EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization.

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

SNT: a unifying toolbox for quantification of neuronal anatomy.

SNT is an end-to-end framework for neuronal morphometry and whole-brain connectomics that supports tracing, proof-editing, visualization, quantification and modeling of neuroanatomy. With an open architecture, a large user base, community-based documentation, support for complex imagery and several model organisms, SNT is a flexible resource for the broad neuroscience community. SNT is both a desktop application and multi-language scripting library, and it is available through the Fiji distribution of ImageJ.

Repetitive aggressive encounters generate a long-lasting internal state in Drosophila melanogaster males.

Multiple studies have investigated the mechanisms of aggressive behavior in Drosophila; however, little is known about the effects of chronic fighting experience. Here, we investigated if repeated fighting encounters would induce an internal state that could affect the expression of subsequent behavior. We trained wild-type males to become winners or losers by repeatedly pairing them with hypoaggressive or hyperaggressive opponents, respectively. As described previously, we observed that chronic losers tend to lose subsequent fights, while chronic winners tend to win them. Olfactory conditioning experiments showed that winning is perceived as rewarding, while losing is perceived as aversive. Moreover, the effect of chronic fighting experience generalized to other behaviors, such as gap-crossing and courtship. We propose that in response to repeatedly winning or losing aggressive encounters, male flies form an internal state that displays persistence and generalization; fight outcomes can also have positive or negative valence. Furthermore, we show that the activities of the PPL1-γ1pedc dopaminergic neuron and the MBON-γ1pedc>α/ß mushroom body output neuron are required for aversion to an olfactory cue associated with losing fights.

The novel gene tank, a tumor suppressor homolog, regulates ethanol sensitivity in Drosophila.

In both mammalian and insect models of ethanol intoxication, high doses of ethanol induce motor impairment and eventually sedation. Sensitivity to the sedative effects of ethanol is inversely correlated with risk for alcoholism. However, the genes regulating ethanol sensitivity are largely unknown. Based on a previous genetic screen in Drosophila for ethanol sedation mutants, we identified a novel gene, tank (CG15626), the homolog of the mammalian tumor suppressor EI24/PIG8, which has a strong role in regulating ethanol sedation sensitivity. Genetic and behavioral analyses revealed that tank acts in the adult nervous system to promote ethanol sensitivity. We localized the function of tank in regulating ethanol sensitivity to neurons within the pars intercerebralis that have not been implicated previously in ethanol responses. We show that acutely manipulating the activity of all tank-expressing neurons, or of pars intercerebralis neurons in particular, alters ethanol sensitivity in a sexually dimorphic manner, since neuronal activation enhanced ethanol sedation in males, but not females. Finally, we provide anatomical evidence that tank-expressing neurons form likely synaptic connections with neurons expressing the neural sex determination factor fruitless (fru), which have been implicated recently in the regulation of ethanol sensitivity. We suggest that a functional interaction with fru neurons, many of which are sexually dimorphic, may account for the sex-specific effect induced by activating tank neurons. Overall, we have characterized a novel gene and corresponding set of neurons that regulate ethanol sensitivity in Drosophila.

Ten-fold Robust Expansion Microscopy.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

Multimodal mapping of cell types and projections in the central nucleus of the amygdala.

The central nucleus of the amygdala (CEA) is a brain region that integrates external and internal sensory information and executes innate and adaptive behaviors through distinct output pathways. Despite its complex functions, the diversity of molecularly defined neuronal types in the CEA and their contributions to major axonal projection targets have not been examined systematically. Here, we performed single-cell RNA-sequencing (scRNA-seq) to classify molecularly defined cell types in the CEA and identified marker genes to map the location of these neuronal types using expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH). We developed new methods to integrate EASI-FISH with 5-plex retrograde axonal labeling to determine the spatial, morphological, and connectivity properties of ~30,000 molecularly defined CEA neurons. Our study revealed spatiomolecular organization of the CEA, with medial and lateral CEA associated with distinct molecularly defined cell families. We also found a long-range axon projection network from the CEA, where target regions receive inputs from multiple molecularly defined cell types. Axon collateralization was found primarily among projections to hindbrain targets, which are distinct from forebrain projections. This resource reports marker gene combinations for molecularly defined cell types and axon-projection types, which will be useful for selective interrogation of these neuronal populations to study their contributions to the diverse functions of the CEA.

