nc82 rrid ab 2314866  (Developmental Studies Hybridoma Bank)

Journal: eLife

Article Title: Inhibitory circuits control leg movements during Drosophila grooming

doi: 10.7554/eLife.106446

Figure Lengend Snippet: Schematic showing segmental distribution of 13 A (green) and 13B (cyan) neurons across pro-, meta-, and meso-thoracic segments ( T1, T2, T3 ) of VNC. Confocal image: Six GABAergic 13 A neurons (green arrowheads) and six 13B neurons (cyan arrowheads) in each VNC hemisegment, labeled with GFP (green) driven by R35G04-GAL4-DBD, GAD-GAL4-AD . Neuropil in magenta (nc82). Panel B’ provides a zoomed-in view of T1 region. EM reconstructions: 62 13 A neurons (green) and 64 13B neurons (cyan) in right T1. Ventral side up. ( A ) Continuous activation of 13 A and 13B neurons labeled by R35G04-GAL4-DBD, GAD-GAL4-AD in dusted flies reduces front leg rubbing and head sweeps and induces unusual leg extensions. Control: AD-DBD EMPTY SPLIT >UAS CsChrimson (gray ). Experiment: R35G04-GAL4-DBD, GAD-GAL4-AD>UAS CsChrimson (red ). Box plots indicate the percentage of time dusted fly engaged in a given behavior over a 4-min assay (n=7). The solid blue line marks the mean, dark shading the 95% confidence interval, red dashed line the median, and light shading ± 1 standard deviation. *** p ≤0.001, * p ≤0.05 . ( E-F ) Continuous activation of 13 A and 13B neuron subsets induces front leg extension in headless flies. ( E, E′ ) Representative video frames showing headless flies (dusted and undusted) with extended front legs (orange arrowhead) following continuous optogenetic activation of neurons labeled with R35G04-GAL4-DBD, GAD-GAL4-AD>UAS- CsChrimson . Dashed box in E highlights the front legs; schematic illustrates the extended posture. ( F ) Quantification of leg extension phenotypes in dusted and undusted headless flies. Bar plots show the percentage of flies displaying leg extension (red) or a normal posture (gray). Percentages are calculated as the number of flies showing each posture divided by the total number of flies per condition. Dusted: n=9; undusted: n = 5. ( G–H ) Silencing 13 A and 13B neuron subsets locks front legs in flexion in headless flies. ( G, G′ ) Representative video frames showing dusted and undusted headless flies with sustained front leg flexion following silencing of neurons labeled with R35G04-GAL4-DBD, GAD-GAL4-AD>UAS TNTe. Blue arrowheads indicate the flexed posture. ( H ) Quantification of leg flexion phenotypes in dusted and undusted headless flies. Bar plots show the percentage of flies displaying sustained flexion (red). All flies (100%) in both dusted (n=13) and undusted (n=9) conditions showed the phenotype. Also see .

Article Snippet: Primary antibodies used were Chicken pAb anti-GFP (Abcam, 1:1000), Rabbit (Rb) anti- GFP (Abcam, 1:1000), mouse (ms) anti-Neuroglian (BP104) (DSHB, 1:40), ms monoclonal anti-Brp antibody (nC82) (DSHB, 1:200).

Techniques: Labeling, Activation Assay, Control, Standard Deviation

Journal: eLife

Article Title: Inhibitory circuits control leg movements during Drosophila grooming

doi: 10.7554/eLife.106446

Figure Lengend Snippet: Confocal image showing two Dbx-positive 13 A neurons/hemisegment labeled by GFP driven by R11C07-DBD, Dbx-AD Split GAL4 in the adult VNC, labeled by GFP (green). nC82 (magenta) labels synaptic neuropil. ( B–G ) Effects of manipulating activity of two 13 A neurons: Silencing and activation experiments in dusted flies using R11C07-DBD, Dbx-AD Split Gal4>UAS GTACR1 and UAS CsChrimson , respectively. Control conditions include AD-DBD Empty Split with UAS GTACR1 for inactivation and with UAS CsChrimson for activation. 13 A inactivation (n=11), activation (n=4). Bar plots in each panel compare control (blue) and experimental (orange) groups. Each dot represents the mean feature value for a single fly. Bars indicate the group mean, and whiskers represent the 95% confidence interval of the group mean. p-Values (raw and FDR–corrected) are shown above each panel. ( C-D’ ) Contour plots (probability distribution of joint positions) of the front legs during grooming actions. Joint positions are significantly altered upon silencing ( C’ ) and activation of 13 A neurons ( D’ ) in dust-covered flies. Joint positions are shown during head sweeps ( C-D’ ). ( E,E’ ) Median frequency (Hz) of the proximal and medial joints decreases upon silencing of two 13 A neurons. ( F ) Maximum angular velocity (° /s*10 [-2] ) of the proximal joint does not significantly reduce upon silencing of two 13 A neurons.

