Neuroscience

Neural substrates of cold nociception in Drosophila larva

Atit A. Patel
Albert Cardona
Daniel N. Cox author has email address

Neuroscience Institute, Georgia State University, Atlanta, GA, USA
HHMI Janelia Research Campus, Ashburn, VA, USA
MRC Laboratory of Molecular Biology, Cambridge, UK
Department of Physiology, Development, and Neuroscience, University of Cambridge, UK

https://doi.org/10.7554/eLife.91582.1

Open access
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Figures and data

CIII md neurons are primary cold sensors and share common post-synaptic partners with mechanosensory and nociceptive Ch and CIV md neurons neurons
(A) Sensory neuron second order connectome analyses. Heatmap plot of synaptic connections between sensory neuron subtypes including chordotonal (Ch), class III (CIII) md, class IV (CIV) md neurons and previously published centrally located neurons. Sensory neurons (SN) Ch, CIII md, and CIV md were analyzed. Multisensory integrator (MSI) neurons include Basin-1, -2, -3, and -4. Neurons downstream of MSI (Post-MSI) include A00c, A05q and Goro. Premotor neurons (PMNs) include Chair-1 (A10a), Down and Back (DnB), A02m and A02n. Lastly, sensory neurons are also connected to several projection neurons including A09e, A10j, A09o, TePn04 and TePn05. Synaptic connectivity data was extracted from Neurophyla LMB Cambridge. (https://neurophyla.mrc-lmb.cam.ac.uk/). (B-C) Calcium imaging of sensory neurons including chordotonal (IAV^GAL4), class III md (19-12^GAL4) and class IV md (ppk^GAL4) neurons using CaMPARI2. There were three conditions for sensory neurons: No photoconversion (PC) control (no stimulus and no photoconversion), photoconversion control (photoconversion and no stimulus) and stimulus (Stim) condition (photoconversion and 6°C stimulus). CaMPARI2 data are reported area normalized intensity ratios for F_red/F_green (mean ± SEM). Average N for each cell type and each condition is n=32. (B) CaMPARI2 response measured at the cell body for each neuron type. (C) Sholl intensity analysis performed using custom FIJI scripts. CaMPARI2 response is measured radially away from the center of the soma. (D-E) Cold-evoked responses of third instar Drosophila larva. Sensory neurons Ch, CIII md or CIV md neurons were silenced by inhibiting neurotransmitter release via cell type specific expression of tetanus toxin (TNT). (D) Instantaneous %CT over time. Heatmap on top represents change in temperature over time. (E) Cumulative %CT response for a duration of 5 seconds. Controls include w1118 and Empty^GAL4>TNT. Significant stars: turquoise stars represent comparison to w1118 and purple stars represent comparison to Empty^GAL4>TNT. Average n=72. (F-G) Neural activation of sensory neurons via cell type specific expression of ChETA relative to Empty^GAL4>ChETA control. Empty^GAL4 n=35. Ch n=20, CIV n=20. & CIII n=143. (F) Instantaneous %CT over time. Blue bar represents optogenetic neural activation. (G) Peak %CT. (H-I) Neural co-activation of sensory neurons and CIII md neurons. Here each condition represents expression of ChETA in CIII md neurons and plus Ch (via IAV-GAL4), CIII (via R83B04^GAL4) or CIV md neurons (via ppk^GAL4). (H) Instantaneous %CT over time. Blue bar represents optogenetic neural activation. (I) Peak %CT response during optogenetic stimulation. CIII n=143 and average experimental n=50 Significant differences indicated via asterisks, where *p<0.05, ***p<0.001, and ****p<0.0001.

