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Cell Imaging and Electrophysiology Core
The laboratory consists of a designated, climate-controlled room for cell imaging and in vitro electrophysiology recording.
Dissociated DRG Neuron Ca2+ Imaging
Dorsal root ganglia from all spinal levels of 4-week old mice or rats are collected in cold DH10 (90% DMEM/F-12, 10% FBS, 100 U/ml penicillin, and 100 µg/ml Streptomycin, Gibco) and treated with enzyme solution (5 mg/ml Dispase, 1 mg/ml Collagenase Type I in HPBS without Ca++ and Mg++, Gibco) at 37°C.
Following trituration and centrifugation, cells are resuspended in DH10, plated on glass cover slips coated with poly-D-lysine (0.5 mg/ml, Stoughton, MA) and laminin (10 µg/ml, Invitrogen), cultured in an incubator (95% O2 and 5% CO2) at 37°C and used within 24 hours.
Neurons are loaded with fura 2-acetomethoxy ester (Molecular Probes) for 30 min in the dark at room temperature or 45 min at 37°C, respectively. After washing, cells are imaged at 340 and 380 nm excitation to detect intracellular free calcium. Calcium imaging assays are performed with an experimenter blind to genotype.
Each experiment was done at least three times and at least 100 neurons are analyzed each time.
Ex vivo Pirt-GCaMP3 Ca2+ Imaging
Two-month-old mice are sacrificed by CO2 asphyxiation. DRG explants are acutely isolated from Pirt-GCaMP3 heterozygote mice, maintaining the anatomical structure and orientation. Explants are then placed in buffer solution for at least one hour for recovery before the imaging is started.
Using standard slice techniques, transverse slices of the spinal cord (300 μm thick) are cut with a vibrating slicer (Leica VT 1000S) using a metal blade in ice-cold N-methyl-D-glucamine (NMDG)-based cutting solution. This solution contained (in mM):
- 135 NMDG
- 1 KCl
- 1.5 MgCl2
- 0.5 CaCl2
- 1.2 KH2PO4
- 24.2 choline bicarbonate
- and 13 glucose, bubbled with 95% O2/5% CO2 to yield pH 7.4.
Transverse spinal cord slices are identified rostrocaudally with reference to the obex (about 1.5 mm caudal to the obex). Ear skin, DRG, and spinal cord slice are perfused with either synthetic interstitial fluid (SIF) (Zimmermann et al., 2009) consisting of (in mM):
- 107.8 NaCl
- 3.5 KCl
- 1.53 CaCl22H2O
- 0.69 MgSO47H2O
- 1.67 NaH2PO42H2O
- 26.2 NaHCO3
- 9.64 C6H11NaO7
- 7.6 sucrose
- 5.55 glucose or artificial cerebrospinal fluid (ACSF) containing (in mM):
- 124 NaCl
- 2.5 KCl, 2.5 CaCl2
- 1.3 MgCl2
- 1 NaH2PO4
- 26.2 NaHCO3
- 20 glucose.
Solutions are bubbled with 95% O2/5% CO2 to yield pH 7.4 and osmolarity is adjusted to 300 to 310 mOsm with sucrose and glucose. Imaging samples (ear skin, DRG, and spinal cord slice) are maintained thereafter in SIF or ACSF at room temperature. The imaging samples are then placed in a submerged chamber that is perfused at 2 ml/min with room temperature SIF or ACSF bubbled with 95% O2 / 5% CO2 to yield pH 7.4. They are visualized on an upright microscope equipped with infrared differential interference contrast (DIC) using a 40X water immersion objective. Capsaicin (Sigma) and isotonic KCl solution are used as agonists.
Capsaicin (50 mg/ml) is dissolved in ethanol, stored at -80°C, and diluted with buffer solution to different concentrations. All experiments are performed at room temperature (~25°C), in a chamber with stimuli applied by bath application.
