Sensory neuron population expansion enhances odor tracking without sensitizing projection neurons.

The evolutionary expansion of sensory neuron populations detecting important environmental cues is widespread, but functionally enigmatic. We investigated this phenomenon through comparison of homologous neural pathways of Drosophila melanogaster and its close relative Drosophila sechellia , an extreme specialist for Morinda citrifolia noni fruit. D. sechellia has evolved species-specific expansions in select, noni-detecting olfactory sensory neuron (OSN) populations, through multigenic changes. Activation and inhibition of defined proportions of neurons demonstrate that OSN population increases contribute to stronger, more persistent, noni-odor tracking behavior. These sensory neuron expansions result in increased synaptic connections with their projection neuron (PN) partners, which are conserved in number between species. Surprisingly, having more OSNs does not lead to greater odor-evoked PN sensitivity or reliability. Rather, pathways with increased sensory pooling exhibit reduced PN adaptation, likely through weakened lateral inhibition. Our work reveals an unexpected functional impact of sensory neuron expansions to explain ecologically-relevant, species-specific behavior.

Odor-regulated oviposition behavior in an ecological specialist.

Colonization of a novel ecological niche can require, or be driven by, evolution of an animal's behaviors promoting their reproductive success. We investigated the evolution and sensory basis of oviposition in Drosophila sechellia, a close relative of Drosophila melanogaster that exhibits extreme specialism for Morinda citrifolia noni fruit. D. sechellia produces fewer eggs than other drosophilids and lays these almost exclusively on noni substrates. We show that visual, textural and social cues do not explain this species-specific preference. By contrast, we find that loss of olfactory input in D. sechellia, but not D. melanogaster, essentially abolishes egg-laying, suggesting that olfaction gates gustatory-driven noni preference. Noni odors are detected by redundant olfactory pathways, but we discover a role for hexanoic acid and the cognate Ionotropic receptor 75b (Ir75b) in odor-evoked oviposition. Through receptor exchange in D. melanogaster, we provide evidence for a causal contribution of odor-tuning changes in Ir75b to the evolution of D. sechellia's oviposition behavior.

Evolution of connectivity architecture in the Drosophila mushroom body.

Brain evolution has primarily been studied at the macroscopic level by comparing the relative size of homologous brain centers between species. How neuronal circuits change at the cellular level over evolutionary time remains largely unanswered. Here, using a phylogenetically informed framework, we compare the olfactory circuits of three closely related Drosophila species that differ radically in their chemical ecology: the generalists Drosophila melanogaster and Drosophila simulans that feed on fermenting fruit, and Drosophila sechellia that specializes on ripe noni fruit. We examine a central part of the olfactory circuit that has not yet been investigated in these species - the connections between the projection neurons of the antennal lobe and the Kenyon cells of the mushroom body, an associative brain center - to identify species-specific connectivity patterns. We found that neurons encoding food odors - the DC3 neurons in D. melanogaster and D. simulans and the DL2d neurons in D. sechellia - connect more frequently with Kenyon cells, giving rise to species-specific biases in connectivity. These species-specific differences in connectivity reflect two distinct neuronal phenotypes: in the number of projection neurons or in the number of presynaptic boutons formed by individual projection neurons. Finally, behavioral analyses suggest that such increased connectivity enhances learning performance in an associative task. Our study shows how fine-grained aspects of connectivity architecture in an associative brain center can change during evolution to reflect the chemical ecology of a species.

Copy number changes in co-expressed odorant receptor genes enable selection for sensory differences in drosophilid species.

Despite numerous examples of chemoreceptor gene family expansions and contractions, how these relate to modifications in the sensory neuron populations in which they are expressed remains unclear. Drosophila melanogaster's odorant receptor (Or) family is ideal for addressing this question because most Ors are expressed in distinct olfactory sensory neuron (OSN) types. Between-species changes in Or copy number may therefore indicate increases or reductions in the number of OSN populations. Here we investigated the Or67a subfamily, which exhibits copy number variation in D. melanogaster and its closest relatives: D. simulans, D. sechellia and D. mauritiana. These species' common ancestor had three Or67a paralogues that had already diverged adaptively. Following speciation, two Or67a paralogues were lost independently in D. melanogaster and D. sechellia, with ongoing positive selection shaping the intact genes. Unexpectedly, the functionally diverged Or67a paralogues in D. simulans are co-expressed in a single neuron population, which projects to a glomerulus homologous to that innervated by Or67a neurons in D. melanogaster. Thus, while sensory pathway neuroanatomy is conserved, independent selection on co-expressed receptors has contributed to species-specific peripheral coding. This work reveals a type of adaptive change largely overlooked for olfactory evolution, raising the possibility that similar processes influence other cases of insect Or co-expression.

