Research ArticleOPTOGENETICS

Optogenetic pacing in Drosophila melanogaster

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Science Advances  09 Oct 2015:
Vol. 1, no. 9, e1500639
DOI: 10.1126/sciadv.1500639
  • Fig. 1 Optogenetic pacing of the Drosophila heart.

    (A) Schematic of the integrated OCM imaging and pacing system. The optogenetic excitation beam was coupled with the sample arm of the spectral domain OCM system using a dichroic beam splitter. (B) Comparison of cardiac-specific mCherry fluorescence expression between ChR2-expressing transgenic flies (24B-GAL4;UAS-H134R-ChR2) and control flies (24B-GAL4/+). (C) M-mode images showing optogenetic pacing in ChR2 and control adult flies. The ChR2 fly heart with an RHR of 6 Hz was successfully paced at three different frequencies: 8, 9, and 10 Hz. In comparison, the control fly heart was not responsive to optical pacing stimulations.

  • Fig. 2 Characterization of cardiac optogenetic pacing.

    (A and B) Different stimulation power levels (A) and pulse widths (B) were used to determine optimal stimulation parameters to achieve successful pacing at different developmental stages (n = 10). On the basis of acquired data, the MLE to achieve successful pacing at different power levels and pulse widths was determined. Results are presented as mean ± SEM. (C) HR measurements in early pupae continuously paced for 1 hour at 3.5 Hz (n = 10). About 50% of early pupae successfully followed the pacing frequency for 1 hour. Results are presented as mean ± SD. (D) No significant differences in the HR of adult flies were observed between flies that were paced for 1 hour during the early pupa stage and control flies (n = 10). The average HR for paced and control flies was 404 ± 23.9 and 386 ± 73.4 beats/min, respectively (P = 0.52). Results are presented as mean ± SD. (E) TEM images of adult fly heart obtained from around the A1 segment of paced and control flies. TEM images show a normal myofibril structure and continuous Z discs in both groups. Results in (D) and (E) demonstrate that optogenetic pacing was safe and that pacing for 1 hour during the early pupa stage did not adversely affect heart development in Drosophila.

  • Fig. 3 Optogenetic pacing of the Drosophila heart at different development stages.

    (A, D, and G) Schematic showing pacing stimulation location and spot size used at the larva, pupa, and adult stages. (B, E, and H) M-mode images showing optogenetic pacing at the larva, pupa, and adult stages. The 1:1 correspondence between pacing pulse and heart contraction can be clearly observed. (C, F, and I) Frequency tuning curves showing the reliable range for optogenetic pacing at different stages (n = 6 at each stage). HR was tuned around the average RHR at the respective stages. In larvae, the fly heart linearly followed the pacing frequency from 4 to 5.2 Hz, where the average RHR was 4.1 Hz. In early pupae, the average RHR was 2.5 Hz, and the heart linearly followed the pacing frequency in the range between 2.8 and 4 Hz. During the adult stage (with a higher average RHR of 6.4 Hz), HR and pacing frequency showed a linear relationship between 7 and 8.6 Hz. The fly heart reliably followed the pacing frequency up to ~25, ~50, and ~35% above the RHR at the larva, pupa, and adult stages, respectively. Results are presented as mean ± SD.

  • Fig. 4 Analysis of cardiac dynamics at different developmental stages.

    (A) M-mode image showing that pacing pulses incident during RP could not initiate a heart contraction (pulses marked with X). (B) The probability that each stimulation pulse would initiate a heart contraction was characterized at different time delays with respect to previously completed systole. RP was estimated as the time delay required after the preceding systole for achieving a 90% probability of successful pacing. It was shortest at the adult stage, intermediate at the larva stage, and longest at the pupa stage. (C) Comparison of heart CT at different developmental stages. CT had a similar trend as RP: shortest at the adult stage, intermediate at the larva stage, and slowest at the pupa stage.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/9/e1500639/DC1

    Fig. S1. Cardiac stimulation of control flies (24B-GAL4/+) at different developmental stages.

    Fig. S2. Estimation of PE at different pacing frequencies.

    Fig. S3. Histogram analysis of RP at different developmental stages.

    Video S1. Tuning the HR of a Drosophila larva at 4, 5, and 6 Hz.

    Video S2. Tuning the HR of a Drosophila early pupa at 3, 3.5, and 4 Hz.

    Video S3. Tuning the HR of an adult Drosophila at 8, 9, and 10 Hz.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Cardiac stimulation of control flies (24B-GAL4/+) at different developmental stages.
    • Fig. S2. Estimation of PE at different pacing frequencies.
    • Fig. S3. Histogram analysis of RP at different developmental stages.
    • Legends for videos S1 to S3

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    Other Supplementary Material for this manuscript includes the following:

    • Video S1 (.mov format). Tuning the HR of a Drosophila larva at 4, 5, and 6 Hz.
    • Video S2 (.mov format). Tuning the HR of a Drosophila early pupa at 3, 3.5, and 4 Hz.
    • Video S3 (.mov format). Tuning the HR of an adult Drosophila at 8, 9, and 10 Hz.

    Files in this Data Supplement:

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