Imaging del due-fotone calcio nei dendriti neuronali nelle fette del cervello

The overall goal of this experiment is to study calcium elevations in neuronal dendrites in response to different stimulation paradigms using whole-cell patch-clamp recordings in neuronal soma in parallel with two-photon calcium imaging in dendrites. This method can be employed to monitor calcium signaling in small dendritic compartments, allowing for close observation of the processes that occurs in dendritic branches during integration of synaptic inputs. The main advantage of this technique is that it enables calcium signaling patterns to be observed in high spatiotemporal resolution and specificity.

Place the mouse brain onto a Petri dish containing continuously oxygenated, cold ACSF-sucrose. To prepare hippocampal slices from the extracted brain, let the brain chill for around two minutes. Afterward, place a piece of filter paper on an ice-packed Petri dish, and transfer the brain onto the filter paper.

Then, dissect the brain into two hemispheres. Glue the dissected hemispheres onto a vibratome sectioning platform, and immerse it into the sectioning tray filled with ice-cold oxygenated ACSF-sucrose. Subsequently, section 300-micrometer-thick slices at one degree Celsius using a vibratome cooler or placing the ice around the vibratome tray.

Using a paint brush, transfer the slices containing the hippocampus to a bath of oxygenated ACSF-recovery heated to 37 degrees Celsius. In this step, transfer a hippocampal slice to a bath under the objective of a laser-scanning two-photon microscope. Continuously perfuse the bath with oxygenated ACSF-normal.

Then, use infrared differential interference contrast microscopy to locate a cell of interest using its size, shape, and position. After that, fill a stimulating pipette with an ACSF-normal solution containing the red fluorophore. Lower it on top of the slice so that the tip is in the same region as the cell of interest.

Following that, fill the patch pipette with patch solution, and attach it to the head stage. Set it over the slice so that its tip is directly above the cell of interest. Subsequently, fit a syringe into the three-way stopcock, and connect it to the patch pipette through a plastic tube.

Inject constant positive pressure into the patch pipette, and keep the stopcock in close position. Next, open the MultiClamp amplifier control software module, and switch to Voltage Clamp mode by clicking on the VC button. Open the electrophysiology Clampex data acquisition software for recording electrophysiological signals, and click on the Membrane Test icon to send out a constant square voltage pulse for monitoring changes in the pipette resistance.

Now, lower the patch pipette until it is right on top of the targeted cell. Upon making contact with the cell membrane, remove the pressure by turning the stopcock into the open position. Then, apply a slight negative pressure to the patch pipette using an empty syringe fitted into the stopcock until the pipette resistance reaches one gigaohm.

In the Membrane Test window of the data acquisition software, clamp the cell at negative 60 millivolts. Continue applying negative pressure until the pipette resistance drops and the whole-cell configuration is achieved. In the MultiClamp amplifier control software module, set the patch configuration to current clamp by clicking on the IC button, and record membrane properties in response to somatic injections of hyperpolarizing and depolarizing currents.

Wait for at least 30 minutes for the cell to be filled with the indicators present in the patch solution. To acquire images, locate a dendrite of interest using the red fluorescence signal. Ensure a visible response by choosing a proximal dendrite shorter than 50 micrometers, as the efficiency of AP backpropagation may significantly decline with distance in GABAergic interneurons, and switch to the xt mode.

With the laser intensity controllers, set the two-photon laser at a minimal power level where baseline green fluorescence is just slightly visible, to avoid phototoxicity. Subsequently, in the Clampex electrophysiology data acquisition software Protocol window and image acquisition software, create a recording trial of the desired duration containing a somatic current injection of the desired amplitude. After that, click the Start Record button, and acquire the fluorescence continuously for one to two seconds.

Repeat the image acquisition three to 10 times. Wait at least 30 seconds between single scans to avoid photodamage. To record dendritic calcium transients induced by electrical stimulation, locate a dendrite of interest using the red fluorescence signal.

Set the stimulating pipette on the surface of the slice above the dendrite of interest. Slowly lower the stimulation pipette 10 to 15 micrometers from the dendrite, minimizing movement to avoid disturbing the whole-cell configuration. To visualize the location of synaptic microdomains in aspiny neurons, in the image acquisition software, switch to the xt mode and position the line along the dendritic branch of interest.

In the Protocol window, create a recording trial that triggers the stimulation. Using the acquisition software, scan along the dendrite continuously for one to two seconds. Repeat acquisition three to five times, waiting 30 seconds between scans to prevent photodamage.

To obtain preliminary information on the cell’s morphology and keep a record of the location of the stimulation pipette, acquire a Z-stack of the cell in the red channel. Using the acquisition software in xyz mode, set the upper and lower stack limits to image the entire cell with all processes included. Then, set the step size at one micrometer, and initiate the stack acquisition using the Start Record button.

When the Z-stack acquisition is complete, use the Maximum Projection option in the software to superimpose all focal plans of the stack and verify the quality of acquisition. Then, slowly retract the patch pipette out of the slice. To fix the slice for post hoc morphological identification, quickly remove the slice from the bath using a paint brush, and place it between two filter papers in an ACSF-filled Petri dish.

Replace ACSF with a 4%paraformaldehyde solution, and leave the dish in a four degrees Celsius overnight. In this figure, the maximum projection of a two-photon Z-stack shows a patched interneuron filled with Alexa-594. White arrowheads point to the location of axonal terminals within the pyramidal layer, suggesting the interneuron is a basket cell.

This figure shows the dendritic branch near which the stimulating electrode was positioned. The dashed line shows the location of the line scan. The images here illustrate the result of a line scan in the red and green channels.

The rise in green fluorescence at the time of stimulation indicates a rise in calcium concentration in that area. The images can be binned into smaller segments, allowing the analysis of the spread of the response. The traces seen here show the calcium transients elicited in different segments in response to electrical stimulation recorded at the somatic level during the imaging trial.

The amplitude of the calcium transients declines with distance from the central hotspot, indicating that the response is spatially localized. Following this procedure, other methods like voltage-sensitive dye imaging or optogenetics can be performed in order to answer additional questions, like local changes in the memory potential within dendritic branches or integration of specific inputs. After watching this video, you should have a good understanding of how to monitor activity-dependent calcium fluctuations in neuronal dendrites using a combination of optophysiological techniques, which will be useful to explore the intricacies of dendritic input integration and plasticity.