The overall goal of this procedure is to study ligand gated ion channel function in neurons from acutely prepared brain slices derived from the adult mice. This is accomplished by first extracting an intact brain from an adult mouse that has been trans Cardi perfused with specialized artificial cerebral spinal fluid. The second step is to prepare brain slices from the brain region of interest in the desired orientation.
Next, an individual brain slices placed in a recording chamber under the microscope and individual neurons within the slice are identified for recording. The final step is whole cell patch clamp recording of a neuron followed by local application of drugs to activate the ligand gated ion channels using a second glass micro pipette. Ultimately, this method of studying ligand gated ion channels is used to show the biophysical properties of native receptors on live neurons.
The main advantage of this technique over existing methods such as bath application or YouTube drug delivery, is that drug delivery to the recorded neuron is very fast and localized. Small quantities of drug can be delivered and multiple neurons can be studied from a single slice. This method can help to answer key questions in the nicotinic acetylcholine receptor field, such as determining the native nicotinic receptors involved in the biophysical cell, biological and behavioral responses to nicotine.
In this procedure, after sacrificing an adult mouse and obtaining the brain, trim the brain in the desired orientation to include the area of interest on an ice chilled metal plate. Next, affix the brain block with super glue to the dry stage surface of a vibrating slicer. Then submerge the brain block in four degrees Celsius NMDG recovery solution and continuously bubble the solution with carbogen.
Subsequently cut the slices of 250 micron thickness each from the brain area of interest. Next, pull a standard patch micro pipette with a resistance of four to six mega ohms using the programmable flaming brown puller. Fill the micro pipette with a suitable intracellular recording solution.
Then transfer a slice to the recording chamber on the stage of an upright microscope continuously super fuse the slice with carbonated 32 degrees Celsius Standard recording A CSF at 1.5 to 2.0 milliliters per minute. Under the microscope, locate and center the brain area of interest using a 10 x air objective. After that, visualize the individual neurons using a 40 x near infrared water immersion objective connected to the IR differential interference contrast optics, and a high speed near infrared charged coupled device or CCD video camera Under IR DIC observation healthy neurons exhibit a smooth light gray plasma membrane following cell visualization.
Perform whole cell recording using conventional techniques. Now pull another standard patch clamp micropipet as before to produce two identical sister tips from the same piece of glass with an optimal resistance of approximately five mega ohms. Backfill one of the micro pipettes with the solution of drug diluted in carbonated standard recording A CSF point the tip downward and flick the drug filled micropipet to remove any air bubbles trapped in the micro pipette.
Next, mount the drug filled micro pipette into a pipette holder attached to the microm manipulator. The pipette holder should be connected with tubing to a suitable pressure ejection system. Manually eject pressure three to four times to the micro pipette in order to build up adequate pressure behind the column of fluid.
Then lower the drug pipette into the slice using the microm manipulator using I-R-D-I-C.Observation. Position the drug micro pipette at the surface of the tissue slice. Execute one pressure ejection and monitor the vicinity of the tip for movement of debris related to the ejection and the micro pipette tip.
For any signs that the tip is clogged or blocked, if the tip is clogged, retract the pipette and replace it with a new one. Approach the recorded cell diagonally from the top of the slice. When it is in the final position, the tip of the drug filled pipette is in the same focal plane as the recorded cell and the tip of the recording electrode, and it is about 10 to 40 microns from the recorded cell.
If the drug filled pipette tip is above the recorded cell, the force from the pressure ejection could disrupt the giga seal of the cell, then record the acetylcholine and or nicotine elicited cellular currents while holding the neurons in voltage clamp mode. Although pressure rejection can be triggered manually when using a pico spritzer three for more reproducible results, it is advisable to trigger the pressure rejection using the acquisition system and a digital TTL pulse. To improve the experiment, the drug filled pipette holder can be mounted to a single dimension pizo electric translator that is capable of accepting an analog voltage input, which is then mounted onto the high precision micro manipulator.
A pizo electric translator is useful because the movement of the drug filled pipette into and out of the slice, including the timing and speed of entry exit can be more easily controlled and reproduced from experiment to experiment. Next, adjust the voltage to its maximal value with the manually controlled potentiometer on the pizo electric voltage controlled amplifier. Use the micro manipulator to position the drug filled pipette in the slice.
Retract the pipette by manually reducing the voltage to zero on the pazo electric voltage controlled amplifier using an analog output signal from the recording acquisition system. Move the drug filled pipette into the slice before the pressure ejection TTL pulse occurs. After the pulse occurs, retract the pipette.
This figure shows the representative response of a dopamine neuron to 100 micromolar acetylcholine applied locally, and this figure shows the representative response of another dopamine neuron to 100 micromolar nicotine applied locally. Here are the spontaneous action potentials of a dopamine neuron in an Alpha six L nine prime S mouse brain slice being held in current clamp mode. The application of one micromolar of nicotine using a 250 millisecond pressure rejection induced changes in action, potential firing rate, and resting membrane potential.
Using the threshold search in P clamp software, instantaneous firing rate was derived to classify the neurons by the types of acetylcholine response individual superior colus neurons. In an Alpha six L nine primes mouse brainin slice were voltage clamped followed by local application of one micromolar nicotine via pressure ejection type one neurons exhibited increased IPCs. Following nicotine application, type two neurons exhibited large inward currents and increased EPCs in response to nicotine application.
The Pizo electric translator offers consistency and protects from drug leakage. A VTA dopamine neuron in a brain slice from an Alpha six L nine prime S mouse was recorded in voltage clap mode. During the application of one micromolar nicotine, the nicotine filled pipette was maneuvered to the final position before the pressure ejection and retracted after the ejection using a pazo electric translator.
A second response was recorded two minutes after the first to demonstrate that no N-A-C-H-R desensitization had occurred. The experiments were repeated with the application of 10 micromolar nicotine. Again, no desensitization was observed in the second response when compared to the first one.
Once mastered, this technique can be performed in as little as three hours if done properly. In some cases, 10 to 15 neurons can be studied from a single brain slice within one day While attempting this procedure, it's important to remember to accurately position the drug filled pipette to maintain a balance between the full activation of surface nicotinic receptors and the giga seal integrity.
