Article Figures & Data
Figures
- Figure 1.
The GF axonal conduction velocity increases 80% during postnatal maturation. A, GFS schematic depiction at 1hPE (Ai) and 24hPE (Aii) control flies. Average latency ± SEM for GF-DLM5–6, GF-TTM, MN5-DLM5–6, and TTMn-TTM branches are indicated between dotted lines. Lower boxes on GF indicate the latency in the GF axon. Upper boxes on GF indicates the GF axonal speed. The percentage of the GF speeding (80%) during the first day of the fly life is presented in the gray-black arrow. B, Ten overlapping sweeps of APs recorded from DLM5–6 muscles, after GF (Bi) or MN5 (Bii) stimulation, and from TTM muscle, after GF (Biii) or TTMn (Biv) stimulation, at 1hPE (gray) and 24hPE (black) flies. Times above double arrows indicate the latency, which is measured as the interval of time between the stimulus artifact (black arrow) and the onset of the initial phase of muscle potential (white arrow). Underlined time values show the difference in latency measurements between the two stages. C, Latency measurements in the GF-DLM5–6 and GF-TTM pathways (Ci), in the MN5-DLM5–6 and TTMn-TTM sub-pathways (Cii), in the GF axon (Ciii), and measurements of the GF axonal speed (Civ), at 1hPE (50% black), 12hPE (70% black), and 24hPE or 48hPE (black) flies. Underlined time values between dotted arrows indicate the latency difference between1hPE and 24hPE. Data are shown as means ± SEM (Ci–Ciii). Dots on box plots showcase the measurements from individual flies (Civ). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., p > 0.19). St, stimulation site; Re, recording site; S, stimulus; R, record.
- Figure 2.
The GF axon diameter does not increase postnatally, and GFP-RNAi decreases GFP fluorescence similarly at 1 h and 24 h post-eclosion. A, Representative confocal image stacks of the GF interneuron axons between the neck connectives and the terminals in the VNC at 1hPE (A) and at 24hPE (B). To avoid potential histology artifacts images were taken live in saline with a 60× water dipping lens from freshly dissected animals with UAS-cd4-tomato expression in the GF. White boxes indicate areas shown as selective enlargements in Ai, Bi. C, Quantification from 14 axons at each stage shows that GF axon diameter is similar at 1hPE and 24hPE (p > 0.6, Student’s t test). VNC, ventral nerve cord. D–G, In comparison to control (D), targeted GFP RNAi knock-down reduces GFP fluorescence at both stages tested, 24hPE (E) and 1hPE (F). G, Quantification of mean gray levels in eight bit tiff images of the GF axon (0–254 gray levels) reveals a significant reduction by ∼70% but no differences between both stages tested (ANOVA with Newman–Keuls post hoc testing); ***p < 0.001; n.s., p > 0.3.
- Figure 3.
Para-RNAi decreases postnatal conduction velocity speeding in the GF. A, GF schematic depiction at 1hPE (gray) and 24hPE (black) in controls (Ai) as compared to 1hPE (orange) and 24hPE (red) in flies with para RNAi (Aii). GF axonal conduction velocity (dotted arrow) is given for each experimental group. The percentage of the GF postnatal speeding in control (gray-black arrow, 80%) is strongly reduced by para RNAi knock-down (orange-red arrow, 47%). B, Representative TTM muscle APs recorded after GF (Bi) or TTMn (Bii) stimulation at 1hPE (gray) and at 24hPE (black) in control flies as compared to para RNAi knock-down (Biii, GF stimulation; Biv, TTMn stimulation) at 1hPE (orange) and at 24hPE (red). Times above double arrows and between dotted lines indicate the latency between GF (or TTMn) and TTM. Latency differences between stages are underlined for control and for para RNAi knock-down. C, Latency measurements in the GF-TTM pathway (Ci), in the TTMn-TTM sub-pathway (Cii), in the GF axon (Ciii) and measurements of the GF axonal speed (Civ), at 1hPE (gray) and 24hPE (black) in controls and at 1hPE (orange) and 24hPE (red) in flies expressing para RNAi transgene. Underlined times between dotted arrows indicate the latency difference between the two stages in control and para RNAi knock-down. Data are shown as means ± SEM (Ci–Ciii). Dots on box plots showcase the measurements from individual flies (Civ). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (*p < 0.05, ***p < 0.001, ****p < 0.0001, n.s., p > 0.29).
