MNI-caged-L-glutamate

This program provides only one guide, please modify it according to your specific needs.
Take a modified ultraviolet (UV) light-induced glutamate release procedure as an example ^[1].
1. Preparation of Solutions
(1) Prepare artificial cerebrospinal fluid (ACSF) for dissection containing (in mM): 125 NaCl, 2.5 KCl, 0.5 CaCl₂, 6 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, and 20 glucose, bubbled with 95% O₂ and 5% CO₂, resulting in a pH of 7.4.
(2) Prepare ACSF for experiments containing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, and 20 glucose, bubbled with 95% O₂ and 5% CO₂, resulting in a pH of 7.4.
(3) Prepare intracellular solution (ICS) containing (in mM): 150 KMeSO₃, 12.5 hydroxy ethyl piperazine ethane sulfonic acid (HEPES), 40 KCl, 5 NaCl, 1.25 ethylene glycol tetraacetic acid (EGTA), 5 Mg-ATP, 0.5 Na-GTP. Adjust pH to 7.3. Store aliquots of 1 ml at -20 °C.
(4) Dilute sodium-binding benzofuran isophthalate (SBFI)-salt in double-distilled water and prepare aliquots of 5 µl of a 10 mM stock solution.
(5) Thaw ICS and add SBFI stock solution at a final concentration of 1 mM. Vortex and micro-filtrate (0.22 µm). Keep at 4 °C until used in experiment. Do not refreeze and only use for one day.
(6) Prepare MNI-caged-L-glutamate stock solution of 50 mM by dissolving the compound in double-distilled water. Dilute MNI-caged-L-glutamate stock solution to a final concentration of 5 mM in normal ACSF. Keep at 4 °C until used in experiment. Do not refreeze and only use for one day. Store aliquots, which are not immediately used in the experiment, at -20 °C.
1. Dissection of Tissue
(1) After decapitation of the mice, the brains were quickly removed from the skulls.
(2) Immediately place the brain in a petri dish with ice-cold dissection ACSF and dissect a hemisphere by performing a sagittal cut along the midline.
(3) Perform a second cut in the desired orientation as a blocking surface and attach this to the cutting stage of a vibratome with superglue.
(4) Take cutting chamber and cooling element (both kept at -20 °C) of the vibratome out of the freezer and place cutting stage with brain section in the chamber. Then put cooling element in the chamber and submerge tissue in ice-cold dissection ACSF. To stabilize the preparation, one may want to counter the tissue block with agar gel.
(5) Cut 250 µm thick, parasagittal slices of the hippocampus with the vibratome. Ensure that all ACS fluids are bubbled at all times.
(6) After slicing, keep tissue on a mesh in a beaker with the normal ACSF and incubate at 34 °C for 30 min. Then keep at room temperature.
2. Preparation of Hardware
(1) Switch on components of the multi-photon system. Test and adjust infrared laser beam alignment.
A: Control beam positioning by flipping in the centering prism instead of an objective. If the beam is dislocated, re-adjust it by the mirrors in the periscope. Note that the centering plane of the prism must be at the same level as the back focal plane of the objective while imaging.
B: Switch on the spectrometer and check for the multi-photon characteristics of the beam.
C: Set parameters of the imaging software to the following values: Choose a frame size of 512 x 512 pixels for overview images of the cell (smaller clip boxes with a zoom factor of 2.5 for high frame rate and high spatial resolution, respectively). Set imaging speed to fast mode and scan mode to XYT. Z-stepping should be 1 µm for overview stacks and 0.2 µm for detailed stacks to match spatial sampling requirements for deconvolution performed later.
(2) Adjust multi-photon laser beam (790 nm) intensity by altering settings of the Pockels’ cell (final power under the objective: ≈ 14 - 16 µW) and the photo-multipliers (PMTs).
(3) Switch on and calibrate uncaging system by placing a fluorescent sample slide under the objective lens.
A: Using the binoculars, adjust the focus of the UV optics and spatially limit the UV laser spot to a size of 2 µm in diameter at maximum by employing the focusing unit at the UGA scan head.
B: Start the calibration routine of the uncaging unit control software.
