.

The fruit fly, Drosophila melanogaster, is a widely used model species in biomedical research. Despite its importance, conducting manual experiments with individual fruit flies can be challenging and time-consuming, especially for studies of individual fly behaviors. Such studies often involve cumbersome preparatory steps, such as manually tethering a fly and then positioning it within an experimental setup1,2. These procedures commonly require the fly to be anesthetized, and, before behavioral assessments begin, the fly must recover from anesthesia. Hence, the introduction of automated phenotyping and behavioral assays would expedite important aspects of fly research, by minimizing manual handling of flies and decreasing the net time needed for experiments. Here, we introduce FlyMAX (Fly Manipulation and Autonomous eXperimentation), an autonomous robotic system for manipulating adult flies without use of anesthesia. FlyMAX collects individual flies from a standard vial, analyzes them with computer vision, and achieves a throughput of >1,000 flies per day for high-throughput inspection and characterization assays. Robotic handling had no detectable adverse effects on fly longevity or our assessments of fly health. Moreover, the behavioral performance of flies, especially of males, was better and less variable than of flies handled manually. Our system employs deep learning-based machine vision for real-time assessments of picking quality and fly phenotypes. This enables fully pipelined, autonomous experimentation for behavioral assays with individual flies in controlled environments, which was previously infeasible. Overall, FlyMAX constitutes a promising technology to enhance the efficiency and reproducibility of research with flies and other insects in fields such as genetics, neuroscience, and drug screening. ### Competing Interest Statement The authors have declared no competing interest.

The development of voltage-sensitive fluorescent probes suggests fluorescence lifetime as a promising readout for electrical activity in biological systems. Existing approaches fail to achieve the speed and sensitivity required for voltage imaging in neuroscience applications. We demonstrated that wide-field electro-optic fluorescence lifetime imaging microscopy (EO-FLIM) allows lifetime imaging at kilohertz frame-acquisition rates, spatially resolving action potential propagation and subthreshold neural activity in live adult Drosophila. Lifetime resolutions of <5 picoseconds at 1 kilohertz were achieved for single-cell voltage recordings. Lifetime readout is limited by photon shot noise, and the method provides strong rejection of motion artifacts and technical noise sources. Recordings revealed local transmembrane depolarizations, two types of spikes with distinct fluorescence lifetimes, and phase locking of spikes to an external mechanical stimulus.

Needle-shaped beams (NBs) featuring a long depth-of-focus (DOF) can drastically improve the resolution of microscopy systems. However, thus far, the implementation of a specific NB has been onerous due to the lack of a common, flexible generation method. Here we develop a spatially multiplexed phase pattern that creates many axially closely spaced foci as a universal platform for customizing various NBs, allowing flexible manipulations of beam length and diameter, uniform axial intensity, and sub-diffraction-limit beams. NBs designed via this method successfully extended the DOF of our optical coherence tomography (OCT) system. It revealed clear individual epidermal cells of the entire human epidermis, fine structures of human dermal-epidermal junction in a large depth range, and a high-resolution dynamic heartbeat of alive Drosophila larvae.

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between targeted cell populations. However, current indicators lack the requisite sensitivity for in vivo multipopulation imaging. We describe next-generation green and red voltage sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants pAce and pAceR. Our indicators enable 0.4- to 1-kilohertz voltage recordings from >50 spiking neurons per field of view in awake mice and ~30-minute continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered brain state–dependent antagonism between neocortical somatostatin-expressing (SST + ) and vasoactive intestinal peptide–expressing (VIP + ) interneurons and contributions to hippocampal field potentials from cell ensembles with distinct axonal projections. By combining three mutually compatible indicators, we performed simultaneous triple-population imaging. These approaches will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution.

To survive in dynamic environments, animals make behavioral decisions based on innate and learnt information about the valences of sensory cues. In the brains of multiple species, innate and learnt sensory valence signals are initially encoded by distinct neural populations1–7 but then reconverge in downstream brain structures that guide behavioral choices6–9. This reconvergence hinges on the prior acquisition of learnt valence information, which in turn might depend upon innate valence signals. However, it remains unknown whether and how innate sensory valence cues shape the acquisition of learnt valence information. Here we show that in the fruit fly brain, interactions between innate and learnt sensory valence signals within interconnected short- and long-term memory units of the mushroom body jointly regulate memory formation and expression via modulation of dopamine teaching signals. By using time-lapse, in vivo optical voltage imaging to record neural spiking with millisecond-resolution in flies undergoing olfactory associative conditioning, we found that PPL1 dopamine neurons (PPL1-DANs) heterogeneously and bi-directionally encode punishment, reward, and innate and learnt odor-valence cues. Learning modulates these representations in a way that combines innate and learnt valence information and allows the PPL1-DANs to regulate memory storage in their downstream targets, mushroom body output neurons (MBONs). PPL1-γ1pedc and PPL1-γ2α’1 neurons control short-term memory formation. After repeated conditioning, feedback signals carrying short-term memory data from MBON-γ1pedc>α/β to PPL1-α’2α2 and PPL1-α3 allow these dopamine cells to encode previously learnt valences and promote long-lasting memory formation. A computational model constrained by the mushroom body connectome and our spiking data explains how dopamine signals integrate innate and learnt valence data to regulate memory storage, extinction, and the interactions between short- and long-term memory traces. The model yields non-intuitive predictions about the effects of different training protocols, which our experiments confirm. Overall, the mushroom body achieves flexible learning through dopamine-mediated integration of innate and learnt valences in parallel sets of DAN/MBON learning units with feedback interconnections. This hybrid physiologic-anatomic mechanism may be a general means by which ecological information regulates learning and memory in other species and brain structures relying on dopaminergic signaling, including the vertebrate basal ganglia10.