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx).

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand 10-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small-molecule stains for both total protein and membranes.

A genetic screen for Drosophila social isolation mutants and analysis of sex pistol.

Prolonged periods of forced social isolation is detrimental to well-being, yet we know little about which genes regulate susceptibility to its effects. In the fruit fly, Drosophila melanogaster, social isolation induces stark changes in behavior including increased aggression, locomotor activity, and resistance to ethanol sedation. To identify genes regulating sensitivity to isolation, I screened a collection of sixteen hundred P-element insertion lines for mutants with abnormal levels of all three isolation-induced behaviors. The screen identified three mutants whose affected genes are likely central to regulating the effects of isolation in flies. One mutant, sex pistol (sxp), became extremely aggressive and resistant to ethanol sedation when socially isolated. sxp also had a high level of male-male courtship. The mutation in sxp reduced the expression of two minor isoforms of the actin regulator hts (adducin), as well as mildly reducing expression of CalpA, a calcium-dependent protease. As a consequence, sxp also had increased expression of the insulin-like peptide, dILP5. Analysis of the social behavior of sxp suggests that these minor hts isoforms function to limit isolation-induced aggression, while chronically high levels of dILP5 increase male-male courtship.

Degradation of arouser by endosomal microautophagy is essential for adaptation to starvation in Drosophila.

Hunger drives food-seeking behaviour and controls adaptation of organisms to nutrient availability and energy stores. Lipids constitute an essential source of energy in the cell that can be mobilised during fasting by autophagy. Selective degradation of proteins by autophagy is made possible essentially by the presence of LIR and KFERQ-like motifs. Using in silico screening of Drosophila proteins that contain KFERQ-like motifs, we identified and characterized the adaptor protein Arouser, which functions to regulate fat storage and mobilisation and is essential during periods of food deprivation. We show that hypomorphic arouser mutants are not satiated, are more sensitive to food deprivation, and are more aggressive, suggesting an essential role for Arouser in the coordination of metabolism and food-related behaviour. Our analysis shows that Arouser functions in the fat body through nutrient-related signalling pathways and is degraded by endosomal microautophagy. Arouser degradation occurs during feeding conditions, whereas its stabilisation during non-feeding periods is essential for resistance to starvation and survival. In summary, our data describe a novel role for endosomal microautophagy in energy homeostasis, by the degradation of the signalling regulatory protein Arouser.

Segmental identity and cerebellar granule cell induction in rhombomere 1.

BACKGROUND: Cerebellar granule cell precursors are specifically generated within the hindbrain segment, rhombomere 1, which is bounded rostrally by the midbrain/hindbrain isthmus and caudally by the boundary of the Hoxa2 expression domain. While graded signals from the isthmus have a demonstrable patterning role within this region, the significance of segmental identity for neuronal specification within rhombomere 1 is unexplored. We examined the response of granule cell precursors to the overexpression of Hoxa2, which normally determines patterns of development specific to the hindbrain. How much does the development of the cerebellum, a midbrain/hindbrain structure, reflect its neuromeric origin as a hindbrain segment? RESULTS: We show that a Gbx2-positive, Otx2-/Hoxa2-negative territory corresponding to rhombomere 1 forms prior to an identifiable isthmic organiser. Early global overexpression of Hoxa2 at embryonic day 0 has no effect on the expression of isthmic signalling molecules or the allocation of rhombomere 1 territory, but selectively results in the loss of granule cell markers at embryonic day 6 and the depletion of cell bodies from the external granule cell layer. By comparison the trochlear nucleus and locus coeruleus form normally in ventral rhombomere 1 under these conditions. Microsurgery, coupled with electroporation, to target Hoxa2 overexpression to rhombic lip precursors, reveals a profound, autonomous respecification of migration. Rhombic lip derivatives, normally destined to occupy the external granule cell layer, violate the cerebellar boundary to form a ventrolateral nucleus in a position comparable to that occupied by rhombic lip derived neurons in rhombomere 2. CONCLUSIONS: Different overexpression strategies reveal that the recognition of migration cues by granule cell precursors is dependent on their identity as rhombomere 1 derivatives. Segmental patterning cues operate autonomously within the rhombic lip precursor pool. By contrast, a subset of coextensive nuclei is refractory to ectopic Hoxa2 and is presumably induced solely by isthmic organiser activity. Thus, graded (isthmic) and segmental mechanisms may operate exclusively of one another in the specification of different neuronal populations within rhombomere 1. The early designation of an Otx2-negative, Hoxa2-negative region, prior to the appearance of the isthmic organiser, is a key initial step in the specification of the cerebellum.

