This article describes a method for visualizing calcium transients in hippocampal neurons using two-photon microscopy. The technique involves the use of stimulation and patch pipettes to manipulate and record from target neurons.
Take a stimulation pipette containing red calcium-insensitive fluorophores in artificial cerebrospinal fluid, or ACSF.
Position the pipette near the target neuron in a hippocampal slice.
Place a patch pipette with a recording electrode bathed in an electrolyte containing red calcium-insensitive and green calcium indicator fluorophores near the target.
Apply constant positive pressure to briefly disturb the tissue surface, indicating proximity to the target.
As the dimple forms on the neuronal membrane, release the pressure to form a seal.
Apply negative pressure to tighten the seal.
Stabilize the membrane potential at a constant negative voltage.
Continue with negative pressure to break the membrane, allowing the fluorophore-containing electrolytes to spread within the neuron.
Visualize the red fluorescence of the target dendritic spine and align the stimulation pipette near it.
Apply electrical pulses to open the calcium channels.
Use two-photon microscopy to visualize transient changes in calcium levels as indicated by green fluorescence.
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 closed position. 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 1 giga ohm. 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.
To record dendritic calcium transients induced by electrical stimulation, locate a dendrite of interest using the red fluorescent 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.
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