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Oral dosing of chemical indicators for in vivo monitoring of Ca2+ dynamics in insect muscle.

- PLoS ONE (2015)

Bottom Line: Advances in circuit miniaturization and insect neuromuscular physiology have enabled the hybridization of living insects and man-made electronic components, such as microcomputers, the result of which has been often referred as a Living Machine, Biohybrid, or Cyborg Insect.We found that there was a positive relationship between the fluorescence intensity of the indicator and the frequency of electrical stimulation which indicates the orally dosed indicator successfully monitored Ca2+ dynamics in the muscle tissue.This oral dosing method has a potential to globally stain tissues including neurons, and investigating various physiological events in insects.

View Article: PubMed Central - PubMed

Affiliation: School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
This paper proposes a remarkably facile staining protocol to visually investigate dynamic physiological events in insect tissues. We attempted to monitor Ca2+ dynamics during contraction of electrically stimulated living muscle. Advances in circuit miniaturization and insect neuromuscular physiology have enabled the hybridization of living insects and man-made electronic components, such as microcomputers, the result of which has been often referred as a Living Machine, Biohybrid, or Cyborg Insect. In order for Cyborg Insects to be of practical use, electrical stimulation parameters need to be optimized to induce desired muscle response (motor action) and minimize the damage in the muscle due to the electrical stimuli. Staining tissues and organs as well as measuring the dynamics of chemicals of interest in muscle should be conducted to quantitatively and systematically evaluate the effect of various stimulation parameters on the muscle response. However, existing staining processes require invasive surgery and/or arduous procedures using genetically encoded sensors. In this study, we developed a non-invasive and remarkably facile method for staining, in which chemical indicators can be orally administered (oral dosing). A chemical Ca2+ indicator was orally introduced into an insect of interest via food containing the chemical indicator and the indicator diffused from the insect digestion system to the target muscle tissue. We found that there was a positive relationship between the fluorescence intensity of the indicator and the frequency of electrical stimulation which indicates the orally dosed indicator successfully monitored Ca2+ dynamics in the muscle tissue. This oral dosing method has a potential to globally stain tissues including neurons, and investigating various physiological events in insects.

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Relationship of Ca2+ dynamics with electrical stimulation frequency.Relative changes in fluorescence intensity ((ΔF/F0)×100%) for leg muscle of (A) beetle orally dosed with Fluo-8 (blue) and Cell Tracker (red) and (B) control beetle measured with the filter setting used for Fluo-8 (blue) and Cell Tracker (red) under varying electrical stimulations (1 Hz, 10 Hz, 50 Hz, and 100 Hz; 10% duty cycle; 2 V). Data were analyzed from the ROI adjacent to the stimulated site (S1B Fig.). The error bars represent the S.D. (N = 8 beetles, n = 24 beetle legs for (A); N = 2 beetles, n = 8 beetle legs for (B)). The small numbers next to each plot indicate the order of stimulation. Cell Tracker data set was compared with Fluo-8 data set at each stimulation frequency evaluated by student’s t-test both for dosed beetles in (A) (1st 50 Hz, p = 9.37×10-4; 10 Hz, p = 7.45×10-3; 1st 1 Hz, p = 2.30×10-1; 100 Hz, p = 4.17×10-4; 2nd 1 Hz, p = 4.49×10-2; and 2nd 50 Hz, p = 8.16×10-3) and for control beetles in (B) (1st 50 Hz, p = 4.10×10-1; 10 Hz, p = 9.30×10-1; 1st 1 Hz, p = 9.29×10-1; 100 Hz, p = 5.69×10-2; 2nd 1 Hz, p = 6.85×10-1; and 2nd 50 Hz, p = 2.67×10-2). The significant differences are displayed by an asterisk (p < 0.05). Fluo-8 intensity dynamics show that Ca2+ dynamics inside the muscle have a positive correlation with electrical stimulation frequency; i.e., higher stimulation frequency induces larger increase in [Ca2+]. On the other hand, Cell Tracker intensity dynamics show that the frequency-dependent intensity change due to muscle displacement is not apparent.
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pone.0116655.g005: Relationship of Ca2+ dynamics with electrical stimulation frequency.Relative changes in fluorescence intensity ((ΔF/F0)×100%) for leg muscle of (A) beetle orally dosed with Fluo-8 (blue) and Cell Tracker (red) and (B) control beetle measured with the filter setting used for Fluo-8 (blue) and Cell Tracker (red) under varying electrical stimulations (1 Hz, 10 Hz, 50 Hz, and 100 Hz; 10% duty cycle; 2 V). Data were analyzed from the ROI adjacent to the stimulated site (S1B Fig.). The error bars represent the S.D. (N = 8 beetles, n = 24 beetle legs for (A); N = 2 beetles, n = 8 beetle legs for (B)). The small numbers next to each plot indicate the order of stimulation. Cell Tracker data set was compared with Fluo-8 data set at each stimulation frequency evaluated by student’s t-test both for dosed beetles in (A) (1st 50 Hz, p = 9.37×10-4; 10 Hz, p = 7.45×10-3; 1st 1 Hz, p = 2.30×10-1; 100 Hz, p = 4.17×10-4; 2nd 1 Hz, p = 4.49×10-2; and 2nd 50 Hz, p = 8.16×10-3) and for control beetles in (B) (1st 50 Hz, p = 4.10×10-1; 10 Hz, p = 9.30×10-1; 1st 1 Hz, p = 9.29×10-1; 100 Hz, p = 5.69×10-2; 2nd 1 Hz, p = 6.85×10-1; and 2nd 50 Hz, p = 2.67×10-2). The significant differences are displayed by an asterisk (p < 0.05). Fluo-8 intensity dynamics show that Ca2+ dynamics inside the muscle have a positive correlation with electrical stimulation frequency; i.e., higher stimulation frequency induces larger increase in [Ca2+]. On the other hand, Cell Tracker intensity dynamics show that the frequency-dependent intensity change due to muscle displacement is not apparent.

