Endocrinol Metab.  2015 Sep;30(3):361-370. 10.3803/EnM.2015.30.3.361.

Effect of Mefloquine, a Gap Junction Blocker, on Circadian Period2 Gene Oscillation in the Mouse Suprachiasmatic Nucleus Ex Vivo

• Koo J
• Choe HK
• Kim HD
• Chun SK
• Son GH
• Kim K

• KMID: 2407088
• DOI: http://doi.org/10.3803/EnM.2015.30.3.361

Abstract

BACKGROUND
In mammals, the master circadian pacemaker is localized in an area of the ventral hypothalamus known as the suprachiasmatic nucleus (SCN). Previous studies have shown that pacemaker neurons in the SCN are highly coupled to one another, and this coupling is crucial for intrinsic self-sustainability of the SCN central clock, which is distinguished from peripheral oscillators. One plausible mechanism underlying the intercellular communication may involve direct electrical connections mediated by gap junctions.
METHODS
We examined the effect of mefloquine, a neuronal gap junction blocker, on circadian Period 2 (Per2) gene oscillation in SCN slice cultures prepared from Per2::luciferase (PER2::LUC) knock-in mice using a real-time bioluminescence measurement system.
RESULTS
Administration of mefloquine causes instability in the pulse period and a slight reduction of amplitude in cyclic PER2::LUC expression. Blockade of gap junctions uncouples PER2::LUC-expressing cells, in terms of phase transition, which weakens synchrony among individual cellular rhythms.
CONCLUSION
These findings suggest that neuronal gap junctions play an important role in synchronizing the central pacemaker neurons and contribute to the distinct self-sustainability of the SCN master clock.

Keyword

Circadian rhythm; Suprachiasmatic nucleus; Gap junctions; Per2; Real-time bioluminescence; Mefloquine

MeSH Terms

Animals
Circadian Rhythm
Electrical Synapses
Gap Junctions*
Hypothalamus
Luminescent Measurements
Mammals
Mefloquine*
Mice*
Neurons
Phase Transition
Suprachiasmatic Nucleus*
Mefloquine

Figure

• 

  Fig. 1 Real-time analyses of circadian Per2::luciferase (PER2::LUC) expression in suprachiasmatic nucleus (SCN) and peripheral tissues explants ex vivo. (A) A representative circadian profile of PER2::LUC expression in the SCN explant culture. Daily periods were calculated and are expressed during 8-day and cultivation periods at the top. (B) Circadian profiles of PER2::LUC expression in peripheral tissues ex vivo. Results from the liver, kidney, lung, heart, pituitary, and adrenal glands are shown. Peripheral tissues were synchronized by a brief application of dexamethasone (DEX, 1 µM) for 2 hours (arrows).

• 

  Fig. 2 Chronic treatment with mefloquine attenuates the circadian rhythm of Per2::luciferase (PER2::LUC) expression in the suprachiasmatic nucleus. Representative bioluminescence recordings are shown in (A) vehicle (0.1% dimethyl sulfoxide), (B) 3 and (C) 10 µM mefloquine-treated groups. Mefloquine was applied directly into the recording medium at a trough after the 3rd or 4th peak. After four more cycles passed, the medium was changed. Daily periods are calculated and expressed during 11- or 12-day cultivation periods at the top of each figure.

• 

  Fig. 3 Effect of mefloquine on Per2::luciferase (PER2::LUC) cycling activities in the suprachiasmatic nucleus. Mean periods (A), period stability (B, SD of mean periods in a cell), and changes in amplitude (C, % of pretreatment) were statistically analyzed among pre- (before treatment), treat- (during treatment), and post- (after washout) periods in three groups (vehicle [n=8], 3 µM [n=6], and 10 µM [n=6] mefloquine) on the rhythmic gene expression of Per2. Data are expressed as mean±standard error of the mean. aP<0.01 vs. pretreatment.

• 

  Fig. 4 Bioluminescence-based real-time imaging of Per2::luciferase (PER2::LUC) reporter activity in the presence of mefloquine. (A) Representative image of the PER2::LUC-expressing suprachiasmatic nucleus (SCN) taken with an intensified CCD camera in the presence of perfused recording medium containing 0.3 mmol/L D-luciferin. (B) Bioluminescence traces measured from a whole PER2::LUC SCN culture. After the 3rd peak of the rhythm, 10 µM mefloquine was applied during the trough. The black bar indicates the period of chronic treatment with 10 µM mefloquine. (C) Time-lapse images show circadian fluctuations of PER2::LUC expression in the SCN culture. Each image was taken by integrating the bioluminescence for 10 minutes. The images were aligned from every six images (every hour) for 7 days.

• 

  Fig. 5 Attenuated circadian Per2::luciferase (PER2::LUC) expression and synchrony among individual suprachiasmatic nucleus (SCN) cells. (A) The video-micrograph illustrates 80 randomly chosen individual cells in the SCN (left panel). The right panel displays the bioluminescence recordings from individual SCN cells. The black bar indicates the chronic treatment with 10 µM mefloquine. (B) Bar charts summarizing the effects of mefloquine at the single-cell level. Mean periods among examined cells, period stability (SD of mean periods in a cell), and changes in amplitude (% of pretreatment) are expressed as the mean±standard error of the mean. (C) A raster plot presentation displaying voxels measured dorsal to ventral throughout the SCN. Red corresponds to the peak of bioluminescence and green to the trough. (D) Phase differences in peak time of each cell compared to the mean value. The X axis represents the order of peaks (seven total peaks are shown). The Y axis represents differences in peak time (hour) of individual cells, shown dotted. SD indicates index variations. aP<0.01 vs. pretreatment.

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