C: The accurate positioning of the uncaging spot is achieved by the import of an image of the imaging system via a network connection between uncaging- and imaging-computers.
D: Turn on micromanipulators, electrophysiology components, and pressure application device for delivery of the caged compound to the target region.
E: Install a focal pressure application device for local perfusion of caged compounds. This will reduce costs enormously as compared to bath perfusion of these substances. Adjust the holding pressure to the given atmospheric pressure to prevent a drag of ACSF into or a leakage of caged substances from the application pipette.
NOTE: The pressure application device hosts a highly precise and ultrafast micro-valve in the pipette holder. This enables to employ minimal application pressures and therefore reduces movement artifacts to a minimum during the local perfusion with the caged compound.
F: Pull pipettes for whole-cell patch-clamp and local perfusion using fire-polished borosilicate glass capillaries. Pipettes should have a tip diameter of ~1 µm and a resistance of R ≈ 3 MΩ (values determined with K-MeSO3-based ICS).
H: Place slice in the experimental bath and affix it with a grid (platinum frame 250 µM thick, spanned with surgical filaments, o.d. 40 µm) Use an inverted, tip-broken, and fire-polished Pasteur pipette (suction ball at the side of the broken tip) for slice transfer. Avoid bending and any other harsh handling of the slice.
G: Place bath at the microscope stage and permanently perfuse slice with ACSF.
3. Whole-cell Patch-clamping
(1) Load patch pipette with ICS containing SBFI and load local perfusion pipette with caged compound. Attach pipettes to corresponding micromanipulators. Place reference electrode in bath. Make sure that you are grounded permanently to avoid damage to the head stage.
(2) Lower both pipettes into bath and place them above the hippocampal CA1 region. Apply gentle pressure to patch pipette (+40 mbar) to avoid dilution of the ICS with ACSF.
(3) Compensate the offset potential of the patch pipette using the electrophysiology software.
(4) Approach a CA1 pyramidal cell with patch pipette employing IR-DIC video microscopy. Apply gentle suction until a Giga-seal is obtained. Choose a cell the soma of which is located 30 -70 µm below the surface of the slice to ensure intact cell morphology on the one hand side and low scattering and attenuation of the uncaging beam on the other hand side.
(5) Compensate for fast capacity. Break membrane and open cell to gain whole-cell configuration.
(6) Compensate for slow capacity and series resistance.
(7) Allow the cell to be dialyzed with the SBFI-ICS for at least 30 min before starting the imaging experiments.
4. Multi-photon imaging and stimulation
(1) Add tetrodotoxin (TTX, 500 nM) to ACSF to prevent activation of voltage-gated sodium channels and generation of action potentials.
(2) Visualize cellular morphology using multi-photon excitation and resulting SBFI fluorescence and choose a spiny dendrite for experiment. Zoom in for images at a higher resolution and place a clip box around the dendrite.
(3) Fill a standard patch pipette with 10 µl ACSF containing caged glutamate. Connect the pipette to the pressure application system and attach it to the micromanipulator.
(4) Place pipette with caged compound near the dendrite (~30 µm). Position the pipette to allow efficient local perfusion of the dendrite of choice. Adjust the uncaging laser: position the uncaging spot close (~1 - 2 µm) to the structure of interest.
(5) Set regions of interest on the chosen dendrite and adjacent spines using imaging software.
(6) Approach the region of interest and focally inject the caged compound for several sec with low pressure (<3 PSI). Start patch clamp and fluorescence recordings (at 790 nm multi-photon excitation) via trigger signal.
NOTE: During the local perfusion of the caged compound, the excitation beam is completely dimmed to prevent bleaching.
(7) Stop local perfusion of the caged compound. Increase intensity of the two-photon laser to enable efficient excitation of SBFI and apply a UV flash to initialize uncaging (uncaging duration 300 msec).
(8) Monitor changes in SBFI fluorescence. Stop recording after SBFI fluorescence has recovered back to baseline.
References:
[1] Kleinhans C, Kafitz KW, Rose CR. Multi-photon intracellular sodium imaging combined with UV-mediated focal uncaging of glutamate in CA1 pyramidal neurons. J Vis Exp. 2014 Oct 8;(92):e52038. doi: 10.3791/52038.