Multisensory integrator second order neurons are required for cold-evoked behaviors and facilitate CIII-evoked behaviors.
(A) Basins (1-4) receive inputs from sensory neurons (Ch, CIII md and CIV md), Basins, premotor neuron Down and Back (DnB) and projection neuron TePn05. Heatmap plot of pre-synaptic connections to Basins. Synaptic connectivity data was extracted from Neurophyla LMB Cambridge. (https://neurophyla.mrc-lmb.cam.ac.uk/). (B-E) Cold-evoked responses of third instar Drosophila. Basin (1-4) neurons were silenced by inhibiting neurotransmitter release via cell type specific expression of tetanus toxin (TNT), where All Basin’ (R72F11^GAL4), All Basin’’ (R57F07^GAL4), Basin-1 (R20B01^GAL4), Basin-2 (SS00739^splitGAL4) and Basin-4 (SS00740^splitGAL4). (B) Instantaneous %CT over time. Heatmap on top represents change in temperature over time. (C) Cumulative %CT response for a duration of 5 seconds. (D) CT duration in seconds. (E) CT magnitude as average percent change in area for the duration of stimulus. Controls include w1118 and Empty^GAL4>TNT. Significant stars: turquoise stars represent comparison to w1118 and purple stars represent comparison to Empty^GAL4>TNT. Average n = 64. (F-I) Neural co-activation of Basin neurons and CIII md neurons. Here each condition represents expression of ChETA in CIII md neurons and plus Basin (1-4) neurons. (F) Instantaneous %CT over time. Blue bar represents optogenetic neural activation. (G) Peak %CT response during optogenetic stimulation.(H) CT duration in seconds during optogenetic stimulation. (I) CT magnitude as average percent change in area for the duration of stimulus. Significant stars: purple stars represent comparison to CIII md + Empty^GAL4>ChETA. Empty^GAL4 n=143 and experimental condition average n =49. (J) Overall percent change from control for either neural silencing or neural co-activation. The metrics for neural silencing include cumulative %CT, CT magnitude, and CT duration. The following metrics were used to calculate percent for neural co-activation: cumulative %CT, peak %CT, CT duration and magnitude. Significant differences indicated via asterisks, where *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Cold- and CIII-evoked calcium responses of Basin neurons.
(A-E) Ca²⁺ responses of Basin neurons upon cold exposure vs. controls (room temperature). Neural responses (CaMPARI2) of Basin neuron cell bodies were analyzed using the following cell type driver lines (A) All Basin’ (R72F11^GAL4) average n=197, (B) All Basin’’ (R57F07^GAL4) average n = 182, (C) Basin-1 (R20B01^GAL4) average n=27, (D) Basin-2 (SS00739^splitGAL4) average n=119 and (E) Basin-4 (SS00740^splitGAL4) average n=46. CaMPARI2 fluorescence ratio is reported as F_red/F_green. We report the data as individual datapoints, where the red line represents mean, and hybrid plots (boxplot & violin) for visualizing the distribution and quartiles of data. Significant stars represent p<0.05, where comparisons were made to their respective no stimulus controls. (F-I) To functionally assess CIII md neuron to Basin-2 or Basin-4 connectivity, we optogenetically activated CIII md neurons (83B04^lexA>CsChrimson) and visualized changes in evoked Ca²⁺ using Basin-2^splitGAL4or Basin-4^splitGAL4>GCaMP6m. Control: No all trans-retinal (ATR) supplemented diet, which is required for optogenetic stimulation in Drosophila. Orange bars indicate optogenetic stimulation. (F,H) Basin-2 and Basin-4 changes in GCaMP reported as ΔF/F_prestimulus, where prestimulus refers to 15 seconds prior to optogenetic stimulation. (G,I) Maximum Basin-2 and Basin-4 neuronal responses (ΔF/F_prestimulus) upon optogenetic stimulation. Average n for each genotype was 13. Comparisons made to relevant controls and significant differences indicated via asterisks, where *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Projection neurons downstream of Basins and sensory neurons function in cold evoked responses.
(A) A00c and A05q primarily receive inputs from Basins and premotor neuron Down and Back (DnB). Goro neurons primarily receive inputs from A05q neurons. Heatmap plot of pre-synaptic connections to downstream neurons. Synaptic connectivity data was extracted from Neurophyla LMB Cambridge. (https://neurophyla.mrc-lmb.cam.ac.uk/). (B-E) Cold-evoked responses of third instar Drosophila. We used cell-type specific driver lines for downstream neurons to drive expression of tetanus toxin (TNT): A00c (R71A10^GAL4), A05q (R47D0 ^GAL4) and Goro (R69F06^GAL4). (B) Instantaneous %CT over time. Heatmap on top represents change in temperature over time. (C) Cumulative %CT response for a duration of 5 seconds. (D) CT duration in seconds. (E) CT magnitude as average percent change in area for the duration of stimulus. Controls include w1118 and Empty^GAL4>TNT. For each genotype average n=64. Significant stars: turquoise stars represent comparison to w1118 and purple stars represent comparison to Empty^GAL4>TNT. (F-I) Neural co-activation of downstream neurons and CIII md neurons. Here each condition represents expression of ChETA in CIII md neurons plus A00c, A05q or Goro neurons. (F) Instantaneous %CT over time. Blue bar represents optogenetic neural activation. (G) Peak %CT response during optogenetic stimulation. (H) CT duration in seconds during optogenetic stimulation. (I) CT magnitude as average percent change in area for the duration of stimulus. Significant purple stars represent comparison to CIII md + Empty^GAL4>ChETA. Empty^GAL4 n=143 and experimental condition average n =33. (J) Overall percent change from control for either neural silencing or neural co-activation. The metrics for neural silencing include cumulative %CT, CT magnitude, and CT duration. The following metrics were used to calculate percent for neural co-activation: cumulative %CT, peak %CT, CT duration and magnitude. Significant differences indicated via asterisks, where *p<0.05, **p<0.01, and ***p<0.001.