Cracking the encoding of human scent in the mosquito brain.

In a recent study, Zhao et al. decipher how the olfactory system encodes human versus animal odors in the mosquito Aedes aegypti. By combining genome engineering, invivo calcium imaging, advanced chemistry, and behavioral analysis, the authors provide compelling evidence that the discriminatory coding of host odors is surprisingly simple - and bridges labeled line with combinatorial coding.

Drosophila sechellia: A Genetic Model for Behavioral Evolution and Neuroecology.

Defining the mechanisms by which animals adapt to their ecological niche is an important problem bridging evolution, genetics, and neurobiology. We review the establishment of a powerful genetic model for comparative behavioral analysis and neuroecology, Drosophila sechellia. This island-endemic fly species is closely related to several cosmopolitan generalists, including Drosophila melanogaster, but has evolved extreme specialism, feeding and reproducing exclusively on the noni fruit of the tropical shrub Morinda citrifolia. We first describe the development and use of genetic approaches to facilitate genotype/phenotype associations in these drosophilids. Next, we survey the behavioral, physiological, and morphological adaptations of D. sechellia throughout its life cycle and outline our current understanding of the genetic and cellular basis of these traits. Finally, we discuss the principles this knowledge begins to establish in the context of host specialization, speciation, and the neurobiology of behavioral evolution and consider open questions and challenges in the field.

Olfactory receptor-dependent receptor repression in Drosophila.

In olfactory systems across phyla, most sensory neurons express a single olfactory receptor gene selected from a large genomic repertoire. We describe previously unknown receptor gene-dependent mechanisms that ensure singular expression of receptors encoded by a tandem gene array [Ionotropic receptor 75c (Ir75c), Ir75b, and Ir75a, organized 5' to 3'] in Drosophila melanogaster Transcription from upstream genes in the cluster runs through the coding region of downstream loci and inhibits their expression in cis, most likely via transcriptional interference. Moreover, Ir75c blocks accumulation of other receptor proteins in trans through a protein-dependent, posttranscriptional mechanism. These repression mechanisms operate in endogenous neurons, in conjunction with cell type-specific gene regulatory networks, to ensure unique receptor expression. Our data provide evidence for inter-olfactory receptor regulation in invertebrates and highlight unprecedented, but potentially widespread, mechanisms for ensuring exclusive expression of chemosensory receptors, and other protein families, encoded by tandemly arranged genes.

Mate discrimination among subspecies through a conserved olfactory pathway.

Communication mechanisms underlying the sexual isolation of species are poorly understood. Using four subspecies of Drosophila mojavensis as a model, we identify two behaviorally active, male-specific pheromones. One functions as a conserved male antiaphrodisiac in all subspecies and acts via gustation. The second induces female receptivity via olfaction exclusively in the two subspecies that produce it. Genetic analysis of the cognate receptor for the olfactory pheromone indicates an important role for this sensory pathway in promoting sexual isolation of subspecies, in combination with auditory signals. Unexpectedly, the peripheral sensory pathway detecting this pheromone is conserved molecularly, physiologically, and anatomically across subspecies. These observations imply that subspecies-specific behaviors arise from differential interpretation of the same peripheral cue, reminiscent of sexually conserved detection but dimorphic interpretation of male pheromones in Drosophila melanogaster. Our results reveal that, during incipient speciation, pheromone production, detection, and interpretation do not necessarily evolve in a coordinated manner.

TReND in Africa: Toward a Truly Global (Neuro)science Community.

TReND is a volunteer-scientist run charity dedicated to promoting research and education on the African continent. Focusing on neuroscience, we discuss approaches to address some of the factors that currently stifle Africa's scientific development and our experience in implementing them.

Olfactory receptor and circuit evolution promote host specialization.

