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 in their chemical ecology: the generalists Drosophila melanogaster and Drosophila simulans and Drosophila sechellia that specializes on ripe noni fruit. We examine a central part of the olfactory circuit that, to our knowledge, has not been investigated in these species-the connections between projection neurons and the Kenyon cells of the mushroom body-and identify species-specific connectivity patterns. We found that neurons encoding food odors connect more frequently with Kenyon cells, giving rise to species-specific biases in connectivity. These species-specific connectivity differences 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.

Mapping Kenyon cell inputs in Drosophila using dye electroporation.

Here, we describe a technique for charting the inputs of individual Kenyon cells in the Drosophila brain. In this technique, a single Kenyon cell per brain hemisphere is photo-labeled to visualize its claw-like dendritic terminals; a dye-filled electrode is used to backfill the projection neuron connected to each claw. This process can be repeated in hundreds of brains to build a connectivity matrix. Statistical analyses of such a matrix can reveal connectivity patterns such as random input and biased connectivity. For complete details on the use and execution of this protocol, please refer to Hayashi et al. (2022).1.

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.

Mushroom body input connections form independently of sensory activity in Drosophila melanogaster.

Associative brain centers, such as the insect mushroom body, need to represent sensory information in an efficient manner. In Drosophila melanogaster, the Kenyon cells of the mushroom body integrate inputs from a random set of olfactory projection neurons, but some projection neurons-namely those activated by a few ethologically meaningful odors-connect to Kenyon cells more frequently than others. This biased and random connectivity pattern is conceivably advantageous, as it enables the mushroom body to represent a large number of odors as unique activity patterns while prioritizing the representation of a few specific odors. How this connectivity pattern is established remains largely unknown. Here, we test whether the mechanisms patterning the connections between Kenyon cells and projection neurons depend on sensory activity or whether they are hardwired. We mapped a large number of mushroom body input connections in partially anosmic flies-flies lacking the obligate odorant co-receptor Orco-and in wild-type flies. Statistical analyses of these datasets reveal that the random and biased connectivity pattern observed between Kenyon cells and projection neurons forms normally in the absence of most olfactory sensory activity. This finding supports the idea that even comparatively subtle, population-level patterns of neuronal connectivity can be encoded by fixed genetic programs and are likely to be the result of evolved prioritization of ecologically and ethologically salient stimuli.

Photo-labeling neurons in the Drosophila brain.

Many genetically encoded tools, including large collections of GAL4 transgenic lines, can be used to visualize neurons of the Drosophila melanogaster brain. However, identifying transgenic lines that are expressed sparsely enough to label individual neurons, or groups of neurons that innervate a particular brain region, remains technically challenging. Here, we provide a detailed procedure in which we used broadly expressed transgenic lines and two-photon microscopy to photo-label neurons with specificity, thereby permitting their morphological characterization. For complete details on the use and execution of this protocol, please refer to Li et al. (2020).

Neuroscience: The Secret of Sauce Béarnaise Syndrome Is in the Circuit.

During conditioned food aversion - a.k.a. sauce béarnaise syndrome - the ingestion of a spoiled food item leads to a lasting aversion towards cues reminiscent of the item. A new study finds that, in Drosophila, taste aversion depends on the immune system and the mushroom body.

Visual Input into the Drosophila melanogaster Mushroom Body.

The patterns of neuronal connectivity underlying multisensory integration, a fundamental property of many brains, remain poorly characterized. The Drosophila melanogaster mushroom body-an associative center-is an ideal system to investigate how different sensory channels converge in higher order brain centers. The neurons connecting the mushroom body to the olfactory system have been described in great detail, but input from other sensory systems remains poorly defined. Here, we use a range of anatomical and genetic techniques to identify two types of input neuron that connect visual processing centers-the lobula and the posterior lateral protocerebrum-to the dorsal accessory calyx of the mushroom body. Together with previous work that described a pathway conveying visual information from the medulla to the ventral accessory calyx of the mushroom body, our study defines a second, parallel pathway that is anatomically poised to convey information from the visual system to the dorsal accessory calyx.

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.

Neuroscience: Intelligence in the Honeybee Mushroom Body.

Intelligence, in most people's conception, involves combining pieces of evidence to reach non-obvious conclusions. A recent theoretical study shows that intelligence-like brain functions can emerge from simple neural circuits, in this case the honeybee mushroom body.

Random convergence of olfactory inputs in the Drosophila mushroom body.

The mushroom body in the fruitfly Drosophila melanogaster is an associative brain centre that translates odour representations into learned behavioural responses. Kenyon cells, the intrinsic neurons of the mushroom body, integrate input from olfactory glomeruli to encode odours as sparse distributed patterns of neural activity. We have developed anatomic tracing techniques to identify the glomerular origin of the inputs that converge onto 200 individual Kenyon cells. Here we show that each Kenyon cell integrates input from a different and apparently random combination of glomeruli. The glomerular inputs to individual Kenyon cells show no discernible organization with respect to their odour tuning, anatomic features or developmental origins. Moreover, different classes of Kenyon cells do not seem to preferentially integrate inputs from specific combinations of glomeruli. This organization of glomerular connections to the mushroom body could allow the fly to contextualize novel sensory experiences, a feature consistent with the role of this brain centre in mediating learned olfactory associations and behaviours.

Cyclin D2 rescues size and function of GATA4 haplo-insufficient hearts.

