Figure 5

Figure 5

Exposure to copper induces migration of neutrophils. (A-A″) Frontal view of Tg(mpx:GFP);Tg(OMP:RFP) larva with OSNs (red) extending into OB. (A) Control. (A′) At 4 h post-copper exposure, there was an increase in neutrophils (green) in the OO and OSNs degenerated (red, asterisk). (A″) One day post treatment (dp) neutrophils decreased and the OSNs (red) were recovering. (B) Quantification of OSN (red fluorescence) in control (gray), copper-treated animals (red), 1 day (green bar) and 2 days posttreatment (blue bar). All fluorescence was normalized using DAPI (n = 3 larvae, two-way ANOVA, Tukey multiple-comparisons test, ****a = P < 0.0001, b = P < 0.01). All scale bars = 100 μm. (C–C^‴) 5-dpf Tg(mpx: GFP);Tg(OMP:RFP) larva, frontal view. Imaging was initiated at time 0′. At 37′ (C′), larvae were exposed to 10 μM of CuSO₄, and imaging continued at times indicated (see Supplementary Video 1 for sequence taken every minute). Boxed areas: Neutrophils (green, asterisks) associated with olfactory organ (OO, C–C″) and OMP:RFP+ OSNs in olfactory bulb (red, asterisk, C^‴). (C″,C^‴) Arrows in (C^‴) non-local neutrophils that do not enter the OO near the ON (see Supplementary Video 4). (D–D^‴) Individual 2D-cell tracking of neutrophils before, during, and after copper exposure. Each color represents a different neutrophil. (E) Number of neutrophils within the OO before and after copper exposure: analysis of six videos from different animals. Unpaired t-test, P < 0.0001). (F) Speed of neutrophils before and after copper exposure (n = 30 neutrophils; Unpaired t-test, P < 0.05). (G) Chemotactic index of neutrophils before and after copper exposure (n = 20 neutrophils; Unpaired t-test, P < 0.05). (H) Time course of neutrophil movement to the OO (n = 48 larvae. ANOVA, Kruskal–Wallis test, P < 0.0001). Scale bar C–C^‴ = 150 μm. Tracking was done using the ImageJ plugin, MTrackJ.