The Role of Even-Skipped in<i>Drosophila</i>Larval Somatosensory Circuit Assembly

Marshall, Zarion D; Heckscher, Ellie S.

doi:10.1523/eneuro.0403-21.2021

Cited by 4 publications

(5 citation statements)

References 47 publications

(104 reference statements)

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“…Techniques that work well in other models -light sheet, lightfield, and confocal microscopy, or various tethering schemes -have not translated to the larva (supplemental discussion 10). Workarounds, including interpreting motor neuron activity in dissected or immobilized animals [61,[65][66][67] and photoswitchable activity markers [68,69], have ameliorated but not eliminated this difficulty. CRASH2p allows functional two photon imaging with ratiometric correction in unrestrained larvae, further advancing the utility of this model organism.…”

Section: Discussionmentioning

confidence: 99%

CRASH2p: Closed-loop Two Photon Imaging in Freely Moving Animals

McNulty,

Wu,

Yamaguchi

et al. 2024

Preprint

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Direct measurement of neural activity in freely moving animals is essential for understanding how the brain controls and represents behaviors. Genetically encoded calcium indicators report neural activity as changes in fluorescence intensity, but brain motion confounds quantitative measurement of fluorescence. Translation, rotation, and deformation of the brain and the movements of intervening scattering or auto-fluorescent tissue all alter the amount of fluorescent light captured by a microscope. Compared to single-photon approaches, two photon microscopy is less sensitive to scattering and off-target fluorescence, but more sensitive to motion, and two photon imaging has always required anchoring the microscope to the brain. We developed a closed-loop resonant axial-scanning high-speed two photon (CRASH2p) microscope for real-time 3D motion correction in unrestrained animals, without implantation of reference markers. We complemented CRASH2p with a novel scanning strategy and a multi-stage registration pipeline. We performed volumetric ratiometrically corrected functional imaging in the CNS of freely movingDrosophilalarvae and discovered previously unknown neural correlates of behavior.

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Section: Discussionmentioning

confidence: 99%

CRASH2p: Closed-loop Two Photon Imaging in Freely Moving Animals

McNulty,

Wu,

Yamaguchi

et al. 2024

Preprint

Self Cite

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“…A Visaton FR12, 4 Ohm speaker (5 inches diameter) and a PYLE PCA2 stereo power amplifier was used to project sound. For further details, refer to Marshall and Heckscher (Marshall & Heckscher, 2022). Images were acquired on a Zeiss LSM 800 confocal microscope using 0.1%–0.2% 488 nm laser power with the pinhole entirely open.…”

Section: Methodsmentioning

confidence: 99%

“…The calcium signal was continuously collected before, during, and after the stimulus. Extracting changes in GCaMP6m fluorescence amplitude was done using Fiji as in Marshall and Heckscher (Marshall & Heckscher, 2022). A region of interest (ROI) that included the larval nerve cord was manually drawn, and the mean fluorescence within the ROI was calculated for each time point.…”

Section: Methodsmentioning

confidence: 99%

Sequential addition of neuronal stem cell temporal cohorts generates a feed-forward circuit in the Drosophila larval nerve cord

Wang

Wreden

Levy

et al. 2022

Preprint

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Understanding how circuits self-assemble starting from neuronal stem cells is a fundamental question in developmental biology. Here, we addressed how neurons from different lineages wire with each other to form a specific circuit motif. To do so, we combined developmental genetics—Twin spot MARCM, Multi-color Flip Out, permanent labeling—with circuit analysis—calcium imaging, connectomics, and network science analyses. We find many lineages are organized into temporal cohorts, which are sets of lineage-related neurons born within a tight time window, and that temporal cohort boundaries have sharp transitions in patterns of input connectivity. We identify a feed-forward circuit motif that encodes the onset of vibration stimuli. This feed-forward circuit motif is assembled by preferential connectivity between temporal cohorts from different neuronal stem cell lineages. Further, connectivity does not follow the often-cited early-to-early, late-to-late model. Instead, the feed-forward motif is formed by sequential addition of temporal cohorts, with circuit output neurons born before circuit input neurons. Further, we generate multiple new tools for the fly community. Ultimately, our data suggest that sequential addition of neurons (with outputs neurons being oldest and input neurons being youngest) could be a fundamental strategy for assembling feed-forward circuits.

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“…A Visaton FR12, 4 Ohm speaker (5 inches diameter) and a PYLE PCA2 stereo power amplifier was used to project sound. For further details, refer to Marshall and Heckscher, 2022 . Images were acquired on a Zeiss LSM 800 confocal microscope using 0.1–0.2% 488 nm laser power with the pinhole entirely open.…”

Section: Methodsmentioning

confidence: 99%

“…The calcium signal was continuously collected before, during, and after the stimulus. Extracting changes in GCaMP6m fluorescence amplitude was done using Fiji as in Marshall and Heckscher, 2022 . A region of interest (ROI) that included the larval nerve cord was manually drawn, and the mean fluorescence within the ROI was calculated for each time point.…”

Section: Methodsmentioning

confidence: 99%

Sequential addition of neuronal stem cell temporal cohorts generates a feed-forward circuit in the Drosophila larval nerve cord

Wang

Wreden

Levy

et al. 2022

eLife

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How circuits self-assemble starting from neuronal stem cells is a fundamental question in developmental neurobiology. Here, we addressed how neurons from different stem cell lineages wire with each other to form a specific circuit motif. In Drosophila larvae, we combined developmental genetics (Twin spot MARCM, Multi-color Flip Out, permanent labeling) with circuit analysis (calcium imaging, connectomics, network science). For many lineages, neuronal progeny are organized into subunits called temporal cohorts. Temporal cohorts are subsets of neurons born within a tight time window that have shared circuit level function. We find sharp transitions in patterns of input connectivity at temporal cohort boundaries. In addition, we identify a feed-forward circuit that encodes the onset of vibration stimuli. This feed-forward circuit is assembled by preferential connectivity between temporal cohorts from different lineages. Connectivity does not follow the often-cited early-to-early, late-to-late model. Instead, the circuit is formed by sequential addition of temporal cohorts from different lineages, with circuit output neurons born before circuit input neurons. Further, we generate new tools for the fly community. Our data raise the possibility that sequential addition of neurons (with outputs oldest and inputs youngest) could be one fundamental strategy for assembling feed-forward circuits.

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The Role of Even-Skipped inDrosophilaLarval Somatosensory Circuit Assembly

Abstract: We would like to thank Chip Ferguson and Paschalis Kratsios (UChicago), Chris Doe (UOregon), and members of the Heckscher lab for comments that improved this manuscript. Also, thank you to Richard Baines (Manchester) for sharing Eve binding site data.

Cited by 4 publications

References 47 publications

CRASH2p: Closed-loop Two Photon Imaging in Freely Moving Animals

CRASH2p: Closed-loop Two Photon Imaging in Freely Moving Animals

Sequential addition of neuronal stem cell temporal cohorts generates a feed-forward circuit in the Drosophila larval nerve cord

Sequential addition of neuronal stem cell temporal cohorts generates a feed-forward circuit in the Drosophila larval nerve cord

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