Mentions: Increase in the fluorescence intensity of Fluo-8 when induced by electrical stimulation was enhanced with increased stimulus frequency, while there was less significant change in that of Cell Tracker, as shown in Figs.4 and 5. The contraction of most muscles is regulated by the rate of physiological neural signal input to the muscle: higher neural input induces larger muscle contraction (tonic contraction) [45, 46]. Similarly, a higher frequency of externally applied electrical stimulus to living muscle induces larger muscle contraction (summation or facilitation) [47–49]. These two types of contraction-enhancement are both due to the elevation of [Ca2+] in muscle cells [19, 50]. Thus, we compared the dependency of fluorescence intensity changes (ΔF/F0 defined as below) of Fluo-8 and Cell Tracker on stimulation frequency in order to examine the effect of electrical stimulus on intracellular Ca2+ dynamics for each beetle leg. ΔF was defined by (F1 —F0), where F1 is the average fluorescence intensity during stimulation and F0 is that of 10 frames before beginning electrical stimulation, both measured from the ROI adjacent to the stimulated sites (electrode implanted sites) as shown in S1B Fig. To compare ΔF of these different chemical indicators, we evaluated the relative changes in ΔF, defined as ΔF/F0, at each stimulation frequency. The ΔF/F0 was then plotted with respect to applied stimulus frequencies in Fig. 4E. Note that the ΔF/F0 of Cell Tracker in some cases displays negative values; for example, during the 100 Hz stimulation and the second 1 Hz stimulation in Fig. 4E, indicating that the muscle displacement in some cases caused decreased intensity.


Oral dosing of chemical indicators for in vivo monitoring of Ca2+ dynamics in insect muscle.

- PLoS ONE (2015)

Relationship of Ca2+ dynamics with electrical stimulation frequency.Relative changes in fluorescence intensity ((ΔF/F0)×100%) for leg muscle of (A) beetle orally dosed with Fluo-8 (blue) and Cell Tracker (red) and (B) control beetle measured with the filter setting used for Fluo-8 (blue) and Cell Tracker (red) under varying electrical stimulations (1 Hz, 10 Hz, 50 Hz, and 100 Hz; 10% duty cycle; 2 V). Data were analyzed from the ROI adjacent to the stimulated site (S1B Fig.). The error bars represent the S.D. (N = 8 beetles, n = 24 beetle legs for (A); N = 2 beetles, n = 8 beetle legs for (B)). The small numbers next to each plot indicate the order of stimulation. Cell Tracker data set was compared with Fluo-8 data set at each stimulation frequency evaluated by student’s t-test both for dosed beetles in (A) (1st 50 Hz, p = 9.37×10-4; 10 Hz, p = 7.45×10-3; 1st 1 Hz, p = 2.30×10-1; 100 Hz, p = 4.17×10-4; 2nd 1 Hz, p = 4.49×10-2; and 2nd 50 Hz, p = 8.16×10-3) and for control beetles in (B) (1st 50 Hz, p = 4.10×10-1; 10 Hz, p = 9.30×10-1; 1st 1 Hz, p = 9.29×10-1; 100 Hz, p = 5.69×10-2; 2nd 1 Hz, p = 6.85×10-1; and 2nd 50 Hz, p = 2.67×10-2). The significant differences are displayed by an asterisk (p < 0.05). Fluo-8 intensity dynamics show that Ca2+ dynamics inside the muscle have a positive correlation with electrical stimulation frequency; i.e., higher stimulation frequency induces larger increase in [Ca2+]. On the other hand, Cell Tracker intensity dynamics show that the frequency-dependent intensity change due to muscle displacement is not apparent.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4295878&req=5