- Figure 4.
Expression of extra sodium channels (NaChBac) increases postnatal conduction velocity speeding in the GF. A, GF schematic depiction at 1hPE (gray) and 24hPE (black) in controls (Ai) as compared to 1hPE (light orange) and 24hPE (neon red) in flies expressing NaChBac channels (Aii). GF axonal conduction velocity (dotted arrow) is given for each experimental group. The percentage of the GF postnatal speeding in control (gray-black arrow, 80%) is strongly increased by NaChBac channels expression (light orange-neon red, 119%). B, Representative TTM muscle APs recorded after GF (Bi) or TTMn (Bii) stimulation at 1hPE (gray) and at 24hPE (black) in control flies as compared to flies expressing NaChBac channels (Biii, GF stimulation; Biv, TTMn stimulation) at 1hPE (light orange) and at 24hPE (neon red). Times above double arrows and between dotted lines indicate the latency between GF (or TTMn) and TTM. Latency differences between stages are underlined for control and for NaChBac channels expression. C, Latency measurements in the GF-TTM pathway (Ci), in the TTMn-TTM sub-pathway (Cii), in the GF axon (Ciii) and measurements of the GF axonal speed (Civ), at 1hPE (gray) and 24hPE (black) in controls and at 1hPE (light orange) and 24hPE (neon red) in flies expressing NaChBac transgene. Underlined times between dotted arrows indicate the latency difference between the two stages in control and flies expressing NaChBac sodium channels. Data are shown as means ± SEM (Ci–Ciii). Dots on box plots showcase the measurements from individual flies (Civ). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (**p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., p > 0.15).
- Figure 5.
DmCa1D-RNAi almost eliminates postnatal conduction velocity speeding in the GF. A, GF schematic depiction at 1hPE (gray) and 24hPE (black) in controls (Ai) as compared to 1hPE (cyan) and 24hPE (blue) in flies with DmCa1D RNAi (Aii). GF axonal conduction velocity (dotted arrow) is given for each experimental group. The percentage of the GF postnatal speeding in control (gray-black arrow, 80%) is extremely reduced by DmCa1D RNAi knock-down (cyan-blue arrow, 15%). B, Representative TTM muscle APs recorded after GF (Bi) or TTMn (Bii) stimulation at 1hPE (gray) and at 24hPE (black) in control flies as compared to DmCa1D RNAi knock-down (Biii, GF stimulation; Biv, TTMn stimulation) at 1hPE (cyan) and at 24hPE (blue). Times above double arrows and between dotted lines indicate the latency between GF (or TTMn) and TTM. Latency differences between stages are underlined for control and for DmCa1D RNAi knock-down. C, Latency measurements in the GF-TTM pathway (Ci), in the TTMn-TTM sub-pathway (Cii), in the GF axon (Ciii) and measurements of the GF axonal speed (Civ), at 1hPE (gray) and 24hPE (black) in controls and at 1hPE (cyan) and 24hPE (blue) in flies expressing DmCa1D RNAi transgene. Underlined time values between dotted arrows indicate the latency difference between the two stages in control and DmCa1D RNAi knock-down. Data are shown as means ± SEM (Ci–Ciii). Dots on box plots showcase the measurements from individual flies (Civ). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., p > 0.05).
- Figure 6.