Article Snippet: Primary antibodies used were Chicken pAb anti-GFP (Abcam, 1:1000), Rabbit (Rb) anti- GFP (Abcam, 1:1000), mouse (ms) anti-Neuroglian (BP104) (DSHB, 1:40), ms monoclonal anti-Brp antibody (nC82) (DSHB, 1:200).

Techniques: Labeling, Activity Assay, Activation Assay, Control

Journal: eLife

Article Title: Inhibitory circuits control leg movements during Drosophila grooming

doi: 10.7554/eLife.106446

Figure Lengend Snippet: ( A-A” ) Intra-joint coordination and muscle synergies . Angular velocities of proximal (P, blue) and medial (M, cyan) joints predominantly move synchronously, while distal (D, purple) can move in or out of phase during leg rubbing. The schematic (right) indicates the corresponding joint angles. ( A’-A” ) The proximal and medial joint movements within a leg occur effectively in phase, with a mean lag of ~0.8 frames (8ms) during leg rubbing (A′) and during head grooming sweeps (A″). Bar plots show the lag; each dot indicates one animal. Frame = 10ms. Neuronal labeling of 13A and 13B neurons. Top: Confocal image of six Dbx positive 13 A neurons per hemisegment labeled by GFP using R35G04-GAL4-DBD, Dbx-GAL4-AD in VNC. Neuroglian (magenta) labels axon bundles. Bottom: Confocal image of three 13B neurons per hemisegment labeled by GFP using R11B07-GAL4-DBD, GAD-GAL4-AD . Nc82 (magenta) labels neuropil. ( C–I ) Effects of neuronal activity manipulation in dusted flies. Silencing and activation of 13 A neurons in dusted flies using R35G04-GAL4-DBD, Dbx-GAL4-AD with UAS Kir or UAS CsChrimson , respectively (n=12 silencing, n=19 activation). Control: AD-GAL4-DBD EMPTY SPLIT with UAS Kir or UAS CsChrimson . For 13B neurons, R11B07-GAL4-DBD, GAD-GAL4-AD with UAS GtACR1 , or UAS CsChrimson, respectively (n=7 silencing, n=9 activation); control: AD-GAL4-DBD EMPTY SPLIT with UAS GtACR1 or UAS CsChrimson . Each panel compares control (blue) and experimental (orange) groups. Each dot represents the mean feature value for a single fly. Bars indicate the group mean, and whiskers represent the 95% confidence interval of the group mean. P -values (raw and false discovery rate [FDR]–corrected) are shown above each panel. ( C–D ) Proximal inter-leg distance : Silencing of 13 A ( C ) or 13B ( D ) neurons during head grooming reduces the distance between the femur-tibia joints of the left and right front legs. ( E–I ) Frequency modulation : Silencing 13 A or 13B neurons reduces mean frequency of proximal joint oscillations in dusted flies. ( F, G ). Activation of 13 A neurons reduced frequency, although this change did not survive FDR correction. However, continuous activation of 13 A and 13B neurons increased variability in frequency. ( H, I ). Mean of the per-animal standard deviation (STD) that reflects variability or spread of data is shown.

Article Snippet: Primary antibodies used were Chicken pAb anti-GFP (Abcam, 1:1000), Rabbit (Rb) anti- GFP (Abcam, 1:1000), mouse (ms) anti-Neuroglian (BP104) (DSHB, 1:40), ms monoclonal anti-Brp antibody (nC82) (DSHB, 1:200).

Techniques: Labeling, Activity Assay, Activation Assay, Control, Standard Deviation