Numb is not a critical regulator of Notch-mediated cell fate decisions in the developing chick inner ear.

The Notch signaling pathway controls differentiation of hair cells and supporting cells in the vertebrate inner ear. Here, we have investigated whether Numb, a known regulator of Notch activity in Drosophila, is involved in this process in the embryonic chick. The chicken homolog of Numb is expressed throughout the otocyst at early stages of development and is concentrated at the basal pole of the cells. It is asymmetrically allocated at some cell divisions, as in Drosophila, suggesting that it could act as a determinant inherited by one of the two daughter cells and favoring adoption of a hair-cell fate. To test the implication of Numb in hair cell fate decisions and the regulation of Notch signaling, we used different methods to overexpress Numb at different stages of inner ear development. We found that sustained or late Numb overexpression does not promote hair cell differentiation, and Numb does not prevent the reception of Notch signaling. Surprisingly, none of the Numb-overexpressing cells differentiated into hair cells, suggesting that high levels of Numb protein could interfere with intracellular processes essential for hair cell survival. However, when Numb was overexpressed early and more transiently during ear development, no effect on hair cell formation was seen. These results suggest that in the inner ear at least, Numb does not significantly repress Notch activity and that its asymmetric distribution in dividing precursor cells does not govern the choice between hair cell and supporting cell fates.

EGFR and FGFR pathways have distinct roles in Drosophila mushroom body development and ethanol-induced behavior.

Epidermal Growth Factor Receptor (EGFR) signaling has a conserved role in ethanol-induced behavior in flies and mice, affecting ethanol-induced sedation in both species. However it is not known what other effects EGFR signaling may have on ethanol-induced behavior, or what roles other Receptor Tyrosine Kinase (RTK) pathways may play in ethanol induced behaviors. We examined the effects of both the EGFR and Fibroblast Growth Factor Receptor (FGFR) RTK signaling pathways on ethanol-induced enhancement of locomotion, a behavior distinct from sedation that may be associated with the rewarding effects of ethanol. We find that both EGFR and FGFR genes influence ethanol-induced locomotion, though their effects are opposite - EGFR signaling suppresses this behavior, while FGFR signaling promotes it. EGFR signaling affects development of the Drosophila mushroom bodies in conjunction with the JNK MAP kinase basket (bsk), and with the Ste20 kinase tao, and we hypothesize that the EGFR pathway affects ethanol-induced locomotion through its effects on neuronal development. We find, however, that FGFR signaling most likely affects ethanol-induced behavior through a different mechanism, possibly through acute action in adult neurons.

The Drosophila surface glia transcriptome: evolutionary conserved blood-brain barrier processes.

Central nervous system (CNS) function is dependent on the stringent regulation of metabolites, drugs, cells, and pathogens exposed to the CNS space. Cellular blood-brain barrier (BBB) structures are highly specific checkpoints governing entry and exit of all small molecules to and from the brain interstitial space, but the precise mechanisms that regulate the BBB are not well understood. In addition, the BBB has long been a challenging obstacle to the pharmacologic treatment of CNS diseases; thus model systems that can parse the functions of the BBB are highly desirable. In this study, we sought to define the transcriptome of the adult Drosophila melanogaster BBB by isolating the BBB surface glia with fluorescence activated cell sorting (FACS) and profiling their gene expression with microarrays. By comparing the transcriptome of these surface glia to that of all brain glia, brain neurons, and whole brains, we present a catalog of transcripts that are selectively enriched at the Drosophila BBB. We found that the fly surface glia show high expression of many ATP-binding cassette (ABC) and solute carrier (SLC) transporters, cell adhesion molecules, metabolic enzymes, signaling molecules, and components of xenobiotic metabolism pathways. Using gene sequence-based alignments, we compare the Drosophila and Murine BBB transcriptomes and discover many shared chemoprotective and small molecule control pathways, thus affirming the relevance of invertebrate models for studying evolutionary conserved BBB properties. The Drosophila BBB transcriptome is valuable to vertebrate and insect biologists alike as a resource for studying proteins underlying diffusion barrier development and maintenance, glial biology, and regulation of drug transport at tissue barriers.

