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Non-invasive in vivo imaging of calcium signaling in mice.

Rogers KL, Picaud S, Roncali E, Boisgard R, Colasante C, Stinnakre J, Tavitian B, Brûlet P - PLoS ONE (2007)

Bottom Line: Rapid and transient elevations of Ca(2+) within cellular microdomains play a critical role in the regulation of many signal transduction pathways.Described here is a genetic approach for non-invasive detection of localized Ca(2+) concentration ([Ca(2+)]) rises in live animals using bioluminescence imaging (BLI).This non-invasive imaging technique opens new avenues for the analysis of Ca(2+) signaling whenever whole body information in freely moving animals is desired, in particular during behavioral and developmental studies.

View Article: PubMed Central - PubMed

Affiliation: Unité d'Embryologie Moléculaire, CNRS URA 2578, Institut Pasteur, Paris, France.

ABSTRACT
Rapid and transient elevations of Ca(2+) within cellular microdomains play a critical role in the regulation of many signal transduction pathways. Described here is a genetic approach for non-invasive detection of localized Ca(2+) concentration ([Ca(2+)]) rises in live animals using bioluminescence imaging (BLI). Transgenic mice conditionally expressing the Ca(2+)-sensitive bioluminescent reporter GFP-aequorin targeted to the mitochondrial matrix were studied in several experimental paradigms. Rapid [Ca(2+)] rises inside the mitochondrial matrix could be readily detected during single-twitch muscle contractions. Whole body patterns of [Ca(2+)] were monitored in freely moving mice and during epileptic seizures. Furthermore, variations in mitochondrial [Ca(2+)] correlated to behavioral components of the sleep/wake cycle were observed during prolonged whole body recordings of newborn mice. This non-invasive imaging technique opens new avenues for the analysis of Ca(2+) signaling whenever whole body information in freely moving animals is desired, in particular during behavioral and developmental studies.

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Distinct mitochondrial Ca2+ responses in newborn mice are linked to different behavioral states.A newborn mouse (P1) was injected (i.p.) with coelenterazine and whole animal bioluminescence was co-registered with the video image with an acquisition rate of 25 Hz using the Video Imager. (A) Graph shown in black is the total light intensity (photons/s) detected during more than 700 s of recording. The thin grey line running through the graph shows the minimum threshold set for scoring Ca2+-responses whose amplitudes reach or go above this limit. The upper blue graph shows the cycling between motion and rest, which was determined by visual analysis of the video frames. Closed black arrow heads show where fast Ca2+-transients (<1 s duration) are correlated to muscular twitches or whole body startles. Sustained Ca2+-responses (>1 s duration) are associated with coordinated movements. The lower grey bar shows periods of baseline Ca2+ activity (below the threshold line set) together with black bars that represent the Ca2+-responses and their durations. (B) Examples of the three behavioral states visually characterized; including (i) whole body startle, (ii) co-ordinated movement and (iii) atonia. Data for each state represents the video image superimposed with the bioluminescence image at the times indicated on each frame and in Figure 5A. (i). Frames represent 40 ms integration of the bioluminescence superimposed to the corresponding video image. Color scale is 0.000725–0.00145 photons/pixel (5 mm smoothing has been applied). (ii & iii) represents 160 ms integration of the bioluminescence superimposed to the corresponding video frame. Color scale is 0.00174–0.00377 photons/pixel (3.5 mm smoothing). The FOV used in these studies was 8×6 cm.
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pone-0000974-g005: Distinct mitochondrial Ca2+ responses in newborn mice are linked to different behavioral states.A newborn mouse (P1) was injected (i.p.) with coelenterazine and whole animal bioluminescence was co-registered with the video image with an acquisition rate of 25 Hz using the Video Imager. (A) Graph shown in black is the total light intensity (photons/s) detected during more than 700 s of recording. The thin grey line running through the graph shows the minimum threshold set for scoring Ca2+-responses whose amplitudes reach or go above this limit. The upper blue graph shows the cycling between motion and rest, which was determined by visual analysis of the video frames. Closed black arrow heads show where fast Ca2+-transients (<1 s duration) are correlated to muscular twitches or whole body startles. Sustained Ca2+-responses (>1 s duration) are associated with coordinated movements. The lower grey bar shows periods of baseline Ca2+ activity (below the threshold line set) together with black bars that represent the Ca2+-responses and their durations. (B) Examples of the three behavioral states visually characterized; including (i) whole body startle, (ii) co-ordinated movement and (iii) atonia. Data for each state represents the video image superimposed with the bioluminescence image at the times indicated on each frame and in Figure 5A. (i). Frames represent 40 ms integration of the bioluminescence superimposed to the corresponding video image. Color scale is 0.000725–0.00145 photons/pixel (5 mm smoothing has been applied). (ii & iii) represents 160 ms integration of the bioluminescence superimposed to the corresponding video frame. Color scale is 0.00174–0.00377 photons/pixel (3.5 mm smoothing). The FOV used in these studies was 8×6 cm.