pone.0116655.g005: Relationship of Ca2+ dynamics with electrical stimulation frequency.Relative changes in fluorescence intensity ((ΔF/F0)×100%) for leg muscle of (A) beetle orally dosed with Fluo-8 (blue) and Cell Tracker (red) and (B) control beetle measured with the filter setting used for Fluo-8 (blue) and Cell Tracker (red) under varying electrical stimulations (1 Hz, 10 Hz, 50 Hz, and 100 Hz; 10% duty cycle; 2 V). Data were analyzed from the ROI adjacent to the stimulated site (S1B Fig.). The error bars represent the S.D. (N = 8 beetles, n = 24 beetle legs for (A); N = 2 beetles, n = 8 beetle legs for (B)). The small numbers next to each plot indicate the order of stimulation. Cell Tracker data set was compared with Fluo-8 data set at each stimulation frequency evaluated by student’s t-test both for dosed beetles in (A) (1st 50 Hz, p = 9.37×10-4; 10 Hz, p = 7.45×10-3; 1st 1 Hz, p = 2.30×10-1; 100 Hz, p = 4.17×10-4; 2nd 1 Hz, p = 4.49×10-2; and 2nd 50 Hz, p = 8.16×10-3) and for control beetles in (B) (1st 50 Hz, p = 4.10×10-1; 10 Hz, p = 9.30×10-1; 1st 1 Hz, p = 9.29×10-1; 100 Hz, p = 5.69×10-2; 2nd 1 Hz, p = 6.85×10-1; and 2nd 50 Hz, p = 2.67×10-2). The significant differences are displayed by an asterisk (p < 0.05). Fluo-8 intensity dynamics show that Ca2+ dynamics inside the muscle have a positive correlation with electrical stimulation frequency; i.e., higher stimulation frequency induces larger increase in [Ca2+]. On the other hand, Cell Tracker intensity dynamics show that the frequency-dependent intensity change due to muscle displacement is not apparent.
Mentions: Increase in the fluorescence intensity of Fluo-8 when induced by electrical stimulation was enhanced with increased stimulus frequency, while there was less significant change in that of Cell Tracker, as shown in Figs.4 and 5. The contraction of most muscles is regulated by the rate of physiological neural signal input to the muscle: higher neural input induces larger muscle contraction (tonic contraction) [45, 46]. Similarly, a higher frequency of externally applied electrical stimulus to living muscle induces larger muscle contraction (summation or facilitation) [47–49]. These two types of contraction-enhancement are both due to the elevation of [Ca2+] in muscle cells [19, 50]. Thus, we compared the dependency of fluorescence intensity changes (ΔF/F0 defined as below) of Fluo-8 and Cell Tracker on stimulation frequency in order to examine the effect of electrical stimulus on intracellular Ca2+ dynamics for each beetle leg. ΔF was defined by (F1 —F0), where F1 is the average fluorescence intensity during stimulation and F0 is that of 10 frames before beginning electrical stimulation, both measured from the ROI adjacent to the stimulated sites (electrode implanted sites) as shown in S1B Fig. To compare ΔF of these different chemical indicators, we evaluated the relative changes in ΔF, defined as ΔF/F0, at each stimulation frequency. The ΔF/F0 was then plotted with respect to applied stimulus frequencies in Fig. 4E. Note that the ΔF/F0 of Cell Tracker in some cases displays negative values; for example, during the 100 Hz stimulation and the second 1 Hz stimulation in Fig. 4E, indicating that the muscle displacement in some cases caused decreased intensity.

Bottom Line: Advances in circuit miniaturization and insect neuromuscular physiology have enabled the hybridization of living insects and man-made electronic components, such as microcomputers, the result of which has been often referred as a Living Machine, Biohybrid, or Cyborg Insect.We found that there was a positive relationship between the fluorescence intensity of the indicator and the frequency of electrical stimulation which indicates the orally dosed indicator successfully monitored Ca2+ dynamics in the muscle tissue.This oral dosing method has a potential to globally stain tissues including neurons, and investigating various physiological events in insects.

View Article: PubMed Central - PubMed

Affiliation: School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
This paper proposes a remarkably facile staining protocol to visually investigate dynamic physiological events in insect tissues. We attempted to monitor Ca2+ dynamics during contraction of electrically stimulated living muscle. Advances in circuit miniaturization and insect neuromuscular physiology have enabled the hybridization of living insects and man-made electronic components, such as microcomputers, the result of which has been often referred as a Living Machine, Biohybrid, or Cyborg Insect. In order for Cyborg Insects to be of practical use, electrical stimulation parameters need to be optimized to induce desired muscle response (motor action) and minimize the damage in the muscle due to the electrical stimuli. Staining tissues and organs as well as measuring the dynamics of chemicals of interest in muscle should be conducted to quantitatively and systematically evaluate the effect of various stimulation parameters on the muscle response. However, existing staining processes require invasive surgery and/or arduous procedures using genetically encoded sensors. In this study, we developed a non-invasive and remarkably facile method for staining, in which chemical indicators can be orally administered (oral dosing). A chemical Ca2+ indicator was orally introduced into an insect of interest via food containing the chemical indicator and the indicator diffused from the insect digestion system to the target muscle tissue. We found that there was a positive relationship between the fluorescence intensity of the indicator and the frequency of electrical stimulation which indicates the orally dosed indicator successfully monitored Ca2+ dynamics in the muscle tissue. This oral dosing method has a potential to globally stain tissues including neurons, and investigating various physiological events in insects.

Show MeSH
Related in: MedlinePlus