GFP signals from Pirt-GCaMP3 het mice are measured to see Ca2+ transients using green emitted light. To minimize phototoxicity, the power and exposure time of laser illumination are maintained as low as possible. Single photon Ca2+ imaging is performed with a 700 Zeiss confocal microscope, using the 488 nm line of a solid state laser for excitation of Pirt-GCaMP3 and a 488 nm laser main dichroic beam splitter (MBS) and a 505-555 nm variable secondary dichroic mirror (VSD) to detect the emission of green fluorescence.
Ca2+ transients are elicited by a capsaicin or KCl stimulus. Pirt-GCaMP3 images are acquired at 2.6Hz in frame-scan mode with a 256 x 256 pixel region of interest. For analysis, raw image stacks (256 × 256 to 1024 × 1024 pixels in the x–y plane and 5–6μm voxel depth; typically 10 optical sections) are imported into Image J (NIH) for further analysis. Optical planes from sequential time points are realigned using Image J to compensate for minor tissue drift during the time course of the acquisition. The contrast is adjusted accordingly and selected optical planes or z-projections of sequential optical sections are used to show final images and to assemble time-lapse movies.
Foreground pixels are determined by thresholding the image, and are spatially averaged to calculate ∆F/F0 for each frame. Each fiber is selected and analyzed with time series analysis. Background correction is performed by subtracting the background fluorescence of a region adjacent to the region of interest. Ca2+ signal amplitudes are expressed as (Ft-F0)/F0 as a function of time; ratio of fluorescence difference (Ft-F0) to basal value (F0). The average fluorescence intensity in the baseline period is taken as F0.
For measurement of activated fiber length in ear skin, each activated fiber is manually selected and traced using Zen (Zeiss) and Imaris (Bitplane) software. Image J or Fiji (NIH) and Zen (Zeiss) software are used to analyze Ca2+ imaging data using standard functions and a custom macro.
In Vivo DRG Pirt-GCaMP3 Imaging
We first surgically expose the right lumbar 4 (L4) DRG, the axons of which innervate the right hindpaw, from the back of an anesthesized Pirt-GCaMP3 mouse. Then the animal is laid with its belly down under Leica LSI single-photon confocal microscope with a long-working distance lens. The vertebrates of the mouse were held by two stationary clamps to minimize the interference of movements from breathing and heart beats.
During the imaging section usually lasting 2-3 hours, the animal is under constant anesthesia and its body temperature was kept at 37 °C with a heat pad. Since the surface of DRG is curved, z-stack imaging is required to cover the entire area of exposed L4 DRG.
Strikingly, more than 1,700 neurons on average per DRG (~12% of total DRG neurons) can be imaged at the speed of ~6 seconds per frame. To increase the number of activated neurons being imaged, we use a Rodent Pincher Analgesia Meter instead of the widely used von Frey filaments which allowed us to accurately apply and monitor mechanical force to a large area of the hindpaws.
In addition, various temperatures (4, 15, 25, 35, 45, 58 °C water bath) and chemicals can be applied onto the hindpaws.
Whole-cell Patch Clamp Recordings of Cultured DRG Neurons
DRG neurons plated on cover slips are transferred into a chamber with medium (the extracellular solution: ECS) of the following composition (in mM):
- NaCl 140
- KCl 4
- CaCl2 2
- MgCl2 2
- HEPES 10
- Glucose 5 with pH adjusted to 7.38 using NaOH.
The intracellular pipette solution (ICS) contains (in mM):
- KCl 135
- MgATP 3
- Na2ATP 0.5
- CaCl2 1.1
- EGTA 2
- Glucose 5 with pH adjusted to 7.38 using KOH and osmolarity adjusted to 300 mOsm with sucrose.
Patch pipettes have resistances of 2-4 MΩ. In current clamp recordings, action potential measurements are performed with an Axon 700B amplifier and the pCLAMP 9.2 software package (Axon Instruments). Electrodes are pulled (Narishige, Model pp-830) from borosilicate glass (WPI, Inc.) All experiments were performed at room temperature (~25°C).
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