Mentions: In subsequent studies, [Ca2+]m responses associated with movement were recorded in non-anaesthetised and unrestrained freely moving CLZN-injected newborn mice (Figure 5A, (n = 6)) using an imaging system that allows co-registration of the video and bioluminescent images. Three major behavioral states, corresponding to the known components of sleep/wake cycles, were identified: (i) whole body startles and myoclonic twitches (see Movie S2 for an example), (ii) coordinated movements (see Movie S3 for an example) and (iii) atonia (Figure 5B). In sleep/wake cycles of newborn rats or mice, myoclonic twitching is the most reliable way to identify the presence of active sleep [31], [32]. In line with the data from contraction/relaxation cycles of the hindlimb muscle, whole body startles or muscular twitches identified in the video recordings were correlated to fast Ca2+-transients having short durations (<1 s) (Figure 5A & 5B (i)). In contrast, coordinated movements were made up of sustained Ca2+ rises occurring across large scale areas of the body (Figure 5A & B (ii)).


Non-invasive in vivo imaging of calcium signaling in mice.

Rogers KL, Picaud S, Roncali E, Boisgard R, Colasante C, Stinnakre J, Tavitian B, Brûlet P - PLoS ONE (2007)

Distinct mitochondrial Ca2+ responses in newborn mice are linked to different behavioral states.A newborn mouse (P1) was injected (i.p.) with coelenterazine and whole animal bioluminescence was co-registered with the video image with an acquisition rate of 25 Hz using the Video Imager. (A) Graph shown in black is the total light intensity (photons/s) detected during more than 700 s of recording. The thin grey line running through the graph shows the minimum threshold set for scoring Ca2+-responses whose amplitudes reach or go above this limit. The upper blue graph shows the cycling between motion and rest, which was determined by visual analysis of the video frames. Closed black arrow heads show where fast Ca2+-transients (<1 s duration) are correlated to muscular twitches or whole body startles. Sustained Ca2+-responses (>1 s duration) are associated with coordinated movements. The lower grey bar shows periods of baseline Ca2+ activity (below the threshold line set) together with black bars that represent the Ca2+-responses and their durations. (B) Examples of the three behavioral states visually characterized; including (i) whole body startle, (ii) co-ordinated movement and (iii) atonia. Data for each state represents the video image superimposed with the bioluminescence image at the times indicated on each frame and in Figure 5A. (i). Frames represent 40 ms integration of the bioluminescence superimposed to the corresponding video image. Color scale is 0.000725–0.00145 photons/pixel (5 mm smoothing has been applied). (ii & iii) represents 160 ms integration of the bioluminescence superimposed to the corresponding video frame. Color scale is 0.00174–0.00377 photons/pixel (3.5 mm smoothing). The FOV used in these studies was 8×6 cm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1991622&req=5

