In vertebrates, stimulus-independent activity accompanies neural circuit maturation throughout the developing brain1,2. The recent discovery of similar activity in the developing Drosophila central nervous system suggests that developmental activity is fundamental to the assembly of complex brains3. How such activity is coordinated across disparate brain regions to influence synaptic development at the level of defined cell types is not well understood. Here we show that neurons expressing the cation channel transient receptor potential gamma (Trpγ) relay and pattern developmental activity throughout the Drosophila brain. In trpγ mutants, activity is attenuated globally, and both patterns of activity and synapse structure are altered in a cell-type-specific manner. Less than 2% of the neurons in the brain express Trpγ. These neurons arborize throughout the brain, and silencing or activating them leads to loss or gain of brain-wide activity. Together, these results indicate that this small population of neurons coordinates brain-wide developmental activity. We propose that stereotyped patterns of developmental activity are driven by a discrete, genetically specified network to instruct neural circuit assembly at the level of individual cells and synapses. This work establishes the fly brain as an experimentally tractable system for studying how activity contributes to synapse and circuit formation.
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The authors declare that all data supporting the findings of this study are available within the paper and its supplementary information files.
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We thank S. L. Zipursky for discussions, support and feedback on the manuscript; C. Montell for sharing fly lines and other reagents; T. R. Clandinin for support in generating the SPARC3-Out-GAL80 flies; J. M. Donlea, V. Hartenstein, D. E. Krantz and Y. Lin for sharing antibodies; J. A. Veenstra for sharing neuropeptide antibodies; D. Hattori for the Kir2.1 flies; A. Nern and G. M. Rubin for the 29C07-FLP flies; the Banerjee Laboratory at UCLA for the UAS-rpr,-hid flies; O. Kanca and H. J. Bellen for the TrpγDropIn-TG4 flies; S. Yamamoto and T. Takano-Shimizu for the UAS-TRPC4 and TRPC5 flies; and members of the Zipursky laboratory for feedback and discussion. B.T.B. is supported by NIH grants F30EY029952 and T32GM008042; OA is supported by NIH grant R01NS123376.
The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Trpγ is necessary for PSINA.
a. Raw values binned by hour for active phase signal amplitude, sweeps/cycle, cycles/hour, and cycle duration for control (black, n=19) and trpγ null (orange, n=31) pupae. Shaded areas, SD. b. Active phase average amplitude (left) and sweeps/cycle (right) binned by hour and normalized to control activity during the turbulent stage, between 65 and 80 hAPF, for control (black, n=19), trpγ null (orange, n=31), and trpγ null with Trpγ-D expressed in Trpγ+ cells (cyan, n=4) pupae. Shaded areas, SD. c. Cycle duration (left) and cycles/hour (right) binned by hour and normalized to control activity between 55 and 65 hAPF. Shaded areas, SD. Genotypes color-matched to B. d. Representative traces of activity in control (black, n=19), trpγ/TrpγG4 (orange, n=5), trpγ/TrpγDropIn-TG4 (gray, n=5), trpγ/Df(2L)1102 (magenta, n=7), and trpγ/Df(2L)1109 pupae (green, n=7). e. Average amplitude (left) and sweeps/cycle (right) binned by hour and normalized to control activity between 55 and 65 hAPF. Shaded areas, SD. Genotypes color-matched to D. f. Active phase average amplitude (left) and sweeps/cycle (right) binned by hour and normalized to control activity during the periodic stage, between 55 and 65 hAPF, for control (black, n=10), trpγ null (orange, n=9), trp null (blue, n=10), and trp null + trpγ null (green, n=10). Shaded areas, SD. g. Active phase average amplitude (left) and sweeps/cycle (right) binned by hour and normalized to control activity during the periodic stage, between 55 and 65 hAPF, for control (black, n=10), trpγ null (orange, n=10), trpL null (blue, n=10), and trpL null+ trpγ null (green, n=10). Shaded areas, SD.
Extended Data Fig. 2 PSINA rescue in trpγ null background.
