The overall goal of the following experiment is to investigate the effects of changes in the ratio and relative timing of excitatory and inhibitory synaptic inputs on the spiking activity of neurons using retinal ganglion cells as model neurons. This is achieved by patching the cell body of a ganglion cell in a whole mount retina, using a glass pipette filled with an intracellular solution that resembles the physiological ionic composition and Lucifer yellow to morphologically identify the cell type as a second step using dynamic clamp, a set of excitatory and inhibitory conductance wave forms are injected into the cell body, which produce a change in membrane potential as a response. Next, various conductance waveforms obtained from physiological experiments in control conditions, or in the presence of drugs are injected into the cell body in order to obtain the corresponding neuronal responses.
The results show the effects of changes in the ratio of excitation and inhibition on the spiking activity of retinal ganglion cells based on the injection of currents composed of excitatory and inhibitory conductances via dynamic clamp recordings. The main advantage of this technique over existing methods like current clamp or voltage clamp, is that it provides an interactive tool by which the ratio and the relative timing of excitatory and inhibitory synaptic conductances can be injected into neurons to investigate the influence on cellular responses. This methodology can provide insights into the function of retinal circuits as well as of any other neuronal networks in the central nervous system.
Begin this procedure by setting up the reperfusion system with 250 milliliters of the carboxy oxygenated aims medium in the electrophysiological setup. Next, smear a thin layer of vacuum grease uniformly onto the bottom of the perfusion chamber. Seal it with a cover slip and make sure it is airtight.
Then place a small amount of grease on the top and bottom ends of the chamber. The grease will hold the grid in place and prevent it from pressing too hard on the retina. Now fix the chamber onto the platform and align both openings after that.
Sacrifice an animal by cervical dislocation after being anesthetized with isof fluorine. Quickly remove its eyes with a pair of small curved scissors. Wash them thoroughly with car oxygenated extracellular fluid in a small beaker, and place them in a Petri dish filled with car oxygenated extracellular fluid.
Under the dissecting microscope, make an insertion hole using a 19 gauge needle in the cornea about two millimeters from the edge on both eyes. This important step should be quick to allow oxygenation of both rein a. After that, remove one cornea with a small pair of iris scissors by cutting parallel to its edge.
Next, remove the iris and lens with a pair of fine forceps and repeat the procedures for the other eye. Transfer one eye cup into a beaker containing the carbox oxygenated extracellular solution. With the scalpel blade, cut one eye cup into half gently separate the retina from the pigment epithelium using a blunt dissecting probe or by gently pulling it off.
Then hold the edge of the detached retinal tissue close to the aura Serta with a pair of fine forceps and grab the aura serta with another pair of fine forceps. Pull it towards the center of the retina. This is a delicate process and may take a few attempts.
If successful aura Serta ciliary body and vitreous humor will be removed and the curvature of the retina will be reduced. Next, trim the edges and then transfer the retina to the perfusion chamber With ganglion cells facing up. Hold the tissue in place by placing the grid onto the grease balls.
Save the remaining tissue with the other eye cup in the carbox oxygenated extracellular solution for later use mount the recording chamber under an upright microscope. Immediately adjust the continuous perfusion of the retina with carbox oxygenated extracellular solution At a rate of three to five milliliters per minute at 35.5 degrees Celsius, make sure the grounding electrode is in place. Now turn on the light source of the microscope and bring its focus onto the cell bodies of ganglion cells through the 40 x of objective.
Locate the center of the hole mount for recording. In this step, fix a glass pipette of five to eight mega ohms in its holder and move it under the microscope using a microm manipulator under a 40 x objective. Slowly lower the pipette until it makes a small dimple on the surface of the inner limiting membrane.
Carefully advance the pipette forward until it catches a small amount of tissue and then move it upward and or sideways to tear a small hole to expose the cell bodies of the ganglion cells. Now fill a clean pipette with eight to 10 microliters, a potassium based intracellular solution with Lucifer yellow. Insert it into the holder, attached to the amplifier's head stage, and make sure the chlorinated silver chloride electrode is submerged.
Select a cell with a large cell body. Slowly lower the pipette tip with positive pressure to make a small dimple on the surface of the membrane. Then stop and release the pressure to obtain a giga seal.
Once a giga seal is achieved, gently apply negative pressure to break the membrane in order to achieve a whole cell patch clamp configuration. This procedure is a delicate step. If too much negative pressure is applied, the connection becomes leaky.
If too little, the membrane remains intact. Over time, Lucifer yellow will fill the cell, allowing visualization of the cell's morphology if stable. This setup should last 30 minutes or more.
Now carry out a current voltage test from minus 75 millivolts to positive 35 millivolts every 10 millivolts using patch master and proceed to later tests. Only if a sodium current larger than one nano amp is present. Next, open lab view and execute the custom written neuro acuma program.
Set the repeat number to eight. Then set the reversal potentials of excitatory and inhibitory conductances at zero and minus 75 millivolts respectively. Select one matching pair of excitatory and inhibitory conductances for testing and the green excitatory conductance trace and the red inhibitory conductance trace will appear in the lower panel.
After that, go back to patch master and select current clamp mode. Immediately switch the amplifier to current clamp mode in neuro acuma. Press the record button.
The cellular response with aphasic burst of action potentials will appear on the upper panel. Then inject current into the cell with low conductance. First if little or no response is observed, increase the current in small increments until it produces a strong response.
Excessive current injection can kill the cell. Once the tests are completed. Select a new pair of conductance waveforms and repeat the procedures for all pairs of physiological and artificial conductances.
After recording, carefully retract the pipette so the cell body stays intact. After afterward, immediately fix the tissue and 4%para formaldehyde for 30 minutes, followed by three 10 minute washes with 0.1 molar phosphate buffer, refrigerate and perform antibodies staining the following day or within a week when ready. Stain against Lucifer yellow filled ganglion cells at room temperature with a primary anti Lucifer yellow rabbit IgG at a dilution of one to 10, 000 for five days.
Then on the sixth day, staying overnight with the secondary goat anti rabbit IgG, add a dilution of one to 500 At the end wet mount the retina in fluorescent preserving media. Place the cover slip and seal with nail polish. Take confocal images of the cell morphology using a Leica SPECT two confocal microscope.
Here are the responses of a ganglion cell to the injection of excitatory and inhibitory conductance Wave forms obtained from the control experiments indicated in green and red respectively. And here are the responses to injection of conductance waveforms recorded after the bath application of tetrodatoxin. Here are the responses of another ganglion cell to various pairs of conductance waveforms arms.
The ratios between the excitatory conductance and inhibitory conductance were changed from one to zero to one to two. As the degree of inhibition increased, the response of the cell decreased, and here are the responses of a ganglion cell to the same level of excitation in which the onset of inhibition was varied. Delta T represents the time difference between the onset of inhibitory and excitatory conductances.
As the delay of inhibition reduced, the response of the cell became weaker. This procedure is a powerful technique which helps reveal the interactions between exci three and inhibitory synap input that generate neuronal output.
