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A method for measuring electrical signals in a primary cilium.

Kleene NK, Kleene SJ - Cilia (2012)

Bottom Line: In 47% of attempts, suction resulted in a seal with high input resistance.In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer and Cell Biology, University of Cincinnati, PO Box 670521, Cincinnati, OH 45267-0521, USA.

ABSTRACT

Background: Most cells in the body possess a single primary cilium. These cilia are key transducers of sensory stimuli, and defects in cilia have been linked to several diseases. Evidence suggests that some transduction of sensory stimuli by the primary cilium depends on ion-conducting channels. However, the tiny size of the cilium has been a critical barrier to understanding its electrical properties. We report a novel method that allows sensitive, repeatable electrical recordings from primary cilia. Adherent cells were grown on small, spherical beads that could be easily moved within the recording chamber. In this configuration, an entire cilium could be pulled into a recording microelectrode.

Results: In 47% of attempts, suction resulted in a seal with high input resistance. Single channels could be recorded while the cilium remained attached to the cell. When the pipette was raised into the air, the cell body was pulled off at the air-bath interface. The pipette retained the cilium and could then be immersed in various solutions that bathed the cytoplasmic face of the membrane. In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.

Conclusions: Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

No MeSH data available.


Related in: MedlinePlus

Photomicrograph of the method for electrical recording from an isolated primary cilium. A glass-coated bead is covered with a confluent layer of mIMCD-3 cells. The yellow line represents the boundary between the bead and the cells. One primary cilium of one cell is visible entering the tip of the recording pipette. Bar = 10 μm.
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Figure 2: Photomicrograph of the method for electrical recording from an isolated primary cilium. A glass-coated bead is covered with a confluent layer of mIMCD-3 cells. The yellow line represents the boundary between the bead and the cells. One primary cilium of one cell is visible entering the tip of the recording pipette. Bar = 10 μm.

Mentions: Images for Figure1A-D were acquired on an inverted, confocal microscope (LSM 710, Karl Zeiss AG, Oberkochen, Germany) and exported as TIFF files using Zen 2009 Light Edition (Zeiss). Figure1A-C were acquired with a 40×/0.75 NA, Plan-Neofluar, phase-contrast objective, 1400 pixels ×1400 pixels, 97 nm × 97 nm × 1,000 nm voxel dimensions, and 8-bit depth. The laser wavelength was 488 nm. For Figure1A,B, the transmission channel was used. For Figure1C, the dichroic mirror was set for 488 nm and the emission filter was 535 to 759 nm. Figure1D was acquired with a 63×/1.20 NA, C-Apochromat, water-immersion objective, 1,188 pixels × 1,188 pixels, 114 nm × 114 nm × 900 nm voxel dimensions, with 8-bit depth. The laser wavelength was 488 nm, the dichroic mirror was set for 488 nm, and the emission filter was 493 to 630 nm. Figure1E was acquired with a Nikon D70 single-lens reflex camera. For Figure1, Adobe Photoshop 6.0 was used for converting to grayscale, cropping, resizing, and adding labels. Photoshop was also used to adjust brightness and contrast, including gamma, via ‘Input Levels’. Figure1A-C were resampled from 97 to 114 nm/pixel X-Y resolution to match the resolution of Figure1D so that the same scale bar could be used for all four images. Inserts for Figure1B-D were resampled to match the greater resolution of the main figures. Figure1E was resampled to match the greater resolution of Figure1A-D. The videos in Additional file 1 were acquired with a camera (WV-CD50, Panasonic, Osaka, Japan), 1× TV relay lens (Nikon), and a digital video recorder (RDR-VX530, Sony, Tokyo, Japan). The videos were processed in mencoder (http://www.mplayerhq.hu) to increase gamma and contrast. Figure2 is the average of two sequential frames from Additional file 1, cell 2. Photoshop was used to crop and add the yellow arc and calibration bar to Figure2.