a. Schematic of Trpγ locus indicating locations of exons (orange rectangles), untranslated regions (gray rectangles), and introns (black lines between exons or untranslated regions) for each isoform. Scale bar, 500 bp. b. Representative traces of activity in: Trpγ-D expression with TrpγG4 (blue, n=8), Trpγ-D expression with TrpγDropIn-TG4 (magenta, n=7), Trpγ-AB expression with TrpγG4 (green, n=9), Trpγ-AB and Trpγ-D expression with TrpγG4 (red, n=10), double Trpγ-D expression with TrpγG4 (cyan, n=4), control (black, n=19), and trpγ mutant (orange, n=31) pupae. c. Active phase average amplitude (left) and sweeps/cycle (right) binned by hour and normalized to control activity between 55 and 65 hAPF. All plots contain data for control (black) and trpγ (orange). Shaded areas, SD. Genotypes color-matched to B. d. Expression control of UAS-Trpγ-D with TARGET (i.e. GAL80ts). In the ‘all-on’ condition, flies are reared at 29oC. In the ‘all-off’ condition, flies are reared at 18oC. e, g. Representative traces of activity in control pupae (black, n=3), trpγ null pupae (orange, n=3), and (E) all-off pupae (blue, n=3) or (G) all-on pupae (red, n=3). f, h. Active phase average amplitude (left) and sweeps/cycle (right) binned by hour and normalized to control activity between 55 and 65 hAPF. Shaded areas, SD. Genotypes color-matched to E and G.
Extended Data Fig. 3 Synapse formation in the visual system depends on PSINA.
a. Table comparing control synapse counts in cells with sparse synaptic density across EM and light microscopy studies. Values are mean synapse count ± SD, with sample size in parentheses. b–g. Left: representative micrographs of R8 (B), L1 (C), L4 (D), L5 (E), Dm9 (F), and Tm9 (G) neurons in control (left set) and trpγ (right set) animals with cell membranes (myr::tdTOM, magenta in merged) and presynaptic sites (BRP-V5, cyan in merged) labeled. Right: Brp puncta counts by layer in heterozygous control (black, n=18-61 per cell type) and trpγ (orange, n=26-65 per cell type) animals. Points indicate individual cells. Box-and-whiskers mark 5th, 25th, 50th, 75th, and 95th percentiles. *, p<0.05; **, p<0.01; ***, p<0.001 by Welch’s t-test following Shapiro-Wilk test. h. Brp puncta counts in Trpγ heterozygotes (black, n=18 for Dm9, n=30 for Tm9), trpγ nulls (orange, n=24 for Dm9, n=30 for Tm9), or trpγ nulls with Trpγ-D expressed in Trpγ+ cells (cyan, n=24 for Dm9, n=30 for Tm9). Boxplots as in B-G. *, p<0.05; **, p<0.01; ***, p<0.001 by Tukey’s post-hoc test following ANOVA for multiple groups. i. Brp puncta counts in control (black, n = 24) or trpγ null (orange, n = 30) L5 clones generated by MARCM. Boxplots as in B-G. *, p<0.05; **, p<0.01; ***, p<0.001 by Welch’s t-test following Shapiro-Wilk test. j. Average Brp puncta through development in control (black, n=64-88 per timepoint) or animals with PSINA blocked with pan-neuronally expressed TNT (magenta, n=35-68 per timepoint). Presynaptic sites assessed at 60, 72, and 84 hAPF. Error bars, SD.
Extended Data Fig. 4 TrpγG4 drives expression in a dynamic neuronal population during development.
a. MIPs of half-brain confocal stacks from different times during pupal development and early adult life. Nuclei of mCherry-NLS expressing Trpγ+ neurons shown (cyan); reference marker is Ncad (magenta). Average, SD, and number of samples for each time point are printed top-right of panels; these values are plotted in Fig. 4a. Dashed yellow lines mark the median plane. CB, central brain. OL, optic lobe. Scale bar, 100µm. b. Top: 13µm-thick MIP of a 72 hAPF brain stained for neuronal nuclei (anti-Elav, yellow), Trpγ+ nuclei (mCherry-NLS, cyan), and a reference marker (Ncad, magenta). Image derived from three stitched confocal stacks. Scale bar, 100µm. Bottom: Expanded views of two regions-of-interest (ROIs) boxed in top panel. Columns are neuronal, Trpγ+, and merged channels, left to right. All Trpγ+ nuclei fully captured in the MIP (red asterisks) co-localize with the neuronal stain. Scale bar, 20µm. c. Top: 13µm-thick MIP of a 72 hAPF brain stained for glial nuclei (anti-Repo, yellow), Trpγ+ nuclei (mCherry-NLS, cyan), and a reference marker (Ncad, magenta). Image derived from three stitched confocal stacks. Scale bar, 100µm. Bottom-left: Histogram of average voxel intensities of segmented Repo+ and Trpγ+ nuclei measured in the anti-Repo channel of the top image. n=5055 (Repo+), 1464 (Trpγ+); half-brain complements analyzed. Inset shows where 9/1464 Trpγ+ cell intensities overlap with the dimmest Repo+ glia. Bottom-right: Histogram of minimum pairwise distance between centroids of 1464 segmented Trpγ+ and Repo+ nuclei. Inset shows all pairs of Trpγ+ and Repo+ nuclei are at least 1µm apart. d. Representative traces of activity in control pupae (black), pan-neuronal control knockdown pupae (gray, n=2), pan-neuronal Trpγ knockdown (magenta, n=3; green, n =3), pan-glial Trpγ knockdown (red, n=2; blue, n=2) in heterozygous Trpγ background. e. Active phase average amplitude (left) and sweeps/cycle (right) binned by hour and normalized to control activity between 55 and 65 hAPF. Shaded areas, SD. Genotypes color-matched to D.
