The overall goal of the following experiment is to record GABA activated currents generated by single molecules, the single channel current, and by specific populations of channels located at synapses or outside of synapses. This is achieved by first preparing hippocampal rat brain slices and making pipettes to be used for patch clamping. As a second step, a pipette is attached to the neuronal membrane.
After the formation of a giga seal, the cell attached patch clamp configuration is obtained. Next, apply suction to the pipette. Once it breaks into the cell, the whole cell patch clamp configuration is established.
Results are obtained that differentiate between synaptic and extra synaptic gabaa channels. The main advantage of the pet clamp technique go with existing methods like emit is that one study the functional properties of the channels. The patch clamp method can help to answer key questions in the field of neuroscience, such as what determine acceptability of neurons and how it is modulate and fine tuned.
The implications of this technology extend towards therapy and the diagnosis of many diseases because every living cell depends on the properly functioning ion channel for survival and the specialized cellular function Demonstrating the procedure will be giant postdoctoral fellow in the lab and young yin, a graduate student in the lab. To begin this procedure, sacrifice a postnatal 16 to 22 days old wistar rat by decapitation and quickly remove the brain with a spatula. Cut the brain along the midline with a surgical blade.
Next, place each hemisphere on a watman filter paper with a cut surface facing the paper. Put a drop of super glue on the specimen disc and place the hemisphere on top of it with a cut surface facing down. Then place the specimen disc in the cutting chamber of the vibrator that is filled with ice cold.
A CSF. Set the slice thickness to 400 micrometers and start cutting the brain. Cut approximately two millimeters of brain tissue off until reaching the hippocampus.
Next, put the hippocampal slices into a glass petri plate filled with a CSF. The plate is placed on a black background to allow easy visualization of the hippocampus. The next step is to isolate the hippocampus from the surrounding tissue using a sharp surgical blade.
Be careful not to push or pull the tissue after that. Incubate the slices and the A CSF solution at 37 degrees Celsius for one hour, and then keep them at room temperature until the experiments are done. Boro silicate glass capillaries are used for the patch pipettes.
Pull the patch pipettes with an automated or manual pipette puller resulting in a resistance of two to four mega ohms for whole cell recordings, or eight to 20 mega ohms for single channel recordings. Polish the pipette with the same boro silicate glass for single channel recording. Using a micro forge melt the boro silicate glass on the platinum wire to make the tip smoother.
Polishing the pipettes this way creates pipettes that form more stable and higher resistant seals as compared to pipettes polished by the heat from an uncoated wire or by a polishing step in the automated puller. The patch clamp equipment consists of microscope on a floating table and enclosed in a faraday cage, an amplifier, a digitizer, and a computer. Together, these equipment are often referred to as a patch clamp rig.
Now insert a pipette into the pipette holder and tighten the cap in order to fasten the pipette. P clamp is used as the patch clamping software. Set the OC clamp amplifier 200 B to V clamp, gained to two, filter to two kilohertz and the holding potential to zero millivolts to create positive pressure in the pipette blow air into the mouthpiece that is attached to the tube which connects to the pipette holder.
Then close the valve between the mouthpiece and the tube. Make sure that there is no leaking of air. Next, lower the pipette into the bath solution.
On the computer screen where the pipette resistance is measured, you'll see a pulse generated by a five millivolt pulse train and a number that tells you the resistance of the pipette. Smaller numbers represent larger pipette tip openings and larger pipettes. Adjust the pipette offset on the amplifier to zero.
Use 10 x magnification on the microscope to identify the region, or 60 x magnification. To identify a parametal neuron that you are going to patch in the hippocampal slices, the dentate gyrus granule cells, the CA one parametal cell body region, and the CA three parametal cell body region each appear as a bright band. Bring the pipette into focus so that you can see both the region or cell and the pipette.
When looking through the microscope eye pieces, place the pipette above the middle of the region using the coarse movement manipulator setting, and lower the pipette until it is just above, but touching the tissue. At this point, lower the pipette very gently onto the center of the cell or region using the fine movement settings of the manipulator. At the same time, watch the computer screen to keep track of the pipette resistance.
Keep lowering the pipette until the resistance of the pipette becomes larger. Release the positive pressure from the pipette by opening the valve. This creates a gentle suction and automatically forms the giga seal.
If the giga seal is not formed, apply suction to the mouthpiece where you previously applied positive pressure. You have now obtained the so-called cell attached patch clamp configuration. Adjust the fast and slow capacitance compensation to cancel the transient coming from the pipette and the pipette holder.
On the computer screen, you will see a flat current trace. The higher the GigE value is, the better signal to noise ratio you will have in the single channel recordings to record single channel currents with a cell attached patch clamp configuration. Set the gain to 500 and the data sampling rate to no slower than 100 microseconds.
Then record the currents. Keep the volume of the bath solution low so that it just covers the slice to minimize the noise in the recordings to perform whole cell recording. Change the pipette potential to minus 60 millivolts or close to the resting membrane potential.
In the cell attached configuration, apply suction to the pipette via the tube that is connected to the pipette holder. At the same time, note the changes in the resistance and the capacitance transient on the computer screen. Once you have broken into the cell.
When this happens, you are in the so-called whole cell configuration or whole cell mode. Allow the pipette solution to equilibrate with the cell interior for five to 10 minutes. In the meantime, keep track of the value of the series resistance.
The current is carried by the populations of open channels in the cell membrane. To demonstrate GABA activated single channels, GABA is added into the pipette solution as it cannot cross the cell membrane. Shown here are the single channel currents activated by 20 nanomolar GABA concentration in a cell attached patch on a dentate gyrus granule neuron.
The patch was depolarized by 40 millivolts. The channel's maximal conductance was 44 Picos semens and its latency of activation was 2.63 minutes. Here's an example of how to differentiate gaba, activated synaptic and extra synaptic currents by the phasic and tonic nature of the currents.
The current through populations of channels was recorded from rat hippocampal neurons using the whole cell voltage clamp method to identify tonic and phasic currents. 100 micromolar of the gabaa antagonist SR 9 55 31 was applied to a dentate gyrus neuron as indicated in BA and to an insulin treated ca. One parametal neuron as indicated in BB SR 9 55 31, blocked the extra synaptic channels and induced a shift in the baseline current, which revealed the tonic current.
Once mastered, obtaining a cell attached patch and then breaking into the cell to establish a whole cell patch can be done within two minutes. Following this procedure, one can patch onto some other tissues like intact pancreatic eyelets or isolated cells like lymphocytes or cells in culture.