Shaker channels are localized in the GF axon and Sh-RNAi decreases postnatal conduction velocity speeding. A–Aii, Projection views of a representative confocal image stack from the GF with UAS-cd4-tomato expression (red) and Shaker channel expression as visualized by an endogenous GFP tag (green). Dotted white boxes in Ai indicate the areas which are selectively enlarged in B, C and shown as z-projections of 1 µm (three optical sections). White arrowheads demark areas with overlap of patches of GF axonal membrane (red) and Shaker-GFP label. The GF axonal lumen is mostly devoid of Shaker-GFP label. D, GF schematic depiction at 1hPE (gray) and 24hPE (black) in controls (Di) as compared to 1hPE (violet) and 24hPE (purple) in flies with Sh RNAi (Dii). GF axonal conduction velocity (dotted arrow) is given for each experimental group. The percentage of the GF postnatal speeding in control (gray-black arrow, 80%) is strongly reduced by Sh RNAi knock-down (violet-purple arrow, 36%). E, Representative TTM muscle APs recorded after GF (Ei) or TTMn (Eii) stimulation at 1hPE (gray) and at 24hPE (black) in control flies as compared to Sh RNAi knock-down (Eiii, GF stimulation; Eiv, TTMn stimulation) at 1hPE (violet) and at 24hPE (purple). Times above double arrows and between dotted lines indicate the latency between GF (or TTMn) and TTM. Latency differences between stages are underlined for control and for Sh RNAi knock-down. F, Latency measurements in the GF-TTM pathway (Fi), in the TTMn-TTM sub-pathway (Fii), in the GF axon (Fiii), and measurements of the GF axonal speed (Fiv), at 1hPE (gray) and 24hPE (black) in controls and at 1hPE (violet) and 24hPE (purple) in flies expressing Sh RNAi. Underlined times between dotted arrows indicate the latency difference between the two stages in control and Sh RNAi knock-down. Data are shown as means ± SEM (Fi–Fiii). Dots on box plots showcase the measurements from individual flies (Fiv). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (*p < 0.05, ***p < 0.001, ****p < 0.0001, n.s., p > 0.15).
- Figure 7.
Shal-RNAi does not affect postnatal conduction velocity speeding in the GF. A, GF schematic depiction at 1hPE (gray) and 24hPE (black) in controls (Ai) as compared to 1hPE (violet) and 24hPE (purple) in flies with Shal RNAi (Aii). GF axonal conduction velocity (dotted arrow) is given for each experimental group. The percentage of the GF postnatal speeding in control (gray-black arrow, 80%) is not significantly reduced by Shal RNAi knock-down (violet-purple arrow, 67%). B, Representative TTM muscle APs recorded after GF (Bi) or TTMn (Bii) stimulation at 1hPE (gray) and at 24hPE (black) in control flies as compared to Shal RNAi knock-down (Biii, GF stimulation; Biv, TTMn stimulation) at 1hPE (violet) and at 24hPE (purple). Times above double arrows and between dotted lines indicate the latency between GF (or TTMn) and TTM. Latency differences between stages are underlined for control and for Shal RNAi knock-down. C, Latency measurements in the GF-TTM pathway (Ci), in the TTMn-TTM sub-pathway (Cii), in the GF axon (Ciii) and measurements of the GF axonal speed (Civ), at 1hPE (gray) and 24hPE (black) in controls and at 1hPE (violet) and 24hPE (purple) in flies expressing Shal RNAi transgene. Underlined time values between dotted arrows indicate the latency difference between the two stages in control and Shal RNAi knock-down. Data are shown as means ± SEM (Ci–Ciii). Dots on box plots showcase the measurements from individual flies (Civ). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (*p < 0.05, ***p < 0.001, ****p < 0.0001, n.s., p > 0.09).
- Figure 8.
Slo-RNAi affects slightly postnatal conduction velocity speeding in the GF. A, GF schematic depiction at 1hPE (gray) and 24hPE (black) in controls (Ai) as compared to 1hPE (mint green) and 24hPE (green) in flies with Slo RNAi (Aii). GF axonal conduction velocity (dotted arrow) is given for each experimental group. The percentage of the GF postnatal speeding in control (gray-black arrow, 80%) is moderately reduced by Slo RNAi knock-down (mint green-green arrow, 56%). B, Representative TTM muscle APs recorded after GF (Bi) or TTMn (Bii) stimulation at 1hPE (gray) and at 24hPE (black) in control flies as compared to Slo RNAi knock-down (Biii, GF stimulation; Biv, TTMn stimulation) at 1hPE (mint green) and at 24hPE (green). Times above double arrows and between dotted lines indicate the latency between GF (or TTMn) and TTM. Latency differences between stages are underlined for control and for Slo RNAi knock-down. C, Latency measurements in the GF-TTM pathway (Ci), in the TTMn-TTM sub-pathway (Cii), in the GF axon (Ciii) and measurements of the GF axonal speed (Civ), at 1hPE (gray) and 24hPE (black) in controls and at 1hPE (mint green) and 24hPE (green) in flies expressing Slo RNAi transgene. Underlined time values between dotted arrows indicate the latency difference between the two stages in control and Slo RNAi knock-down. Data are shown as means ± SEM (Ci–Ciii). Dots on box plots showcase the measurements from individual flies (Civ). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (**p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., p > 0.1).
