A connectome and analysis of the adult Drosophila central brain.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly’s brain.

Experience alters brain structure, but the underlying mechanism remained unknown. Structural plasticity reveals that brain function is encoded in generative changes to cells that compete with destructive processes driving neurodegeneration. At an adult critical period, experience increases fiber number and brain size in Drosophila. Here, we asked if Toll receptors are involved. Tolls demarcate a map of brain anatomical domains. Focusing on Toll-2, loss of function caused apoptosis, neurite atrophy and impaired behaviour. Toll-2 gain of function and neuronal activity at the critical period increased cell number. Toll-2 induced cycling of adult progenitor cells via a novel pathway, that antagonized MyD88-dependent quiescence, and engaged Weckle and Yorkie downstream. Constant knock-down of multiple Tolls synergistically reduced brain size. Conditional over-expression of Toll-2 and wek at the adult critical period increased brain size. Through their topographic distribution, Toll receptors regulate neuronal number and brain size, modulating structural plasticity in the adult brain.

While tau and aging have highly overlapping differential gene expression signatures, they diverge in the affected cell types, with aging having a wide-ranging impact and tau-triggered changes instead polarized to excitatory neurons and glia.

Decision letter: Visual projection neurons in the Drosophila lobula link feature detection to distinct behavioral programs

General anaesthesia is a drug-reversible state of unconsciousness associated with a loss of functional connectivity in the human brain. Whether effects are similar across all animal brains is unknown. In this thesis we examine general anaesthesia in one of the smallest animal brains, in the fruit fly, to determine if there may be some general underlying principles. To address functional connectivity in the fly brain during anaesthesia, we examine different methods of quantifying neural timing relationships among brain regions, such as coherence and phase locking. To better understand how information processing in the fly brain might be altered during states of altered or reduced consciousness, we compare these electrophysiological measures during general anaesthesia and spontaneous sleep.To compare states of sleep and general anaesthesia, we conducted in vivo experiments in Drosophila melanogaster flies, which share anaesthesia targets with mammals and have recently been found to display dynamic sleep stages. We recorded local field potentials (LFPs) from multiple brain regions using a 16-channel linear probe, which allowed inferences about neural communication to be made during spontaneous sleep, and during isoflurane anaesthesia. Neural synchrony was calculated pairwise between channel signals during spontaneous periods where no stimuli were presented, or between channels in relation to the timing of a visual stimulus. Theories of consciousness such as cognitive binding and integrated information theory hypothesise that one reason for loss of perception during sleep or general anaesthesia is that a level of coordinated activity among brain regions is lost, and information from multiple sensory streams are no longer integrated. This can be studied using spontaneous or evoked activity, where we expect the processing of either the environment (spontaneous) or of a visual stimulus (evoked) is impaired, and neural synchrony decreased.In this thesis, following a general introduction (Chapter 1), we first conducted a behavioural study to determine if prolonged isoflurane anaesthesia affected subsequent sleep (Chapter 2). Following this, to better understand sleep dynamics across the fly brain, we used the multichannel preparation to investigate how brain activity changes between wake and spontaneous sleep states over hours of time (Chapter 3). Following this, we studied evoked brain responses to visual stimuli, to determine how information processing is affected by isoflurane anaesthesia (Chapter 4). By stimulating the fly with visual flicker between 1-100Hz, we first asked if there were frequency-specific responses in the fly brain. We then determined which frequency responses were most affected by isoflurane. We next investigated how evoked responses and spontaneous LFP activity were affected across the fly brain at a lower and higher dose of isoflurane to determine if either of these resembled fly sleep (Chapter 5). Finally, we tested a more complex periodic visual stimulus to determine if LFP responses were differentially affected across the fly brain under isoflurane anaesthesia (Chapter 6).Behaviourally, we found daytime isoflurane exposure does not lead to changes in night-time sleep. In terms of electrophysiology, we find that while sleep and isoflurane anaesthesia both cause global decreases in LFP power across the brain, fly sleep differs in its functional connectivity. Fly sleep involves increased phase locking across low-frequency domains and within the central brain, while isoflurane anaesthesia causes a decrease in phase locking across all frequencies for all brain regions. Contrary to mammals and humans, flies do not show increased activity in lower frequency bands during general anaesthesia. This is likely a consequence of the differing neural circuits between mammals and flies, the result being that fly sleep shows less similarity to general anaesthesia under these conditions, possibly owing to the simpler frequency structure in LFPs.We find this global depression of brain activity under isoflurane applies similarly to evoked potentials, with lower frequencies more depressed both in power and phase locking. This indicates that evoked potentials might be used as a proxy for studying global brain activity under anaesthesia, which might be strategically applied to other brain states such as sleep to study perceptual processing, as similar studies have been done in mammals. One area of future investigation is to identify the neural circuits underlying the frequency changes identified during sleep. This could be accomplished using an experimental manipulation of sleep circuits to track the propagation of signals between the central brain and optic lobes, where we observe the most change during sleep. Studying how circuit-level changes alter LFP activity in the fly brain might eventually help explain how neural dynamics evolve into broader oscillation bands, including how these are involved with a loss of perceptual processing during altered brain states.