Premotor neurons downstream of sensory neurons and Basin neurons are required for cold-evoked responses.
(A) Chair-1 (A10a), A02m/n (predicted to be mCSI neurons) and Down and Back (DnB, A09l) primarily receive inputs from Basins, CIII md and CIV md neurons. Heatmap plot of pre-synaptic connections to premotor neurons. Synaptic connectivity data was extracted from Neurophyla LMB Cambridge. (https://neurophyla.mrc-lmb.cam.ac.uk/). (B-E) Cold-evoked responses of third instar Drosophila. Premotor neurons were silenced by inhibiting neurotransmitter release via cell type specific expression of tetanus toxin (TNT), where Chair-1 (SS00911^splitGAL4), DnB’ (IT4015^GAL4), DnB’’ (IT412^GAL4) and mCSI (R94B10^GAL4). (B) Instantaneous %CT over time. Heatmap on top represents change in temperature over time. (C) Cumulative %CT response for a duration of 5 seconds. (D) CT duration in seconds. (E) CT magnitude as average percent change in area for the duration of stimulus. Controls include w1118 and Empty^GAL4>TNT. Average n = 69. Significant stars: turquoise stars represent comparison to w1118 and purple stars represent comparison to Empty^GAL4>TNT. (F-I) Neural co-activation of premotor neurons and CIII md neurons. Here each condition represents expression of ChETA in CIII md neurons plus premotor neurons. (F) Instantaneous %CT over time. Blue bar represents optogenetic neural activation. (G) Peak %CT response during optogenetic stimulation. (H) CT duration in seconds during optogenetic stimulation. (I) CT magnitude as average percent change in area for the duration of stimulus. Empty^GAL4 n=143 and experimental condition average n =35. Significant stars represent p<0.05, where purple stars represent comparison to CIII md + Empty^GAL4>ChETA. (J) Overall percent change from control for either neural silencing or neural co-activation. The metrics for neural silencing include cumulative %CT, CT magnitude, and CT duration. The following metrics were used to calculate percent for neural co-activation: cumulative %CT, peak %CT, CT duration and magnitude. Significant differences indicated via asterisks, where *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Cold- and CIII-evoked calcium responses of premotor neurons.
(A-D) Ca²⁺ responses of premotor neurons upon cold exposure vs. controls (room temperature). Neural responses (CaMPARI2) of premotor neuron cell bodies were analyzed using the following cell type driver lines DnB’ (IT4015^GAL4) average n=250, (B) DnB’’ (IT412^GAL4) average n=245, (C) mCSI (R94B10^GAL4) average n=116, and (D) Chair-1 (SS00911^GAL4) DnB’ (IT4015^GAL4) average n=250, (B) DnB’’ (IT412^GAL4) average n=245, (C) mCSI (R94B10^GAL4) average n=116, and (D) Chair-1 (SS00911^splitGAL4) average n=66. CaMPARI2 fluorescence ratio is reported as F_red/F_green. We report the data as individual datapoints, where the red line represents mean, and hybrid plots (boxplot & violin) for visualizing the distribution and quartiles of data. Significant stars represent p<0.05, where comparisons were made to their respective no stimulus controls. (E-F) To functionally assess CIII md neuron to DnB connectivity, we optogenetically activated CIII md neurons (83B04^lexA>CsChrimson) and visualized changes in evoked Ca²⁺ using DnB-GAL4>GCaMP6m. Control: No ATR supplemented diet, which is required for optogenetic stimulation in Drosophila. Orange bars indicate optogenetic stimulation. (E) DnB changes in GCaMP reported as ΔF/F_prestimulus, where prestimulus refers to 15 seconds prior to optogenetic stimulation. (F) Maximum DnB neuronal responses (ΔF/F_prestimulus) upon optogenetic stimulation. Average n=10. Significant differences indicated via asterisks, where *p<0.05, **p<0.01, and ****p<0.0001.