The evolution of animal behaviour is poorly understood1,2. Despite numerous correlations between interspecific divergence in behaviour and nervous system structure and function, demonstrations of the genetic basis of these behavioural differences remain rare3-5. Here we develop a neurogenetic model, Drosophila sechellia, a species that displays marked differences in behaviour compared to its close cousin Drosophila melanogaster6,7, which are linked to its extreme specialization on noni fruit (Morinda citrifolia)8-16. Using calcium imaging, we identify olfactory pathways in D. sechellia that detect volatiles emitted by the noni host. Our mutational analysis indicates roles for different olfactory receptors in long- and short-range attraction to noni, and our cross-species allele-transfer experiments demonstrate that the tuning of one of these receptors is important for species-specific host-seeking. We identify the molecular determinants of this functional change, and characterize their evolutionary origin and behavioural importance. We perform circuit tracing in the D. sechellia brain, and find that receptor adaptations are accompanied by increased sensory pooling onto interneurons as well as species-specific central projection patterns. This work reveals an accumulation of molecular, physiological and anatomical traits that are linked to behavioural divergence between species, and defines a model for investigating speciation and the evolution of the nervous system.

Publisher Correction: An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An interhemispheric neural circuit allowing binocular integration in the optic tectum.

Binocular stereopsis requires the convergence of visual information from corresponding points in visual space seen by two different lines of sight. This may be achieved by superposition of retinal input from each eye onto the same downstream neurons via ipsi- and contralaterally projecting optic nerve fibers. Zebrafish larvae can perceive binocular cues during prey hunting but have exclusively contralateral retinotectal projections. Here we report brain activity in the tectal neuropil ipsilateral to the visually stimulated eye, despite the absence of ipsilateral retinotectal projections. This activity colocalizes with arbors of commissural neurons, termed intertectal neurons (ITNs), that connect the tectal hemispheres. ITNs are GABAergic, establish tectal synapses bilaterally and respond to small moving stimuli. ITN-ablation impairs capture swim initiation when prey is positioned in the binocular strike zone. We propose an intertectal circuit that controls execution of the prey-capture motor program following binocular localization of prey, without requiring ipsilateral retinotectal projections.

Dynactin1 depletion leads to neuromuscular synapse instability and functional abnormalities.

BACKGROUND: Dynactin subunit 1 is the largest subunit of the dynactin complex, an activator of the molecular motor protein complex dynein. Reduced levels of DCTN1 mRNA and protein have been found in sporadic amyotrophic lateral sclerosis (ALS) patients, and mutations have been associated with disease, but the role of this protein in disease pathogenesis is still unknown. METHODS: We characterized a Dynactin1a depletion model in the zebrafish embryo and combined in vivo molecular analysis of primary motor neuron development with live in vivo axonal transport assays in single cells to investigate ALS-related defects. To probe neuromuscular junction (NMJ) function and organization we performed paired motor neuron-muscle electrophysiological recordings and GCaMP calcium imaging in live, intact larvae, and the synapse structure was investigated by electron microscopy. RESULTS: Here we show that Dynactin1a depletion is sufficient to induce defects in the development of spinal cord motor neurons and in the function of the NMJ. We observe synapse instability, impaired growth of primary motor neurons, and higher failure rates of action potentials at the NMJ. In addition, the embryos display locomotion defects consistent with NMJ dysfunction. Rescue of the observed phenotype by overexpression of wild-type human DCTN1-GFP indicates a cell-autonomous mechanism. Synaptic accumulation of DCTN1-GFP, as well as ultrastructural analysis of NMJ synapses exhibiting wider synaptic clefts, support a local role for Dynactin1a in synaptic function. Furthermore, live in vivo analysis of axonal transport and cytoskeleton dynamics in primary motor neurons show that the phenotype reported here is independent of modulation of these processes. CONCLUSIONS: Our study reveals a novel role for Dynactin1 in ALS pathogenesis, where it acts cell-autonomously to promote motor neuron synapse stability independently of dynein-mediated axonal transport.

An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing.

Through analysis of the Drosophila ionotropic receptors (IRs), a family of variant ionotropic glutamate receptors, we reveal that most IRs are expressed in peripheral neuron populations in diverse gustatory organs in larvae and adults. We characterise IR56d, which defines two anatomically-distinct neuron classes in the proboscis: one responds to carbonated solutions and fatty acids while the other represents a subset of sugar- and fatty acid-sensing cells. Mutational analysis indicates that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological responses to carbonation and fatty acids, but not sugars. We further demonstrate that carbonation and fatty acids both promote IR56d-dependent attraction of flies, but through different behavioural outputs. Our work provides a toolkit for investigating taste functions of IRs, defines a subset of these receptors required for carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory molecules in distinct neurons to coordinate behaviour.

The dual developmental origin of spinal cerebrospinal fluid-contacting neurons gives rise to distinct functional subtypes.

