Biology subjects

Le, J. Q.

Publications and source records attributed to Le, J. Q..

6 recordsLinked to original sources

bioRxiv2024

Four SpsP neurons are an integrating sleep regulation hub in Drosophila

Sleep is an essential and conserved behavior, yet the mechanisms underlying sleep regulation remain largely unknown. To address the neural mechanisms of sleep drive, here we carry out whole brain calcium-modulated photoactivatable ratiometric integrator (CaMPARI) imaging of Drosophila and show that the activity of the protocerebral bridge (PB), a part of the central complex, correlates with sleep drive. Through a neural activation screen followed by anatomical and functional connectivity assays, we further narrow down the key player of sleep regulation in the PB to a three-layer circuit composed of 4 SpsP neurons and their upstream and downstream synaptic partners: the 4 SpsP neurons act as an integrating hub by responding to ellipsoid body (EB) signals from EPG neurons, and by sending signals back to the EB through PEcG neurons. Moreover, sleep deprivation enriches the presynaptic active zones of SpsP neurons and strengthens the connections of the EPG-SpsP-PEcG circuit, indicating plasticity gating in the circuit in response to sleep drive change. As the SpsP neurons also receive input from the sensorimotor brain region and given their known role in navigation, these neurons potentially further integrate sleep drive with other sensorimotor cues. The data taken together indicate that the four SpsP neurons and their sleep regulatory circuit play an important and dynamic role in sleep regulation.

neuroscience↗

bioRxiv2024

FlyBox: A Flexible Open-Source Behavior Monitoring System

Over the past two decades, the vast majority of circadian behavior in Drosophila has been recorded in Drosophila Activity Monitor (DAM) boards. Though simple and robust, locomotor behavior recording via DAM boards can be prohibitively expensive, especially when taking incubator costs into consideration. Furthermore, their simplicity limits their experimental options and resolution. Here, we present the FlyBox: a simple, open-source benchtop locomotor activity recording system. FlyBox was designed to monitor activity in animals loaded into a standard laboratory multi-well plate. It features light-tight construction and multiple programmable LEDs for simulating day/night cycles and optogenetic manipulation. In total, a single FlyBox costs approximately $750 to build and around two days of labor. In addition, we also present the FlyBoxScanner software to simplify activity monitoring while maintaining compatibility with DAM analysis software. FlyBox is an attractive and affordable package for behavior monitoring that also offers considerable room for customization. Materials and instructions for the FlyBox are available at https://github.com/Rosbash-Lab-FlyBox/FlyBox, and FlyBoxScanner is available at https://github.com/jose-elias-alvarez/flybox-scanner.

neuroscience↗

bioRxiv2024

Novel clock neuron subtypes regulate temporal aspects of sleep

Circadian neurons within animal brains orchestrate myriad physiological processes and behaviors, But the contribution of these neurons to the regulation of sleep is not well understood. To address this deficiency, we leveraged single-cell RNA sequencing to generate a new and now comprehensive census of transcriptomic cell types of Drosophila clock neurons. We focused principally on the enigmatic DN3s, which constitute about half of the 75 pairs of clock neurons in the fly brain and were previously almost completely uncharacterized. These DN3s are organized into 12 clusters with unusual gene expression features compared to the more well-studied clock neurons. We further show that different DN3 subtypes with distinct projection patterns promote sleep at specific times of the day through a common G protein-coupled receptor, TrissinR. Our findings indicate an intricate regulation of sleep behavior by clock neurons and highlight their remarkable diversity in gene expression, projection patterns and functional properties.