Projection neurons downstream of CIII md neurons are required for cold-evoked responses.
(A) A09e, A09o, A10j, TePn04, TePn05 and A08n primarily receive inputs from CIII md, CIV md, Basin-1, Basin-2 and DnB neurons. Heatmap plot of pre-synaptic connections to projection neurons. Synaptic connectivity data was extracted from Neurophyla LMB Cambridge. (https://neurophyla.mrc-lmb.cam.ac.uk/). (B-E) Cold evoked responses of third instar Drosophila. Projection neurons were silenced by inhibiting neurotransmitter release via cell type specific expression of tetanus toxin (TNT), where A09e (SS00878^splitGAL4), R61A01^GAL4 (labels A09o, A10j, TePn04, and TePn05) and A08n (R82E12^GAL4). (B) Instantaneous %CT over time. Heatmap on top represents change in temperature over time. (C) Cumulative %CT response for a duration of 5 seconds. (D) CT duration in seconds. (E) CT magnitude as average percent change in area for the duration of stimulus. Controls include w1118 and Empty^GAL4>TNT. Average n=73. Significant stars: turquoise stars represent comparison to w1118 and purple stars represent comparison to Empty^GAL4>TNT. (F-I) Neural co-activation of projection neurons and CIII md neurons. Here each condition represents expression of ChETA in CIII md neurons plus projection neurons. (F) Instantaneous %CT over time. Blue bar represents optogenetic neural activation. (G) Peak %CT response during optogenetic stimulation. (H) CT duration in seconds during optogenetic stimulation. (I) CT magnitude as average percent change in area for the duration of stimulus. Empty^GAL4n=143 and experimental condition average n =35. Significant purple stars represent comparison to CIII md + Empty^GAL4>ChETA. (J) Overall percent change from control for either neural silencing or neural co-activation. The metrics for neural silencing include cumulative %CT, CT magnitude, and CT duration. The following metrics were used to calculate percent for neural co-activation: cumulative %CT, peak %CT, CT duration and magnitude. Significant differences indicated via asterisks, where *p<0.05, **p<0.01, and ***p<0.001.

Cold- and CIII-evoked calcium responses of projection neurons.
Neural responses of A09e (SS00878^GAL4) (A-C) and terminally located TePns (−04, -05) were analyzed using R61A01^GAL4(E-G). (A,E) Ca²⁺ responses of projection neurons upon cold exposure vs. controls (room temperature). Cold evoked neural responses (CaMPARI2) of projection neuron cell bodies were analyzed for (A) A09e (n=27) and (E) TePns04, & TePn05 (n=42). CaMPARI2 fluorescence ratio is reported as F_red/F_green. We report the data as individual datapoints, where the red line represents mean, and hybrid plots (boxplot & violin) for visualizing the distribution and quartiles of data. Significant stars represent p<0.05, where comparisons were made to their respective no stimulus controls. (B-C, F-G) To assess, if A09e or TePns functions downstream of CIII md neurons, we optogenetically activated CIII md neurons (83B04^lexA>CsChrimson) and visualized changes in evoked Ca²⁺ using projection neuron specific GAL4>GCaMP6m. Control: No ATR supplemented diet, which is required for optogenetic stimulation in Drosophila. Orange bars indicate optogenetic stimulation. (B, F) Changes in GCaMP reported as ΔF/F_prestimulus, where prestimulus refers to 15 seconds prior to optogenetic stimulation. (F) Maximum neuronal responses (ΔF/F_prestimulus) upon optogenetic stimulation. A09e average n=22. TePns average n=8. Significant differences indicated via asterisks, where *p<0.05, ***p<0.001, and ****p<0.0001.

