Subcelulares patch-clamp grabaciones desde el dominio somatodendrítica de la sustancia nigra dopamina Neuronas

This method can help answer key questions in the neurobiology field, such as how individual voltage-gated ion channels contribute to the functions of a specific neuron. The main advantage of this technique, is that it allows the function of ion channels to be mapped at the subcellular level in neurons. Begin this procedure by placing the brain on a 9 centimeter petri dish with the bottom covered with a layer of SiliGuard.

Next, surround the brain with ice cold sucrose-ACSF. To prepare coronal slices, make two cuts to remove the cerebellum and the rostral part of the brain. Then apply cyanoacrylate glue on the specimen tray, and glue the brain block to the tray such that the rostral part faces the slicing stage.

Subsequently, apply a few drops of sucrose-ACSF on the brain and slowly submerge the tray into the cutting chamber. Afterward, adjust the razorblade at an angle of 15 degrees with reference to the horizontal plane. Cut coronal brain slices of about 500 micrometers in the cottle nasal direction before reaching the substantia nigra.

Then adjust the thickness to cut 300 micrometer slices containing the region of interest. Separate the slices from the tissue block with a very thin profusion needle attached to a syringe parallel to the edge of the razor blade. Following that, transfer the slices to the reserve chamber at 34 degrees Celsius for one hour.

In this procedure, transfer a brain slice to the recording chamber. Anchor the slice at the bottom of the recording chamber with a platinum ring, and ensure that the slice is of good quality with a smooth, even surface. Then select a neuron with a dendrite extending over a long distance in the same plane, and ensure that the dendrite of interest can be followed to a well-defined soma.

Next, fill the patch pipette with electrode solution. To patch the dendrite in cell-attached mode, identify and focus on a portion of the dendrite. Apply positive pressure to the pipette, and lower it using the micro manipulator.

Then position the pipette close to the membrane and adjust the pressure in order to create a small dimple. Subsequently, release the pressure on the pipette tip and patch the dendrite, while controlling the pipette resistance. Try to obtain a seal resistance larger than one gigaohm, at best between three and ten gigaohms, for cell-attached recordings.

Afterward, retract the pipette away from the membrane, by a couple of micrometers, to avoid deformation of the dendrite. The action currents seen here represent the spontaneous action potentials of nigral neurons. To suppress the action currents, apply calcium and sodium channel blockers to ACSF.

Then apply a voltage step of 90 millivolts, from a holding potential of zero millivolts, to evoke the hyperpolarization-activated cation current. At the end of the recording, rupture the patch to achieve the whole cell mode. After that, slowly and gently withdraw the dendritic pipette from the neuronal membrane to obtain an outside-out patch.

To record from the corresponding soma in the whole-cell mode, locate the dendrite's corresponding soma. Lower a pipette with resistance between five to ten megaohms with a potassium-based intracellular solution to the cell body. Then apply a brief negative pressure to the pipette tip to rupture the membrane in order to achieve the whole-cell mode.

Check for the identity of the neuron in current-clamp mode by applying long hyper and depolarizing current steps, and allow biocytin from the pipette, to diffuse along the dendrites. After 10 minutes, withdraw the pipette to obtain an outside-out patch. Use a sequence of short lateral upward movements to favor the proper closing of the cell membrane, once the pipette is above the slice, to get completely out of the recording chamber.

Keep the slice in the recording chamber for an additional 15 minutes in standard ACSF to allow equilibration of the biocytin and the recorded neuron. Then gently transfer the slice into a 25 milliliter amber glass bottle containing standard ACSF for histology. In this procedure, roughly locate the flourescently labeled neuron in the slice with epiflourescence.

Briefly examine dendritic arbor of neurons and locate the axon and axonal bleb using a 10X objective. Next, switch to the confocal microscope, and select the 488 nanometer laser diode to excite the labeled neuron. Follow the extension of the axon and dendrites in the Z axis, and record successive images in order to obtain a Z stack for the entire cell.

Adjust the resolution of the Z axis and collect the confocal images of the cell at a low and a high magnification. At the end, open the image file of the neuron with an imaging software. Compare the Z projection image with an IR-DGC image by eye to locate the precise position of the patch pipette during the electrophysiological recording.

Shown here is the IR-DGC image of a dopamine neuron and a pipette on the proximal dendrite. The current trace shows capacity of the EN leak currents in response to a five millivolt voltage command, in cell-attached configuration, with the seal resistance of five gigaohms. Hyperpolarizing voltage step, from a membrane potential of zero millivolts, induced a slowly activating hyperpolarization-activated cation current.

The current trace was inverted and fitted with a single exponential function, giving a time constant of 632 milliseconds. Here are the voltage responses of the whole cell recorded soma to one second hyper and depolarizing current pulses in the presence of voltage gated sodium and calcium current blockers to remove action potentials during cell attached recording. Once mastered, this technique can be done in one day, if it is performed properly.

While attempting this procedure, it's important to remember to obtain healthy tissue and stable recording conditions. Following this procedure, other methods, like immunohistochemistry, can be performed in order to answer additional questions like the identity of the recorded neuron. After its development this technique paved the way for researchers in the field of neurophysiology to explore the function of properties and role of voltage-gated ion channels in excitable cells.

After watching this video, you should have a good understanding of how to record and examine the distribution of currents flowing through voltage-gated ion channels in dendrites.