neuroscience↗

bioRxiv2024

Light and dopamine impact two circadian neurons to promote morning wakefulness

In both mammals and flies, circadian brain neurons orchestrate physiological oscillations and behaviors like wake and sleep; these neurons can be subdivided by morphology and by gene expression patterns. Recent single-cell sequencing studies identified 17 Drosophila circadian neuron groups. One of these include only two lateral neurons (LNs), which are marked by the expression of the neuropeptide ion transport peptide (ITP). Although these two ITP+ LNs have long been grouped with five other circadian evening activity cells, inhibiting the two neurons alone strongly reduces morning activity; this indicates that they are prominent morning neurons. As dopamine signaling promotes activity in Drosophila like in mammals, we considered that dopamine might influence this morning activity function. Moreover, the ITP+ LNs express higher mRNA levels than other LNs of the type 1-like dopamine receptor Dop1R1. Consistent with the importance of Dop1R1, CRISPR/Cas9 mutagenesis of this receptor only in the two ITP+ LNs renders flies significantly less active in the morning, and ex vivo live imaging shows that dopamine increases cAMP levels in these two neurons; cell-specific mutagenesis of Dop1R1 eliminates this cAMP response to dopamine. Notably, the response is more robust in the morning, reflecting higher morning Dop1R1 mRNA levels in the two neurons. As morning levels are not elevated in constant darkness, this suggests light-dependent upregulation of morning Dop1R1 transcript levels. Taken together with enhanced morning cAMP response to dopamine, the data indicate how light stimulates morning wakefulness in flies, which mimics the important effect of light on morning wakefulness in humans.

neuroscience↗

bioRxiv2023

Dissecting cell-specific functions of circadian genes using modified cell-specific CRISPR approaches

Circadian behavioral rhythms in Drosophila melanogaster are regulated by about 75 pairs of brain neurons. They all express the core clock genes but have distinct functions and gene expression profiles. To understand the importance of these distinct molecular programs, neuron-specific gene manipulations are essential. Although RNAi based methods are standard to manipulate gene expression in a cell-specific manner, they are often ineffective, especially in assays involving smaller numbers of neurons or weaker Gal4 drivers. We and others recently exploited a neuron-specific CRISPR-based method to mutagenize genes within circadian neurons. Here we further explore this approach to mutagenize three well-studied clock genes: the transcription factor gene vrille, the photoreceptor gene Cryptochrome (cry) and the neuropeptide gene Pdf. The CRISPR-based strategy not only reproduced their known phenotypes but also assigned cry function for different light mediated phenotypes to discrete, different subsets of clock neurons. We further tested two recently published methods for temporal regulation in adult neurons, inducible Cas9 and auxin-inducible gene expression system (AGES). The results were not identical, but both approaches successfully showed that the adult-specific knockout of the neuropeptide Pdf reproduces the canonical loss-of-function mutant phenotypes. In summary, a CRISPR-based strategy is a highly effective, reliable, and general method to temporally manipulate gene function in specific adult neurons. Significance statementMost animals have specific brain neurons that regulate sleep-wake cycles and other aspects of circadian behavior. Drosophila has only about 150 of these clock neurons. Despite their small numbers, they have remarkably diverse anatomy and gene expression profiles. To address the different functions of these neurons, we used highly specific and efficient CRISPR-based methods to create cell type-specific disruptions of three traditional circadian genes. We were able to assign the function of the photoreceptor cryptochrome to two tiny subsets of clock neurons. In addition, two independent methods assigned the neuropeptide PDF to the adult stage. In summary, we find that the CRISPR-based methods are very efficient at studying adult specific functions of genes in small, discrete sets of neurons.

genetics↗

bioRxiv2022

Neural connectivity molecules best identify the heterogeneous clock and dopaminergic cell types in the Drosophila adult brain

Our recent single cell sequencing of most adult Drosophila circadian neurons indicated striking gene expression heterogeneity, about 2-3 cells per clock neuron group. To extend this characterization to other adult fly brain neurons, we used the identical plate-based methods to generate single cell data from a similar subset of dopaminergic neurons. To minimize batch effects and to apply an additional sequencing strategy, we also assayed these two populations together with 10X Chromium. An unsupervised clustering algorithm indicates that dopaminergic neurons are comparably heterogeneous, suggesting that the transcriptomic diversity of adult fly brain neurons parallels its EM connectome. The results here further indicate that connectivity molecules like cell surface molecules best characterize all neuron groups. We suggest that these surprising features are general and make major contributions to neuronal identity and connectivity of the adult central brain as well as underlie the complex behavioral repertoire of Drosophila.

neuroscience↗