Dimensional reduction analysis of Drosophila larval behavioral responses and synaptic connectivity informs functional connectivity assessed via Ca²⁺ imaging
(A-B) Instantaneous CT proportions for all genotypes in this study. (A) Neural co-activation experiments, where CIII plus additional neuronal types were simultaneously optogenetically activated. Controls for optogenetic experiments were tested with or without ATR supplement and include the following conditions: background strain (w1118), background strain crossed to UAS-ChETA, and Empty^GAL4 crossed to UAS-ChETA. Blue bar represents optogenetic stimulation. (B) Neurotransmitter release inhibition of individual neuronal types using cell-type specific expression of TnT. (C-D) t-distributed stochastic neighbor embedding (t-SNE) analysis of all neuronal subtypes role in both cold nociception (neural silencing data) and CIII md neuron evoked CT facilitation (co-activation data). (C) 2D plot of t-SNE analysis, where post-hoc clustering analysis based on “Euclidian complete” method revealed 5 unique groups. The following percent change from control (Empty^GAL4) data were included in the analysis: For neural co-activation (peak % CT response, cumulative % CT response, average % change in area, and CT duration) and for neural silencing (cumulative % CT response, average % change in area, and CT duration). (D) Average percent change from control for each cluster in ‘C’ across all neural co-activation or neural silencing metrics. (E-G) Analyses of connectivity upon select circuit components and comparative CIII md neuron evoked calcium responses in post-synaptic neurons. (E) Proportion of synaptic inputs amongst neurons are plotted. A09e neurons integrate responses from multiple pathways originating from CIII md neurons. Network map created using Cytoscape (Shannon et al., 2003) (F-G) CIII md neuron evoked calcium responses in post-synaptic neurons. (F) ΔF/F_baseline over time. (G) Average max ΔF/F_baseline. Averages ± SEM of all trial are plotted. Significant differences indicated via asterisks, where *p<0.05, **p<0.001, and ****p<0.0001.

Drosophila melanogaster strains used in this study

Sensory neuron connectivity matrix.
Synaptic connectivity matrix for neurons whose role in cold nociception was assessed. The number in brackets indicates the total number of neurons analyzed for each cell-type. (A) Absolute number of synaptic connections between pre- and post-synaptic neurons. (B) Connectivity represented as proportion of synaptic input to the post-synaptic neurons.

Stimulus evoked calcium responses of Drosophila larval ventral nerve cord.
Representative images of Drosophila larval in vivo intact animal ventral nerve cord calcium responses assessed via pan-neural CaMPARI expression. Freely moving larvae were exposed innocuous touch, noxious heat (45°C) or noxious cold (6°C) stimulus for 20 seconds and simultaneously exposed to 20 seconds of photo-converting light. Z-stacks of ventral nerve cord are shown with 2µm steps. Briefly, all animals were from the same batch, imaging was conducted using the same settings, and larvae were exposed to same levels of photo-converting light. For better visualization, only photoconverted red CaMPARI fluorescence is reported, and images were pseudo-colored for enhancing signal-to-noise ratio, where highest intensity represented as white and lowest intensity with dark blue. Scale bar represents 50µm.

Somatosensory neural dendritic morphology and representative images of sensory neurons expressing CaMPARI2.
(A) Representative images of somatosensory neurons (Ch, CIII md and CIV md). Note, all images have separate magnification. Each image’s scale bar represents 50µm. Drosophila larva and larval brain graphic was created with BioRender.com. (B) Confocal images of chordotonal (Ch: IAV^GAL4), class III md (CIII: 19-12^GAL4) and class IV md (CIV: ppk^GAL4) neurons expressing CaMPARI2. There were three conditions for sensory neurons: No photoconversion control (no stimulus (Stim) and no photoconversion (PC)), photoconversion control (photoconversion and no stimulus) and stimulus condition (photoconversion and 6°C stimulus). Top row shows merge of F_red and F_green, second row is F_green, third row is F_red and last row contains F_redLUT, which is pseudo-colored with highest intensity being white and lowest intensity being dark blue (color scale bar on bottom right). Cell body and dendrites are outlined in white for Ch and cell body are outline in white for CIII and CIV neurons. Scale bar represents 10µm.