pone-0000974-g005: Distinct mitochondrial Ca2+ responses in newborn mice are linked to different behavioral states.A newborn mouse (P1) was injected (i.p.) with coelenterazine and whole animal bioluminescence was co-registered with the video image with an acquisition rate of 25 Hz using the Video Imager. (A) Graph shown in black is the total light intensity (photons/s) detected during more than 700 s of recording. The thin grey line running through the graph shows the minimum threshold set for scoring Ca2+-responses whose amplitudes reach or go above this limit. The upper blue graph shows the cycling between motion and rest, which was determined by visual analysis of the video frames. Closed black arrow heads show where fast Ca2+-transients (<1 s duration) are correlated to muscular twitches or whole body startles. Sustained Ca2+-responses (>1 s duration) are associated with coordinated movements. The lower grey bar shows periods of baseline Ca2+ activity (below the threshold line set) together with black bars that represent the Ca2+-responses and their durations. (B) Examples of the three behavioral states visually characterized; including (i) whole body startle, (ii) co-ordinated movement and (iii) atonia. Data for each state represents the video image superimposed with the bioluminescence image at the times indicated on each frame and in Figure 5A. (i). Frames represent 40 ms integration of the bioluminescence superimposed to the corresponding video image. Color scale is 0.000725–0.00145 photons/pixel (5 mm smoothing has been applied). (ii & iii) represents 160 ms integration of the bioluminescence superimposed to the corresponding video frame. Color scale is 0.00174–0.00377 photons/pixel (3.5 mm smoothing). The FOV used in these studies was 8×6 cm.
Mentions: In subsequent studies, [Ca2+]m responses associated with movement were recorded in non-anaesthetised and unrestrained freely moving CLZN-injected newborn mice (Figure 5A, (n = 6)) using an imaging system that allows co-registration of the video and bioluminescent images. Three major behavioral states, corresponding to the known components of sleep/wake cycles, were identified: (i) whole body startles and myoclonic twitches (see Movie S2 for an example), (ii) coordinated movements (see Movie S3 for an example) and (iii) atonia (Figure 5B). In sleep/wake cycles of newborn rats or mice, myoclonic twitching is the most reliable way to identify the presence of active sleep [31], [32]. In line with the data from contraction/relaxation cycles of the hindlimb muscle, whole body startles or muscular twitches identified in the video recordings were correlated to fast Ca2+-transients having short durations (<1 s) (Figure 5A & 5B (i)). In contrast, coordinated movements were made up of sustained Ca2+ rises occurring across large scale areas of the body (Figure 5A & B (ii)).

Bottom Line: Rapid and transient elevations of Ca(2+) within cellular microdomains play a critical role in the regulation of many signal transduction pathways.Described here is a genetic approach for non-invasive detection of localized Ca(2+) concentration ([Ca(2+)]) rises in live animals using bioluminescence imaging (BLI).This non-invasive imaging technique opens new avenues for the analysis of Ca(2+) signaling whenever whole body information in freely moving animals is desired, in particular during behavioral and developmental studies.

View Article: PubMed Central - PubMed

Affiliation: Unité d'Embryologie Moléculaire, CNRS URA 2578, Institut Pasteur, Paris, France.

ABSTRACT
Rapid and transient elevations of Ca(2+) within cellular microdomains play a critical role in the regulation of many signal transduction pathways. Described here is a genetic approach for non-invasive detection of localized Ca(2+) concentration ([Ca(2+)]) rises in live animals using bioluminescence imaging (BLI). Transgenic mice conditionally expressing the Ca(2+)-sensitive bioluminescent reporter GFP-aequorin targeted to the mitochondrial matrix were studied in several experimental paradigms. Rapid [Ca(2+)] rises inside the mitochondrial matrix could be readily detected during single-twitch muscle contractions. Whole body patterns of [Ca(2+)] were monitored in freely moving mice and during epileptic seizures. Furthermore, variations in mitochondrial [Ca(2+)] correlated to behavioral components of the sleep/wake cycle were observed during prolonged whole body recordings of newborn mice. This non-invasive imaging technique opens new avenues for the analysis of Ca(2+) signaling whenever whole body information in freely moving animals is desired, in particular during behavioral and developmental studies.

Show MeSH
Related in: MedlinePlus