A method for measuring electrical signals in a primary cilium.

Kleene NK, Kleene SJ - Cilia (2012)

Photomicrograph of the method for electrical recording from an isolated primary cilium. A glass-coated bead is covered with a confluent layer of mIMCD-3 cells. The yellow line represents the boundary between the bead and the cells. One primary cilium of one cell is visible entering the tip of the recording pipette. Bar = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539729&req=5

Figure 2: Photomicrograph of the method for electrical recording from an isolated primary cilium. A glass-coated bead is covered with a confluent layer of mIMCD-3 cells. The yellow line represents the boundary between the bead and the cells. One primary cilium of one cell is visible entering the tip of the recording pipette. Bar = 10 μm.
Mentions: Images for Figure1A-D were acquired on an inverted, confocal microscope (LSM 710, Karl Zeiss AG, Oberkochen, Germany) and exported as TIFF files using Zen 2009 Light Edition (Zeiss). Figure1A-C were acquired with a 40×/0.75 NA, Plan-Neofluar, phase-contrast objective, 1400 pixels ×1400 pixels, 97 nm × 97 nm × 1,000 nm voxel dimensions, and 8-bit depth. The laser wavelength was 488 nm. For Figure1A,B, the transmission channel was used. For Figure1C, the dichroic mirror was set for 488 nm and the emission filter was 535 to 759 nm. Figure1D was acquired with a 63×/1.20 NA, C-Apochromat, water-immersion objective, 1,188 pixels × 1,188 pixels, 114 nm × 114 nm × 900 nm voxel dimensions, with 8-bit depth. The laser wavelength was 488 nm, the dichroic mirror was set for 488 nm, and the emission filter was 493 to 630 nm. Figure1E was acquired with a Nikon D70 single-lens reflex camera. For Figure1, Adobe Photoshop 6.0 was used for converting to grayscale, cropping, resizing, and adding labels. Photoshop was also used to adjust brightness and contrast, including gamma, via ‘Input Levels’. Figure1A-C were resampled from 97 to 114 nm/pixel X-Y resolution to match the resolution of Figure1D so that the same scale bar could be used for all four images. Inserts for Figure1B-D were resampled to match the greater resolution of the main figures. Figure1E was resampled to match the greater resolution of Figure1A-D. The videos in Additional file 1 were acquired with a camera (WV-CD50, Panasonic, Osaka, Japan), 1× TV relay lens (Nikon), and a digital video recorder (RDR-VX530, Sony, Tokyo, Japan). The videos were processed in mencoder (http://www.mplayerhq.hu) to increase gamma and contrast. Figure2 is the average of two sequential frames from Additional file 1, cell 2. Photoshop was used to crop and add the yellow arc and calibration bar to Figure2.

Bottom Line: In 47% of attempts, suction resulted in a seal with high input resistance.In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer and Cell Biology, University of Cincinnati, PO Box 670521, Cincinnati, OH 45267-0521, USA.

ABSTRACT

Background: Most cells in the body possess a single primary cilium. These cilia are key transducers of sensory stimuli, and defects in cilia have been linked to several diseases. Evidence suggests that some transduction of sensory stimuli by the primary cilium depends on ion-conducting channels. However, the tiny size of the cilium has been a critical barrier to understanding its electrical properties. We report a novel method that allows sensitive, repeatable electrical recordings from primary cilia. Adherent cells were grown on small, spherical beads that could be easily moved within the recording chamber. In this configuration, an entire cilium could be pulled into a recording microelectrode.

Results: In 47% of attempts, suction resulted in a seal with high input resistance. Single channels could be recorded while the cilium remained attached to the cell. When the pipette was raised into the air, the cell body was pulled off at the air-bath interface. The pipette retained the cilium and could then be immersed in various solutions that bathed the cytoplasmic face of the membrane. In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.

Conclusions: Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

No MeSH data available.


Related in: MedlinePlus