Drosophila larval cold plate assay and quantitative analysis.
(A) A schematic of cold plate assay. Briefly, 3^rd instar Drosophila larvae are plated on a thin metal plate, then we expose the larvae to noxious temperature by transferring the plate onto pre-chilled Peltier cold plate. (B) Behavior videos are automatically processed using custom macros in Fiji, where behavioral videos are cropped, and background is removed to improve quantitative analysis. (C) Larval surface is area is measured using Noldus Ethovision. The following larval cold evoked contraction (CT) behavioral metrics are calculated using r: Instantaneous CT%, cumulative CT%, CT duration and CT magnitude. We define CT behavioral response as a reduction in surface area less than -10%.

Drosophila larval neural activation assays using optogenetics.
To perform neural activation experiments, we created a custom built optogenetic experimental setup, which has a very high spatial resolution and signal to noise ratio by using principles of dark field illumination. (A) Schematic of custom optogenetic rig, where stimulus and video recording are controlled via computer using Noldus Ehtovision. Individual Drosophila larva are plated on clear glass plate and illuminated from below with white light. Neuron activating blue light is also delivered from the below. Individual behavioral videos are automatically cropped and stabilized using custom Fiji macros. (B-C) We measured to two variables Drosophila larval surface area and mobility. (B) Top, image stills from various timepoints before and during optogenetic stimulation of cold sensitive CIII md neurons resulting in contraction (CT) behavioral response. Bottom, percent change in area over time of an individual animal. During baseline, larval locomotion and turns results brief changes in surface area (±2-4%). Whereas upon neural stimulation, there is a distinct lasting reduction in surface area of less than -10%. (C) Larval mobility refers to changes in larval postures as measured by changes in occupied space. Since the amount of larval mobility is a function of various intrinsic and extrinsic factors, we normalized larval mobility to baseline period, where there was no neural activation. Top, conceptual framework of how larval mobility is measured. Original videos are motion stabilized in the XY axis and thresholded. Next, we perform image calculations (Mobility=Frame_{timepoint 2} – Frame_{timepoint 1}) to get just the red shaded portions denoting changes in postural locations (red arrowheads). Drosophila larval time series mobility data are reported as percent change in mobility, time spent being immobile and percent of immobile animals, or for a genotype percent of animals that are immobile over time, immobility is defined as -25% or more reduction in mobility.

Drosophila larval mobility pipeline and effects of neural activation and co-activation of sensory neurons on larval mobility
(A-F) Drosophila larval mobility following somatosensory neuron optogenetic activation (A-C) and CIII md neuron plus co-activation of Ch, additional CIII GAL4 driver (R83B04^GAL4) or CIV md neurons (D-F). (A, D) Instantaneous percent immobility. Blue bar represents optogenetic neural activation. (B, E) Average percent change in mobility for each genotype, where greater percent immobility results in larger changes in average mobility. (C, F) Immobility duration in seconds during stimulation. Neural activation: Empty^GAL4 n=35. Ch n=20, CIV n=20. & CIII n=143. Neural co-activation: CIII n=143 and average experimental n=50.Significant differences indicated via asterisks, where ****p<0.0001.

Neural reconstructions and larval mobility for multisensory integrator neurons.
(A) Individual Basin neuron subtypes (magenta) and CIII md neuron axons (blue). Neural reconstruction data was extracted from Neurophyla LMB Cambridge. (https://neurophyla.mrc-lmb.cam.ac.uk/). (B-D) Drosophila larval mobility for Basin plus CIII md neuron optogenetic co-activation. (B) Instantaneous percent immobility. Blue bar represents optogenetic neural activation. (C) Average percent change in mobility for each genotype, where greater percent immobility results in larger changes in average mobility. (D) Immobility duration in seconds during stimulation. Empty^GAL4 n=143 and experimental condition n =49. Comparisons to CIII md + Empty^GAL4>ChETA. Significant differences indicated via asterisks, where **p<0.01, and ****p<0.0001.