Tables
- Table 1.
Latency measurements in the GF-TTM pathways, in the TTMn-TTM Sub-pathways, and in the GF axon at 1hPE (gray) and 24hPE (black) control as compared to 1hPE and 24hPE in flies where genes encoding ion channels were expressed or knocked down specifically in the GF interneurons
GF-TTM; mean ± SEM (n) TTMn-TTM; mean ± SEM (n) GF=(GF-TTM)-(TTMn-TTM); mean ± SEM (n) Genotype 1hPE 24hPE 1hPE 24hPE 1hPE 24hPE Control 1.32 ± 0.02 ms
(7)1.13 ± 0.02 ms
(9)0.80 ± 0.04 ms
(5)0.84 ± 0.02 ms
(8)0.52 ± 0.04 ms
(7)0.29 ± 0.03 ms
(9)Para-RNAi 1.52 ± 0.03 ms
(8)1.29 ± 0.03 ms
(8)0.83 ± 0.03 ms
(5)0.82 ± 0.03 ms
(5)0.69 ± 0.04 ms
(8)0.47 ± 0.03 ms
(8)NaChBac 1.21 ± 0.02 ms
(9)1.02 ± 0.01 ms
(8)0.86 ± 0.02 ms
(6)0.86 ± 0.02 ms
(7)0.35 ± 0.02 ms
(9)0.16 ± 0.03 ms
(8)DmCa1D-RNAi 1.59 ± 0.03 ms
(9)1.48 ± 0.02 ms
(9)0.86 ± 0.02 ms
(5)0.84 ± 0.02 ms
(6)0.73 ± 0.05 ms
(9)0.64 ± 0.04 ms
(9)Sh-RNAi 1.43 ± 0.04 ms
(8)1.25 ± 0.02 ms
(8)0.87 ± 0.03 ms
(5)0.84 ± 0.03 ms
(5)0.56 ± 0.05 ms
(8)0.41 ± 0.04 ms
(8)Shal-RNAi 1.42 ± 0.03 ms
(9)1.17 ± 0.02 ms
(8)0.87 ± 0.02 ms
(6)0.84 ± 0.03 ms
(7)0.55 ± 0.03 ms
(9)0.33 ± 0.03 ms
(8)Slo-RNAi 1.58 ± 0.04 ms
(9)1.31 ± 0.02 ms
(10)0.87 ± 0.01 ms
(5)0.86 ± 0.02 ms
(7)0.71 ± 0.04 ms
(9)0.45 ± 0.03 ms
(10)The latency in the GF axon is estimated by subtracting the TTMn-TTM latency from the GF-TTM latency. Data are shown as means ± SEM; n, number of preparation tested.
- Table 2.
% Increase (+) or decrease (–) in GF axonal conduction speed due to targeted expression or knock-down of genes encoding ion channels in the GF interneurons at 1hPE (gray) and 24hPE (black) flies, or during the first day of the fly life (1–24hPE)
% Change due to “knock-down” or “expression” of genes encoding ionic channels % Change due to postnatal maturation Genotype 1hPE 24hPE 1–24hPE Control ------- ------ 80%*** Para-RNAi –32%* –62%*** 47%*** NaChBac +49%*** +81%** 119%**** DmCa1D-RNAi –40%** –120%**** 15%; ns Sh-RNAi –7%; ns –42%* 36%*** Shal-RNAi –6%; ns –14%; ns 67%*** Slo-RNAi –35%** –56%** 56%**** Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., p > 0.05).
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