mg 2i  (Med Chem 101)

Journal: Science Advances

Article Title: Functional interrogation of neuronal connections by chemoptogenetic presynaptic ablation

doi: 10.1126/sciadv.aeb6755

Figure Lengend Snippet: ( A ) Transgenic y252-Gal4, UAS:dL5-mCer larva at 3 dpf. Merged fluorescent and visible light exposures. ( B ) Maximum projection of confocal images of y252-Gal4, UAS:dL5-mCer 6 dpf larvae. Untreated (left), MG-2I only, light only, and combined light and MG-2I as indicated. Puncta in ablated larvae are cellular debris. Scale bar, 100 μm. (Ba) A square ROI was selected from each panel in (B) and is presented as higher-magnification images in (Ba). Scale bar, 20 μm. ( C ) Cerulean fluorescence intensity (a.u., arbitrary units) in y252-Gal4, UAS:dL5-mCer immediately before and after photoablation and at the indicated time points. Points show values from individual larvae normalized to nonfluorescent sibling larvae. h, hours. ( D ) Maximum projection (D) and matched single horizontal confocal slices (Da) through part of the diencephalon of y252:Gal4, UAS:dL5-mCer, vglut2a:DsRed larvae immediately before exposure to NIR light (top panels) and 24 hours later (bottom panels). The region in (Da) is rotated from the boxed area in (D). Scale bars, 20 μm.

Article Snippet: For dL5 ablations, we used a stock solution of MG-2I (gift from M. Bruchez, Carnegie Mellon University) or custom-synthesized MG-2I ( www.medchem101.com ) that was dissolved at 1 mM in ethanol and stored in aliquots at −20°C.

Techniques: Transgenic Assay, Fluorescence

Journal: Science Advances

Article Title: Functional interrogation of neuronal connections by chemoptogenetic presynaptic ablation

doi: 10.1126/sciadv.aeb6755

Figure Lengend Snippet: ( A ) Patterned illumination from a DMD used to selectively illuminate part of the y252-Gal4 expression pattern. Larvae were tested for behavior 48 hours after MG-2I treatment and photoablation. ( B to D ) Confocal 40-μm substack maximum projections showing dL5-mCer expression and differential interference contrast background for 5-dpf untreated y252-Gal4, UAS:dL5-mCer larva (B) and larva 48 hours after selective illumination of R1 (C) or R4 (D) neurons. Scale bar, 100 μm. ( E and F ) SLC and LLC escape behavior and PPI in y252-Gal4, UAS:dL5-mCer larvae after selective photoablation of R1 neurons (E) or R4 neurons (F). MWU, significant P values indicated. The increase in PPI in (E) was not present in replication experiment [analysis of variance (ANOVA), main effect treatment F 1,54 = 2.9, P = 0.094 for combined dataset]. In (E), n = 24 and 25 for ctl (dL5 − /Mg2I + ) and abl (dL5 + /Mg2I + ) for SLC and LLC; n = 22 and 17 for PPI. In (F), n = 27 and 23 for SLC; n = 22 and 21 for LLC; n = 16 and 17 for PPI.

Article Snippet: For dL5 ablations, we used a stock solution of MG-2I (gift from M. Bruchez, Carnegie Mellon University) or custom-synthesized MG-2I ( www.medchem101.com ) that was dissolved at 1 mM in ethanol and stored in aliquots at −20°C.

Techniques: Expressing

Journal: Science Advances

Article Title: Functional interrogation of neuronal connections by chemoptogenetic presynaptic ablation

doi: 10.1126/sciadv.aeb6755

Figure Lengend Snippet: ( A ) Schematic of the construct used to make the UAS:syp-dL5-mNeonGreen transgenic line. ( B ) Horizontal confocal section through the spinal cord of 3-dpf y417-Gal4, UAS:syp-dL5-mNeonGreen embryo (green), stained with anti-sv2 (magenta). Scale bar, 20 μm. Arrows show synaptic puncta from y417 neurons. “A” and “D” denote anterior-dorsal orientation. The boxed region is shown at a higher magnification in the right panel. Scale bar, 5 μm. ( C and D ) Maximum projection from the confocal z -stack of GFP and differential interference contrast channels in 4 dpf y252-Gal4, UAS:syp-dL5-mNeonGreen larvae: untreated control larva (C) and an MG-2I–treated larva exposed for 20 min to wide-field illumination at 3 dpf (D). Scale bar, 100 μm. A square ROI was cropped from each panel in (C) and (D) and is shown at a higher magnification in (Ca) and (Da). ( E ) SLC [ n = 25 and 25 for ctl (dL5 − /Mg2I + ) and abl (dL5 + /Mg2I + )] and LLC ( n = 25 and 20) escape responsiveness and PPI ( n = 14 and 16) in control and wide-field light–exposed y252-Gal4, UAS:syp-dL5-mNeonGreen larvae. MWU, significant P value indicated and * P < 0.001. ( F ) Maximum projection of 80-μm confocal stacks from 4-dpf tph2:Gal4, UAS:syp-dL5, UAS:Kaede Red untreated larvae and in larvae exposed to NIR illumination in the indicated region of the left hindbrain at 3 dpf. Scale bars, 100 μm. Ra, raphe neurons. ( G ) Number of neurons in dorsal raphe 24 hours after photoablation of neuropil in the region indicated in (F) in untreated [ctl (dL5 − /Mg2I + ), n = 9] and photoablated larvae [abl (dL5 + /Mg2I + ), n = 8]. Welch t test, P = 0.50.

Article Snippet: For dL5 ablations, we used a stock solution of MG-2I (gift from M. Bruchez, Carnegie Mellon University) or custom-synthesized MG-2I ( www.medchem101.com ) that was dissolved at 1 mM in ethanol and stored in aliquots at −20°C.

Techniques: Construct, Transgenic Assay, Staining, Control