Segmentation of center brains and optic lobes in 3D confocal images of adult fruit fly brains

Dedication of Retinal Special Issue to: Harvey J. Karten, M.D.

Motor Control in a Drosophila Taste Circuit

Optogenetic activation reveals a larger role for the fly brain 'sleep switch' neurons in controlling both waking and sleeping behavioral responsiveness, partly via a parallel channel involving innexin6 electrical synapses.

Neuronal cell types, projections, and spatial organization of the central amygdala.

We use time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging to investigate the effects of orally administrated methylphenidate on lipids in the brain of Drosophila melanogaster (fruit fly), a major invertebrate model system in biological study and neuroscience. TOF-SIMS imaging was carried out using a recently designed high energy 40 keV Ar4000(+) gas cluster ion gun which demonstrated improved sensitivity for intact lipids in the fly brain compared to the 40 keV C60(+) primary ion gun. In addition, correlation of TOF-SIMS and SEM imaging on the same fly brain showed that there is specific localization that is related to biological functions of various biomolecules. Different lipids distribute in different parts of the brain, central brain, optical lobes, and proboscis, depending on the length of the carbon chain and saturation level of fatty acid (FA) branches. Furthermore, data analysis using image principal components analysis (PCA) showed that methylphenidate dramatically affected both the distribution and abundance of lipids and their derivatives, particularly fatty acids, diacylglycerides, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol in the fly brains. Our approach using TOF-SIMS imaging successfully visualizes the effects of methylphenidate on the chemical structure of the fly brain.

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Decision letter: Visual and motor signatures of locomotion dynamically shape a population code for feature detection in Drosophila

The mammalian brain is composed of millions to billions of cells that are organized into numerous cell types with specific spatial distribution patterns and structural and functional properties. An essential step towards understanding brain function is to obtain a parts list, i.e., a catalog of cell types, of the brain. Here, we report a comprehensive and high-resolution transcriptomic and spatial cell type atlas for the whole adult mouse brain. The cell type atlas was created based on the combination of two single-cell-level, whole-brain-scale datasets: a single-cell RNA-sequencing (scRNA-seq) dataset of ~7 million cells profiled, and a spatially resolved transcriptomic dataset of ~4.3 million cells using MERFISH. The atlas is hierarchically organized into five nested levels of classification: 7 divisions, 32 classes, 306 subclasses, 1,045 supertypes and 5,200 clusters. We systematically analyzed the neuronal, non-neuronal, and immature neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell type organization in different brain regions, in particular, a dichotomy between the dorsal and ventral parts of the brain: the dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. We also systematically characterized cell-type specific expression of neurotransmitters, neuropeptides, and transcription factors. The study uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types across the brain, suggesting they mediate a myriad of modes of intercellular communications. Finally, we found that transcription factors are major determinants of cell type classification in the adult mouse brain and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole-mouse-brain transcriptomic and spatial cell type atlas establishes a benchmark reference atlas and a foundational resource for deep and integrative investigations of cell type and circuit function, development, and evolution of the mammalian brain.

In Vivo Clonal Analysis Reveals Self-Renewing and Multipotent Adult Neural Stem Cell Characteristics