Chemical and mechanical cues from the cerebrospinal fluid (CSF) can affect the development and function of the central nervous system (CNS). How such cues are detected and relayed to the CNS remains elusive. Cerebrospinal fluid-contacting neurons (CSF-cNs) situated at the interface between the CSF and the CNS are ideally located to convey such information to local networks. In the spinal cord, these GABAergic neurons expressing the PKD2L1 channel extend an apical extension into the CSF and an ascending axon in the spinal cord. In zebrafish and mouse spinal CSF-cNs originate from two distinct progenitor domains characterized by distinct cascades of transcription factors. Here we ask whether these neurons with different developmental origins differentiate into cells types with different functional properties. We show in zebrafish larva that the expression of specific markers, the morphology of the apical extension and axonal projections, as well as the neuronal targets contacted by CSF-cN axons, distinguish the two CSF-cN subtypes. Altogether our study demonstrates that the developmental origins of spinal CSF-cNs give rise to two distinct functional populations of sensory neurons. This work opens novel avenues to understand how these subtypes may carry distinct functions related to development of the spinal cord, locomotion and posture.

Second-Generation Drosophila Chemical Tags: Sensitivity, Versatility, and Speed.

Labeling and visualizing cells and subcellular structures within thick tissues, whole organs, and even intact animals is key to studying biological processes. This is particularly true for studies of neural circuits where neurons form submicron synapses but have arbors that may span millimeters in length. Traditionally, labeling is achieved by immunofluorescence; however, diffusion of antibody molecules (>100 kDa) is slow and often results in uneven labeling with very poor penetration into the center of thick specimens; these limitations can be partially addressed by extending staining protocols to over a week (Drosophila brain) and months (mice). Recently, we developed an alternative approach using genetically encoded chemical tags CLIP, SNAP, Halo, and TMP for tissue labeling; this resulted in >100-fold increase in labeling speed in both mice and Drosophila, at the expense of a considerable drop in absolute sensitivity when compared to optimized immunofluorescence staining. We now present a second generation of UAS- and LexA-responsive CLIPf, SNAPf, and Halo chemical labeling reagents for flies. These multimerized tags, with translational enhancers, display up to 64-fold increase in sensitivity over first-generation reagents. In addition, we developed a suite of conditional reporters (4xSNAPf tag and CLIPf-SNAPf-Halo2) that are activated by the DNA recombinase Bxb1. Our new reporters can be used with weak and strong GAL4 and LexA drivers and enable stochastic, intersectional, and multicolor Brainbow labeling. These improvements in sensitivity and experimental versatility, while still retaining the substantial speed advantage that is a signature of chemical labeling, should significantly increase the scope of this technology.

Homology-Independent Integration of Plasmid DNA into the Zebrafish Genome.

Targeting nucleases like zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system have revolutionized genome-editing possibilities in many model organisms. They allow the generation of loss-of-function alleles by the introduction of double-strand breaks at defined sites within genes, but also more sophisticated genome-editing approaches have become possible. These include the integration of donor plasmid DNA into the genome by homology-independent repair mechanisms after CRISPR/Cas9-mediated cleavage. Here we present a protocol outlining the most important steps to target a genomic site and to integrate a donor plasmid at this defined locus.

2C-Cas9: a versatile tool for clonal analysis of gene function.

CRISPR/Cas9-mediated targeted mutagenesis allows efficient generation of loss-of-function alleles in zebrafish. To date, this technology has been primarily used to generate genetic knockout animals. Nevertheless, the study of the function of certain loci might require tight spatiotemporal control of gene inactivation. Here, we show that tissue-specific gene disruption can be achieved by driving Cas9 expression with the Gal4/UAS system. Furthermore, by combining the Gal4/UAS and Cre/loxP systems, we establish a versatile tool to genetically label mutant cell clones, enabling their phenotypic analysis. Our technique has the potential to be applied to diverse model organisms, enabling tissue-specific loss-of-function and phenotypic characterization of live and fixed tissues.

Sexual circuitry in Drosophila.

The sexual behavior of Drosophila melanogaster is an outstanding paradigm to understand the molecular and neuronal basis of sophisticated animal actions. We discuss recent advances in our knowledge of the genetic hardwiring of the underlying neuronal circuitry, and how pertinent sensory cues are differentially detected and integrated in the male and female brain. We also consider how experience influences these circuits over short timescales, and the evolution of these pathways over longer timescales to endow species-specific sexual displays and responses.