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between distinct, targeted cell populations. However, current indicator families lack the requisite sensitivity for in vivo multipopulation imaging. We describe high-performance green and red sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants, pAce and pAceR. Our indicators enable 0.4-1 kHz voltage recordings from >50 neurons per field-of-view in awake mice and ∼30-min continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered behavioral state-dependent interactions between distinct neocortical subclasses, as well as contributions to hippocampal field potentials from non-overlapping projection neuronal ensembles. By combining three mutually compatible indicators, we demonstrate concurrent triple-population voltage imaging. Our approach will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution. One Sentence Summary A new suite of voltage sensors enables simultaneous cellular-resolution activity imaging from multiple, targeted neuron-types in awake animals.

A facile one-step reaction of [60]fullerene with cinnamaldehydes and amines promoted by magnesium perchlorate/ferric perchlorate under air conditions afforded a series of rare amino-substituted cyclopentafullerenes in moderate to good yields. Stereoselectivity was readily achieved. Secondary amines exclusively produced N,N-disubstituted cyclopentafullerenes as cis isomers, while arylamines gave N-monosubstituted cyclopentafullerenes with a preference of cis isomers as major products. N-Monosubstituted cyclopentafullerenes could be further converted into other scarce cyclopentafullerenes in the presence of acid chloride or paraformaldehyde. A possible reaction pathway was proposed to elucidate the formation of amino-substituted cyclopentafullerenes.

Genetically encoded voltage indicators (GEVIs) are emerging optical tools for acquiring brain-wide cell-type-specific functional data at unparalleled temporal resolution. To broaden the application of GEVIs in high-speed multispectral imaging, we used a high-throughput strategy to develop voltage-activated red neuronal activity monitor (VARNAM), a fusion of the fast Acetabularia opsin and the bright red fluorophore mRuby3. Imageable under the modest illumination intensities required by bright green probes (<50 mW mm-2), VARNAM is readily usable in vivo. VARNAM can be combined with blue-shifted optical tools to enable cell-type-specific all-optical electrophysiology and dual-color spike imaging in acute brain slices and live Drosophila. With enhanced sensitivity to subthreshold voltages, VARNAM resolves postsynaptic potentials in slices and cortical and hippocampal rhythms in freely behaving mice. Together, VARNAM lends a new hue to the optical toolbox, opening the door to high-speed in vivo multispectral functional imaging.

N-alkylation of fulleropyrrolidines in the presence of polyformaldehyde and p-toluenesulfonic acid was systematically investigated. With the addition of ketones/arylboronic acids, a series of novel N-alkyl fulleropyrrolidines were formed. Nevertheless, in the absence of ketones/arylboronic acids, scarce N-methyl fulleropyrrolidines were obtained with the aid of benzyl alcohol. Possible reaction pathways for the formation of N-alkyl fulleropyrrolidines were proposed to elucidate the above reaction process.

Time-lapse in vivo microscopy studies of cellular morphology and physiology are crucial toward understanding brain function but have been infeasible in the fruit fly, a key model species. Here we use laser microsurgery to create a chronic fly preparation for repeated imaging of neural architecture and dynamics for up to 50 days. In fly mushroom body neurons, we track axonal boutons for 10 days and record odor-evoked calcium transients over 7 weeks. Further, by using voltage imaging to resolve individual action potentials, we monitor spiking plasticity in dopamine neurons of flies undergoing mechanical stress. After 24 h of stress, PPL1-α’3 but not PPL1-α’2α2 dopamine neurons have elevated spike rates. Overall, our chronic preparation is compatible with a broad range of optical techniques and enables longitudinal studies of many biological questions that could not be addressed before in live flies.

A series of scarce tetrahydropyridinofullerenes were synthesized by the metal-free-mediated reaction of [60]fullerene with cheap and easily available α-methyl-substituted arylmethanamines and aldehydes in the presence of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 4-dimethylaminopyridine (DMAP) in moderate to good yields comparable to the previously reported data for most monoadducts. The in situ generation of azadienes played a crucial role in the successful synthesis of tetrahydropyridinofullerenes. A plausible reaction mechanism was proposed to elucidate the reaction process.

