Preparaten en protocollen voor de hele cel Patch klem opname van Xenopus laevis Tectal neuronen

The overall goal of this procedure is to obtain whole cell electrophysiological recordings from neurons of the tadpole optic tectum in order to learn about their pattern of connectivity during development and ultimately to understand how neural circuits form and function. This method can help answer key questions in the field of neural development such as how do neurons change over the course of development and how do circuits function to give rise to behavior. The main advantage of these preparations is that they allow for the electrophysiological recording of optic tectum neurons to provide insight about the function of the developing neural circuits.

Demonstrating the procedure will be Zhenyu Lui, a graduate student in my lab. Using a disposal transfer pipette, move the anesthetized tadpole to a dissection recording dish containing external recording solution. Secure the anesthetized tadpole to a submerged silicone block on the floor of the dissecting recording dish.

For a clear view of the brain, remove the skin overlying the brain by making a superficial incision along the midline using a sterile 25 gauge needle. Filet the brain along the same midline axis by inserting the needle into the neural tube and by gently pulling upward such that the dorsal portion of the tube is cleanly cut while leaving the floor plate intact. To isolate the brain, first use a 25 gauge needle to sever the hindbrain.

Then gently run the needle underneath the brain in a caudal to rostral direction to sever all lateral and ventral connective tissue and nerve fibers. Next, secure the brain to a block of silicone elastomer by placing one pin through one of the olfactory bulbs and another pin through the hindbrain. This is the optimal configuration for recording from tectal neurons.

Then move the dish containing the pinned whole brain preparation from the dissecting scope to the electrophysiology rig. Remove the ventricular membrane using a broken glass pipette. To directly activate the RGC axons, gently lower a bipolar stimulating electrode onto the optic chiasm such that a small dent is formed in the tissue.

The bipolar stimulating electrode should be rostral and almost adjacent to the large middle ventricle where the optic chiasm is located. The bipolar electrode is driven by a pulse stimulator which allows the strength of stimulation to be precisely controlled. To prepare the horizontal brain slice, start with the whole brain preparation.

Then use a razor blade to excise the most lateral fourth of one side of one optic tectum. This cut is made parallel to the rostral caudal plane. Afterward, re-pin the brain to the side of the silicone elastomer with the sliced side facing upward so that the somata and neuropil can be directly accessed for recording and the ventricular surface of the brain facing away from the silicone elastomer block so that a bipolar electrode can be placed on the optic chiasm.

Then perform the whole cell patch clamp or local field potential recording. Here is the schematic showing the configuration of a horizontal brain slice preparation. Note that although the stimulating electrode remains at the optic chiasm, the recording pipette is now positioned to access cells across all the tectal layers exposed by the horizontal brain slice preparation.

This is an RGC-evoked response from a neuron residing in the superficial layer of the tectum. In this figure, the recorded field potentials across the neuropil and the converted current source densities are shown by image plot. Once mastered, this technique can be done within 10 minutes and will typically for up to three or four hours.

After watching this video, you should have a good understanding of how to perform whole brain and horizontal brain slice preparation for whole cell recordings in order to quantify the electrical properties of the tectal neurons and their connectivity.