Transcription factor GATA4 is a key regulator of cardiomyocyte growth, and differentiation and 50% reduction in GATA4 levels results in hypoplastic hearts. Search for GATA4 targets/effectors revealed cyclin D(2) (CD2), a member of the D-type cyclins (D(1), D(2), and D(3)) that play a vital role in cell growth and differentiation as a direct transcriptional target and a mediator of GATA4 growth in postnatal cardiomyocytes. GATA4 associates with the CD2 promoter in cardiomyocytes and is sufficient to induce endogenous CD2 transcription and to dose-dependently activate the CD2 promoter in heterologous cells. Cardiomyocyte-specific overexpression of CD2 results in enhanced postnatal cardiac growth because of increased cardiomyocyte proliferation. When these transgenic mice are crossed with Gata4 heterozygote mice, they rescue the hypoplastic cardiac phenotype of Gata4(+/-) mice and enhance cardiomyocyte survival and heart function. The data uncover a role for CD2 in the postnatal heart as an effector of GATA4 in myocyte growth and survival. The finding that postnatal upregulation of a cell-cycle gene in GATA4 haplo-insufficient hearts may be protective opens new avenues for maintaining or restoring cardiac function in GATA4-dependent cardiac disease.

BAPTI and BAPTISM birthdating of neurons in zebrafish.

This protocol describes how the photoconvertible protein Kaede can be used to determine the birthdates of neurons in live zebrafish. The methods used are birthdating analysis by photoconverted fluorescent protein tracing in vivo (BAPTI) and BAPTI combined with subpopulation markers (BAPTISM). Because Kaede can be converted from green to red fluorescence at any developmental time point, it serves as a temporal landmark for cell birth. When it is used in combination with subpopulation markers, the eventual fate of a cell can be correlated with its birthdate. We describe how we used this method to study the development of trigeminal sensory neurons and discuss how the technique can be extended to the study of other organs.

Zebrafish TRPA1 channels are required for chemosensation but not for thermosensation or mechanosensory hair cell function.

Transient receptor potential (TRP) ion channels have been implicated in detecting chemical, thermal, and mechanical stimuli in organisms ranging from mammals to Caenorhabditis elegans. It is well established that TRPA1 detects and mediates behavioral responses to chemical irritants. However, the role of TRPA1 in detecting thermal and mechanical stimuli is controversial. To further clarify the functions of TRPA1 channels in vertebrates, we analyzed their roles in zebrafish. The two zebrafish TRPA1 paralogs are expressed in sensory neurons and are activated by several chemical irritants in vitro. High-throughput behavioral analyses of trpa1a and trpa1b mutant larvae indicate that TRPA1b is necessary for behavioral responses to these chemical irritants. However, TRPA1 paralogs are not required for behavioral responses to temperature changes or for mechanosensory hair cell function in the inner ear or lateral line. These results support a role for zebrafish TRPA1 in chemical but not thermal or mechanical sensing, and establish a high-throughput system to identify genes and small molecules that modulate chemosensation, thermosensation, and mechanosensation.

In vivo birthdating by BAPTISM reveals that trigeminal sensory neuron diversity depends on early neurogenesis.

Among sensory systems, the somatic sense is exceptional in its ability to detect a wide range of chemical, mechanical and thermal stimuli. How this sensory diversity is established during development remains largely elusive. We devised a method (BAPTISM) that uses the photoconvertible fluorescent protein Kaede to simultaneously analyze birthdate and cell fate in live zebrafish embryos. We found that trigeminal sensory ganglia are formed from early-born and late-born neurons. Early-born neurons give rise to multiple classes of sensory neurons that express different ion channels. By contrast, late-born neurons are restricted in their fate and do not form chemosensory neurons expressing the ion channel TrpA1b. Accordingly, larvae lacking early-born neurons do not respond to the TrpA1b agonist allyl isothiocyanate. These results indicate that the multimodal specification and function of trigeminal sensory ganglia depends on the timing of neurogenesis.

Members of the miRNA-200 family regulate olfactory neurogenesis.

MicroRNAs (miRNAs) are highly expressed in vertebrate neural tissues, but the contribution of specific miRNAs to the development and function of different neuronal populations is still largely unknown. We report that miRNAs are required for terminal differentiation of olfactory precursors in both mouse and zebrafish but are dispensable for proper function of mature olfactory neurons. The repertoire of miRNAs expressed in olfactory tissues contains over 100 distinct miRNAs. A subset, including the miR-200 family, shows high olfactory enrichment and expression patterns consistent with a role during olfactory neurogenesis. Loss of function of the miR-200 family phenocopies the terminal differentiation defect observed in absence of all miRNA activity in olfactory progenitors. Our data support the notion that vertebrate tissue differentiation is controlled by conserved subsets of organ-specific miRNAs in both mouse and zebrafish and provide insights into control mechanisms underlying olfactory differentiation in vertebrates.

Fingerprinting techniques as tools towards molecular quality control of Pseudozyma flocculosa.

In an effort to meet the stringent requirements towards registration of microorganisms as biopesticides, several molecular techniques were tested as part of a strategy to develop a quality control system for Pseudozyma flocculosa, the active ingredient of Sporodex, a biofungicide used for the control of powdery mildew fungi. In the first approach, multiplex PCR fingerprints generated by three sets of primers allowed differentiation of several isolates of P. flocculosa from closely related species, or genera such as Tilletiopsis. The same set of primers was used in quality control experiments and revealed fungal contamination that was otherwise not observed by standard culture and microscopy techniques. In addition, the use of random amplified microsatellites generated by (GT)n and (CCA)n primers was applied as a measure of possible genetic variation over 65 repeated subcultures of P. flocculosa. Finally, a novel technique was developed and named reverse intron PCR (RIP), based on the presence of an intron revealed by partial sequencing of mtLSU of P. flocculosa. RIP allowed not only differentiation of P. flocculosa from other species but also separated the isolates of different origins within P. flocculosa. This new technique could find useful applications in phylogenetic studies of closely related fungal species and isolates.