Reaction of [60]fullerene with β-substituted ethylamines under air conditions generated a series of scarce fulleropyrrolines with trisubstituted alkene functional groups in moderate yields.

Genetically encoded voltage indicators (GEVIs) are a promising technology for fluorescence readout of millisecond-scale neuronal dynamics. Previous GEVIs had insufficient signaling speed and dynamic range to resolve action potentials in live animals. We coupled fast voltage-sensing domains from a rhodopsin protein to bright fluorophores through resonance energy transfer. The resulting GEVIs are sufficiently bright and fast to report neuronal action potentials and membrane voltage dynamics in awake mice and flies, resolving fast spike trains with 0.2-millisecond timing precision at spike detection error rates orders of magnitude better than previous GEVIs. In vivo imaging revealed sensory-evoked responses, including somatic spiking, dendritic dynamics, and intracellular voltage propagation. These results empower in vivo optical studies of neuronal electrophysiology and coding and motivate further advancements in high-speed microscopy.

We present a robot that enables high-content studies of alert adult Drosophila by combining operations including gentle picking; translations and rotations; characterizations of fly phenotypes and behaviors; microdissection; or release. To illustrate, we assessed fly morphology, tracked odor-evoked locomotion, sorted flies by sex, and dissected the cuticle to image neural activity. The robot's tireless capacity for precise manipulations enables a scalable platform for screening flies' complex attributes and behavioral patterns.

The mushroom body (MB), a bilateral brain structure possessing about 2000–2500 neurons per hemisphere, plays a central role in olfactory learning and memory in Drosophila melanogaster. Extensive studies have demonstrated that three major types of MB neurons (α/β, α′/β′ and Γ) exhibit distinct functions in memory processing, including the critical role of approximately 1000 MB α/β neurons in retrieving long-term memory. Inspired by recent findings that MB α/β neurons can be further divided into three subdivisions (surface, posterior and core) and wherein the α/β core neurons play an permissive role in long-term memory consolidation, we examined the functional differences of all the three morphological subdivisions of MB α/β by temporally precise manipulation of their synaptic outputs during long-term memory retrieval. We found the normal neurotransmission from a combination of MB α/β surface and posterior neurons is necessary for retrieving both aversive and appetitive long-term memory, whereas output from MB α/β posterior or core subdivision alone is dispensable. These results imply a specific requirement of about 500 MB α/β neurons in supporting long-term memory retrieval and a further functional partitioning for memory processing within the MB α/β region.

Extensive investigations show several molecular and neuroanatomical mechanisms underlying short‐lived and long‐lasting memory in Drosophila . At the molecular level, the genetic pathway of memory formation, which was obtained through mutant research, seems to occur sequentially. So far, studies of Drosophila mutants appear to support the idea that mutants defective in short‐term memory ( STM ) are always associated with long‐term memory ( LTM ) impairment. At the neuroanatomical level, distinct memory traces are partially independently distributed. However, whether memory phase dissociation also exists at the molecular level remains unclear. Here, we report on molecular separation of STM and consolidated memory through genetic dissection of rugose mutants. Mutants in the rugose gene, which encodes an evolutionarily conserved A‐kinase anchor protein, show immediate memory defects as assayed through aversive olfactory conditioning. Intriguingly, two well‐defined consolidated memory components, anesthesia‐resistant memory and protein synthesis‐dependent LTM , are both normal in spite of the defective immediate memory after 10‐session massed and spaced training. Moreover, rugose genetically interacts with cyclic AMP ‐protein kinase A signaling during STM formation. Considering our previous study that AKAP Yu specifically participates in LTM formation, these results suggest that there exists a molecular level of memory phase dissociation with distinct AKAP s in Drosophila .

Extensive studies of Drosophila mushroom body in formation and retrieval of olfactory memories allow us to delineate the functional logic for memory storage and retrieval. Currently, there is a questionable disassociation of circuits for memory storage and retrieval during Drosophila olfactory memory processing. Formation of the initial aversive olfactory memory involves mushroom body γ lobe, whereas α/β lobes are reported to be necessary for the retrieval of such memory. In contrast, formation and retrieval of the short-term appetitive olfactory memory appears to involve γ lobe. With the help of newly identified Gal4 lines and of focusing on 3-h memory for both aversive and appetitive conditionings, our reexamination of the retrieval of aversive and appetitive olfactory memories suggests a new view. Blocking γ lobe output led to severe deficiency of aversive early memory retrieval and partial impairment of appetitive early memory retrieval. Interrupting α/β lobe output impaired the retrieval of both aversive and appetitive early memories. The contribution of the γ lobe and α/β lobes appeared to be additive for the retrieval of appetitive early memory. Thus, these results suggest that the retrieval of aversive and appetitive olfactory early memories requires the synaptic outputs from both γ lobe and α/β lobe neurons. This discovery may help us to rethink how aversive and appetitive memories are processed from memory formation to memory retrieval.