Extended Data Fig. 5 Trpγ+ neurons are a diverse population.
a–i. Top: 13µm-thick (A-C) or full (D-I) MIPs of 72 hAPF brains stained for neuronal class marker (yellow), Trpγ+ nuclei (mCherry-NLS or GFP-NLS, cyan), and a reference marker (Ncad, magenta). Images (A-C, E-H) derived from three stitched confocal stacks. Scale bar, 100µm. Bottom: Expanded view(s) of ROI(s) boxed in top panel. Columns (D, rows) are class marker, Trpγ+, and merged channels, left to right (D, top to bottom). Marked Trpγ+ nuclei (red asterisks) co-localize with the neuronal class marker. ROI 2 in (G) shows transient PDF-TRI cells (38). Scale bar, 20µm.
Extended Data Fig. 6 SPARC3-Out-GAL80 reveals morphologies of individual Trpγ+ neurons.
a. Schematic of the SPARC3-Out-GAL80 cassette. PhiC31 recombines one of two competing attP target sequences with one attB target sequence. Rxn 1 leads to loss of the GAL80 ORF, disinhibiting GAL4-driven effector expression. Rxn 2 preserves Tubulin promoter driven GAL80 expression, maintaining GAL4 inhibition. Three progressively truncated variants for the first attP sequence were designed (25) to bias the recombination in favor of Rxn 2, resulting in frequent (Dense), sporadic (Intermediate), or rare (Sparse) loss of GAL80 and disinhibition of GAL4>UAS expression. b. Map of pHD-3xP3-DsRed-ΔattP (a CRISPR-HDR-donor precursor) showing multiple cloning sites for homology arm insertion (right). c. Map of pHD-3xP3-DsRed-ΔattP-CRISPR-donor (example includes homology arms targeting the Su(Hw)AttP5 region of the Drosophila genome). d. Assembled SPARC3-Out-GAL80 cassette; see Materials and Methods for details. MCS, multiple cloning site. gRNA, guide RNA. HDVR, hepatitis delta virus ribozyme sequence. e, g. Single Trpγ+ neuron (orange, manually segmented) in the context of others (cyan) labeled using SPARC. Neurons expressing myr::SM-V5. Reference marker (magenta), Ncad. Image MIP of stitched confocal stacks of 72 hAPF brain. Scale bar, 100µm. f. Trpγ+ visual processing neurons identified in 72 hAPF brains using SPARC. We observed a given neuron up to three times in 30 sparsely labeled brains.
Extended Data Fig. 7 Additional characterization of Trpγ+ neuron activity.
a. Representative autocorrelograms from pan-neuronal GCaMP6s in control (empty-GAL4, black), panN-GAL4>Kir2.1 (blue), and TrpγG4>Kir2.1 (orange) pupae. b, c. Representative micrographs (B) and traces (C) from 2PM imaging of pan-neuronal GCaMP6s in control (empty-GAL4, black), panN-GAL4>Kir2.1 (blue), and TrpγG4>Kir2.1 (orange) pupae. Scale bar, 40µm. d. Inset: expanded view showing fewer sweeps in panN-GAL4 and TrpγG4 conditions. e. Representative traces for Trpγ+ neurons expressing GCaMP6s (cyan, n=10) and pan-neuronal expression of GCaMP6s (black, n=10) by wide-field imaging with a ROI encompassing the head. f. Active phase average amplitude for Trpγ+ neurons expressing GCaMP6s (cyan) binned by hour and normalized to pan-neuronal expression of GCaMP6s (black). Shaded areas, SD. g, h. AIP of pupae expressing pan-neuronal GCaMP6s (g). ROIs indicate regions used to calculate traces (h) from optic lobes. Scale bar, 200µm. i. 0-lag correlation between traces in each optic lobe in control (empty-GAL4, black, n=4), and TrpγG4>Kir2.1 (orange, n=4) pupae. Round markers are values from individual time series, bars are averages for each genotype. j. Correlogram between traces in each optic lobe in TrpγG4>Kir2.1 pupa. k. Cell-type-specific Brp puncta counts in control (empty-GAL4>Kir2.1 pupae, black, n=35 per cell type), PanN-GAL4>Kir2.1 pupae (cyan, n=40 for Dm9, n=25 for Tm9) and in TrpγG4>Kir2.1 pupae (orange, n=40 cells for Dm9, n=35 cells for Tm9).