A genetic screen for olfactory habituation mutations in Drosophila: analysis of novel foraging alleles and an underlying neural circuit.

Habituation is a form of non-associative learning that enables animals to reduce their reaction to repeated harmless stimuli. When exposed to ethanol vapor, Drosophila show an olfactory-mediated startle response characterized by a transient increase in locomotor activity. Upon repeated exposures, this olfactory startle attenuates with the characteristics of habituation. Here we describe the results of a genetic screen to identify olfactory startle habituation (OSH) mutants. One mutation is a transcript specific allele of foraging (for) encoding a cGMP-dependent kinase. We show this allele of for reduces expression of a for-T1 isoform expressed in the head and functions normally to inhibit OSH. We localize for-T1 function to a limited set of neurons that include olfactory receptor neurons (ORNs) and the mushroom body (MB). Overexpression of for-T1 in ORNs inhibits OSH, an effect also seen upon synaptic silencing of the ORNs; for-T1 may therefore function in ORNs to decrease synaptic release upon repeated exposure to ethanol vapor. Overall, this work contributes to our understanding of the genes and neurons underlying olfactory habituation in Drosophila.

arouser reveals a role for synapse number in the regulation of ethanol sensitivity.

A reduced sensitivity to the sedating effects of alcohol is a characteristic associated with alcohol use disorders (AUDs). A genetic screen for ethanol sedation mutants in Drosophila identified arouser (aru), which functions in developing neurons to reduce ethanol sensitivity. Genetic evidence suggests that aru regulates ethanol sensitivity through its activation by Egfr/Erk signaling and its inhibition by PI3K/Akt signaling. The aru mutant also has an increased number of synaptic terminals in the larva and adult fly. Both the increased ethanol sensitivity and synapse number of the aru mutant are restored upon adult social isolation, suggesting a causal relationship between synapse number and ethanol sensitivity. We thus show that a developmental abnormality affecting synapse number and ethanol sensitivity is not permanent and can be reversed by manipulating the environment of the adult fly.

The genetic relationships between ethanol preference, acute ethanol sensitivity, and ethanol tolerance in Drosophila melanogaster.

The relationship between alcohol consumption, sensitivity, and tolerance is an important question that has been addressed in humans and rodent models. Studies have shown that alcohol consumption and risk of abuse may correlate with (1) increased sensitivity to the stimulant effects of alcohol, (2) decreased sensitivity to the depressant effects of alcohol, and (3) increased alcohol tolerance. However, many conflicting results have been observed. To complement these studies, we utilized a different organism and approach to analyze the relationship between ethanol consumption and other ethanol responses. Using a set of 20 Drosophila melanogaster mutants that were isolated for altered ethanol sensitivity, we measured ethanol-induced hyperactivity, ethanol sedation, sedation tolerance, and ethanol consumption preference. Ethanol preference showed a strong positive correlation with ethanol tolerance, consistent with some rodent and human studies, but not with ethanol hyperactivity or sedation. No pairwise correlations were observed between ethanol hyperactivity, sedation, and tolerance. The evolutionary conservation of the relationship between tolerance and ethanol consumption in flies, rodents, and humans indicates that there are fundamental biological mechanisms linking specific ethanol responses.

GSK-3/Shaggy regulates olfactory habituation in Drosophila.

Habituation is a universal form of nonassociative learning that results in the devaluation of sensory inputs that have little information content. Although habituation is found throughout nature and has been studied in many organisms, the underlying molecular mechanisms remain poorly understood. We performed a forward genetic screen in Drosophila to search for mutations that modified habituation of an olfactory-mediated locomotor startle response, and we isolated a mutation in the glycogen synthase kinase-3 (GSK-3) homolog Shaggy. Decreases in Shaggy levels blunted habituation, whereas increases promoted habituation. Additionally, habituation acutely regulated Shaggy by an inhibitory phosphorylation mechanism, suggesting that a signal transduction pathway that regulates Shaggy is engaged during habituation. Although shaggy mutations also affected circadian rhythm period, this requirement was genetically separable from its role in habituation. Thus, shaggy functions in different neuronal circuits to regulate behavioral plasticity to an olfactory startle and circadian rhythmicity.