Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

The overall goal of this procedure is to evoke and analyze sodium dynamics in small cellular compartments, such as spines and dendrites of central neurons in the intact tissue. This is accomplished by first preparing an acute mouse hippocampal slice, loading a pyramidal neuron with the sodium sensitive fluorescent dye SBFI through a patch pipette, and visualizing the cellular morphology using multi photon excitation. The next steps are to choose a spiny dendrite for the experiment and to Foley inject a photo activated compound such as caged glutamate close to this dendrite.

A UV flash is then applied to Foley uncaged glutamate using a UV laser that has been accurately positioned to target a small volume close to the dendrite of choice. The final step is to monitor the resulting changes in SBFI fluorescence in the dendrite, and to monitor the resulting changes in sodium in the adjacent spines. The accompanying membrane currents are also recorded.

Ultimately using the appropriate pharmacological tools such as receptor blockers will enable the study of the mechanisms generating the evoked intracellular sodium signals. This method provides a reliable tool for the investigation of intracellular sodium signals in small cellular compartments and in intact brain tissue. Here we combine whole cell patch clamp and multiphoton sodium imaging with a modified procedure for UV light induced uncaging.

With allows us to monitor cellular responses upon precise and focal applications of neuroactive compounds. The procedure will be demonstrated by Karl KA and by Kaan Kleinhans, a grad student in my lab. To begin dissection of the tissue, immediately place the brain in a Petri dish with ice cold dissection, artificial cerebral spinal fluid, or A CSF and dissect a hemisphere by performing a sagittal cut along the midline.

Perform a second cut in the desired orientation as a blocking surface. Then attach this to the cutting stage of a vibram with super glue. After taking the cutting chamber and cooling element of the vibram out of the freezer, place the cutting stage with the brain section in the chamber.

Then put the cooling element in the chamber and submerge the tissue in ice cold dissection A CSF. Next cut 250 micrometer thick. Para sagittal slices of the hippocampus with a viome.

Ensure that a CS fluids are bubbled at all times. After slicing, keep the tissue on a mesh in a beaker with a normal A CSF and incubate at 34 degrees Celsius for 30 minutes. Following incubation, keep the tissue at room temperature.

This experiment should be run in a dark environment, but is shown here under dim light conditions for filming purposes. To prepare hardware first, switch on the components of the multi photon system and adjust the infrared laser beam alignment as described in the text protocol. Then adjust the multi photon laser wavelength and beam intensity by altering the settings of the pcal cell and the photo multipliers.

Next, switch on and calibrate the uncaging system by placing a fluorescent sample slide under the objective lens. Start the calibration routine of the uncaging unit control software. Set the UV laser to several points within its scan range while the fluorescence is captured with a CCD camera by clicking on the UV laser spot.

At each point, adjust the positioning of the galvanic scan mirrors to correspond to a certain coordinate in the software. The accurate positioning of the uncaging spot is achieved by importing an image of the imaging system via a network connection between uncaging and imaging computers. Using screen grabber software continuously read out the frames of the imaging software and adjust the imaging.

And in done caging frames congruently export every 10th frame from the imaging software as a reference image into the flash unit to ensure proper adjustment during the entire experiment. Next, turn on the micro manipulators electrophysiology components and the pressure application device for delivery of the caged compound to the target region. Pull pipettes for whole cell patch clamp and local profusion using fire polished boro silicate glass capillaries.

Then place the slice in the experimental bath and to fix it with a grid, place the bath at the microscope stage and permanently perfuse the slice with A CSF. Begin whole cell patch clamping by loading the patch pipette with intracellular solution or ICS containing SBFI then load the local perfusion pipette with a caged compound. Attach the pipettes to the corresponding micro manipulators and place the reference electrode in the bath.

Choose a CA one pyramidal cell with a soma that is located 30 to 70 micrometers below the surface of the slice to ensure intact cell morphology as well as low scattering and attenuation of the uncaging beam. Approach this cell with a patch pipette employing IR DIC video microscopy. Apply gentle suction until a giga seal is obtained.

Compensate for fast capacity. Then break the membrane and open the cell to gain cell configuration. Finally, assess cell viability in current clamp mode.

Inject a depolarizing current and check for resulting spiking activity to perform imaging and stimulation. First, add tetro DOIND to A CSF to prevent activation of voltage gated sodium channels and generation of action potentials. Visualize cellular morphology using multi photon excitation and resulting SBFI fluorescence.

Choose a spiny dendrite for the experiment. Zoom in for images at a higher resolution and place a clip box around the dendrite. Then place the pipette with the caged compound near the dendrite.

Position the pipette to allow efficient local perfusion of the dendrite of choice. Adjust the uncaging laser to position the uncaging spot close to the structure of interest. Next, set regions of interest on the chosen dendrite and adjacent spines using imaging software.

Approach the region of interest and fo inject the caged compound for several seconds with low pressure start patch, clamp and fluorescence recordings via the trigger signal. During the local profusion of the caged compound, the excitation beam is completely dimmed to prevent bleaching. Next, stop local profusion of the caged compound.

Then increase the intensity of the two photon laser to enable efficient excitation of SBFI and apply a UV flash to initialize on caging monitor changes in SBFI fluorescence as well as somatic current proceed to stop recording after SBFI fluorescence has recovered back to baseline. To obtain morphology record an XY, Z stack of the clip box region set for the performed measurements. Ensure that this stack is over sampled spatially to enable optimal image deconvolution and to increase image quality and resolution.

Loading neurons with SBFI through the patch pipette enabled the visualization of the entire cell, including fine dendrites and adjacent spines by employing multi photon excitation after local perfusion with caged glutamate. Applying a UV flash close to a dendrite resulted in a transient decrease in fluorescent emission of SBFI reflecting an increase in the intracellular sodium concentration. At the same time, an inward current was recorded at the SOMA application of a UV flash to slices, which had not been pre perfused with caged glutamate.

Never elicited changes in SBFI fluorescence nor inward currents indicating that these signals are due to the uncaging of glutamate. Peak amplitudes were slightly higher in spines close to the uncaging spot, whereas patterns for further spines resembled the parent dendrite. The pathway for sodium influx into dendrites and spines in response to uncaging of glutamate was studied using selective blockers for the sodium permeable ionotropic glutamate receptors, glutamate induced intracellular sodium signals, and the elicited somatic currents were omitted in the presence of these blockers.

Upon washout of the blockers, the signals are regained, demonstrating that uncaging of glutamate activates cytotropic glutamate receptors on ca one pyramidal neurons resulting in intracellular sodium transient and inward currents. While attempting this procedure, it's important to remember to choose the lowest possible intensity for the infrared laser to minimize any damage to the preparation. After watching this video, you should have a good understanding of the critical steps of our experimental approach to study sodium signaling in cellular micro remains Once mastered, this technique enables high resolution imaging of activity evoked intracellular ion signals in the intact tissue, which is free of movement artifacts as introduced by other techniques.