Extended Data Fig. 8 Silencing Trpγ+ neurons in the central brain, but not the optic lobes, attenuates PSINA.
a. Schematic of spatially-targeted Kir2.1 expression. Both experimental genotypes carry two variants of tubP-GAL80: GAL80ts and one of two FLP-responsive conditional alleles. In the optic lobes, ey-FLP either turns on GAL80 expression by removing the interruption cassette (‘-STOP-‘, top) or turns it off by locally excising the FRT-flanked ORF (bottom). Animals are reared at 18oC and shifted 29oC at 40 hAPF to unmask these differential GAL80 expression domains (blue) just prior to PSINA onset; GAL4-driven Kir2.1 expression is disinhibited in the complementary domains (yellow). CB, central brain. OL, optic lobe. b, c. MIPs of half-brains (top) or optic lobes (bottom) at 60 hAPF in which TrpγG4 is driving Kir2.1 expression in the CB (B) or the OL (C). The OL condition also includes expression in the antennal lobes and a small number of CB neurons. Kir2.1 expression domain detected by staining against 3xHA tagged co-cistronic tdTOM. Scale bar, 100µm. d, g. AIP of ~60 hAPF pupa expressing GCaMP6s pan-neuronally. CB (D) and OL (G) ROIs used for measuring PSINA outlined (cyan). Scale bar, 200µm. e, h. PSINA traces from CB (E) and OL (H) for control (no Kir2.1, black, n=5), TrpγG4(CB)>Kir2.1 (red, n=3), and TrpγG4(OL)>Kir2.1 (blue, n=3) genotypes. f, i. Average amplitude measured in CB (F) and OL (G) normalized to corresponding control activity. Shaded areas, SD. Genotypes color-matched to E.
Extended Data Fig. 9 Trpγ+ neurons are necessary for PSINA.
a. Temporal expression control with TARGET; animals shifted from 18oC to 29oC at 40 hAPF. b. PSINA traces from pan-neuronal GCaMP6s in control (empty-GAL4, black, n=3), panN-GAL4>hid, rpr (blue, n=3), and TrpγG4>hid, rpr (orange, n=3) pupae. c. Average amplitude normalized to control activity between 55 and 75 hAPF. Shaded areas, SD. Genotypes color-matched to B. d. PSINA traces from pan-neuronal GCaMP6s in control (empty-GAL4, black, n=7), panN-GAL4>TNT (blue, n=7), and TrpγG4>TNT (orange, n=8) pupae. e. Average amplitude normalized to control activity between 55 and 75 hAPF. Shaded areas, SD. Genotypes color-matched to D. f. Representative traces of pupae expressing pan-neuronal GCaMP6s, with TNT expressed in expression domains of the indicated neuronal class. n=4 tested for each genotype.
Extended Data Fig. 10 Activation of Trpγ+ neurons increases brain-wide activity frequency.
a, b. Left: PSINA traces from pan-neuronal GCaMP6s in control (empty-GAL4, black, n=3), panN-GAL4>TrpA1 (blue, n=3), and TrpγG4>TrpA1 (orange, n=3) pupae at 18oC (A) or 29oC (B). Right: representative auto-correlograms calculated from the first trace shown for each genotype. Inset (B): expanded view of boxed region. Scale bar, 2min. c. Activity from pan-neuronal GCaMP6s in control (empty-GAL4, black, n=6), panN-GAL4>TRpA1 (blue, n=6), VGlut-GAL4>TrpA1 (red, n=6), Gad1-GAL4 (green, n=5), TrpγG4>TrpA1 (orange, n=6) pupae at 60 hAPF. Pupae reared at 18oC and shifted to 29oC. d. MIPs of 60 hAPF brains stained for a nuclear marker (Cherry-NLS, cyan) driven by Gad1- or Vglut-GAL4, and a reference marker (Ncad, magenta). Images derived from three stitched confocal stacks. Scale bar, 100µm. e. Conceptual circuit organizations for coordinating and propagating PSINA. Metronomes indicate CPGs. Colored arrows indicate Trpγ+ and other relay neurons.
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Primers used to amplify the Trpγ coding sequence
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Plasmids used in the generation of SPARC3-Out-GAL80 flies
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gRNA sequences used in pCFD5-U6-3-t-Su(Hw)attP5
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Bajar, B.T., Phi, N.T., Isaacman-Beck, J. et al. A discrete neuronal population coordinates brain-wide developmental activity. Nature 602, 639–646 (2022). https://doi.org/10.1038/s41586-022-04406-9
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