Optogenetic activation of individual neuronal cell-types.
Heatmap represents optogenetically evoked instantaneous contraction (CT) proportions of Drosophila larvae. Individual neuronal cell-types were optogenetically activated using cell-type specific expression of ChETA. Black arrow indicates optogenetic activation of primary cold somatosensory CIII md neurons, which are the only the genotype with high proportions CT. Average n= 31.

Summary of behavioral and functional roles of multisensory integrators in cold nociception.
Blue arrows indicate strength of synaptic connectivity between the CIII md neurons and second order neurons. In behavioral analysis column, the arrow direction indicates reduction or enhancement of CT response and shading indicates magnitude of change from control. In neural activity column: NC denotes no significant change in Ca²⁺ response, the arrow direction indicates reduction or enhancement in evoked Ca²⁺ response and shading indicates magnitude of change from control. Empty spaces indicate experimental analyses were not performed.

Neural reconstructions and larval mobility for A00c, A05q, and Goro neurons.
(A) Individual interneuron subtypes (magenta) and CIII md neuron axons (blue). Neural reconstruction data was extracted from Neurophyla LMB Cambridge. (https://neurophyla.mrc-lmb.cam.ac.uk/). (B-D) Drosophila larval mobility for A00c, A05q or Goro neurons plus CIII md neuron optogenetic coactivation. (B) Instantaneous percent immobility. Blue bar represents optogenetic neural activation. (C) Average percent change in mobility for each genotype, where greater percent immobility results in larger changes in average mobility. (D) Immobility duration in seconds during stimulation. Significant stars represent p<0.05, where stars represent comparison to CIII md + Empty^GAL4>ChETA. Empty^GAL4 n=143 and experimental condition average n =33.

Neural reconstructions and larval mobility for premotor neurons.
(A) Individual premotor neuron subtypes (magenta/green) and CIII md neuronal axons (blue). Neural reconstruction data was extracted from Neurophyla LMB Cambridge. (https://neurophyla.mrc-lmb.cam.ac.uk/). (B-D) Drosophila larval mobility observed with premotor neurons plus CIII md neuron optogenetic coactivation. (B) Instantaneous percent immobility. Blue bar represents optogenetic neural activation. (C) Average percent change in mobility for each genotype, where greater percent immobility results in larger changes in average mobility. (D) Immobility duration in seconds during stimulation. Empty^GAL4 n=143 and experimental condition average n =35. Comparison to CIII md + Empty^GAL4>ChETA. Significant differences indicated via asterisks, where *p<0.05, and ****p<0.0001.

Summary of behavioral and functional roles of premotor neurons in cold nociception.
Blue arrows indicate strength of synaptic connectivity between the CIII md neurons and second order neurons. In behavioral analysis column, the arrow direction indicates reduction or enhancement of CT response and shading indicates magnitude of change from control. In neural activity column: NC denotes no significant change in Ca²⁺ response, the arrow direction indicates reduction or enhancement in evoked Ca²⁺ response and shading indicates magnitude of change from control. Empty spaces indicate experimental analyses were not performed.

Neural reconstructions and larval mobility for projection neurons.
(A) Individual projection neuron subtypes (magenta) and CIII md neuron axons (blue). Neural reconstruction data was extracted from Neurophyla LMB Cambridge. (https://neurophyla.mrc-lmb.cam.ac.uk/). (B-D) Drosophila larval mobility observed with projection neurons plus CIII md neuron optogenetic coactivation. (B) Instantaneous percent immobility. Blue bar represents optogenetic neural activation. (C) Average percent change in mobility for each genotype, where greater percent immobility results in larger changes in average mobility. (D) Immobility duration in seconds during stimulation. Significant stars represent p<0.05, where stars represent comparison to CIII md + Empty^GAL4>ChETA. Empty^GAL4 n=143 and experimental condition average n =35. Significant differences indicated via asterisks, where **p<0.01.

Summary of behavioral and functional roles of projection neurons in cold nociception.
Blue arrows indicate strength of synaptic connectivity between the CIII md neurons and second order neurons. In behavioral analysis column, the arrow direction indicates reduction or enhancement of CT response and shading indicates magnitude of change from control. In neural activity column: NC denotes no significant change in Ca²⁺ response, the arrow direction indicates reduction or enhancement in evoked Ca²⁺ response and shading indicates magnitude of change from control. Empty spaces indicate experimental analyses were not performed.

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