The overall aim of this procedure is to prepare agar embedded retinal slices for the study of the microcircuits located in the presynaptic bipolar cell nerve terminals. This is accomplished by first obtaining the retina from a dark adapted goldfish eye and cutting a small rectangular piece of this retina. The retinal piece is then immersed in a transparent and low melting point agar solution, which is then solidified in a nice cold slice solution.
The next step is to slice the solid agar block containing the rectangular piece of retina using a vibrator. The final step is to visualize, identify, and patch. Clamp a bipolar cell nerve terminal.
Ultimately, results show the pre-synaptic calcium current and synaptic physical exocytosis with membrane capacitance measurements from atomized and intact bipolar cell terminals. Hi, my name is Han Kim. I am postdoc in PCO of lab at Burham Institute.
Today I'm going to introduce AGA embedded slice preparation of cold facial retina and demonstrate patch clamp capacitance measurement at single pipeline of. To begin this procedure, hemis each eye of a goldfish by cutting evenly around the front of the eye with a pair of spring scissors. Then place the eye cups in chilled slice solution with no calcium.
If necessary, remove the lens with tweezers. Next, remove the pigment epithelium attached to the retina by peeling the retina away from the eye cup with a pair of 45 degree angled tip fine forceps. After that, cut the optic nerve with either a pair of fine forceps or spring scissors.
Then apply suction with a modified pasta pipette or a large tip transfer pipette. To remove the retina, gently remove the rest of the pigment epithelium attached to the retina. The surface of the cleaned and healthy retina should appear smooth.
Then treat the isolated retina with about 0.03%higher IDE for 15 to 30 minutes at room temperature. To remove the vitreous humor, note that the procedure should be performed in low light. In the next step, cut a rectangular piece of retina about two by two millimeters, which contains the full thickness of the retina by removing the curved edges with a razor blade.
Then transfer the retinal piece to a small container filled with agar solution and try to minimize the amount of solution around the retina as it is placed into the liquid agar. Subsequently immerse the retina with the agar directly in the ice cold slice solution in order to solidify the agar. Next, cut the solid agar block containing the rectangular piece of retina into a one by one by one centimeter cube.
Glue the block to the surface of the slicing plate in the preco slice chamber. Then slice the block into slices with the thickness of 200 to 250 microns in the ice cold slice solution. Using a vibrato, a total of five to 10 slices can be obtained, which are viable for about five to six hours.
After that, transfer one of the slices to the recording chamber. Place a U-shaped platinum frame with a grid of nylon threads on top of the slice. Perfuse the slice continuously at the rate of one to two milliliters per minute with recording solution bubbled with carbogen.
In this step, use a syringe with a 0.2 micron filter tip to fill the glass patch pipette with internal solution to a level one to two centimeters from the back of the pipette. Then remove air bubbles from the tip afterward. Secure the pipette in the holder.
Apply positive pressure to the pipette and move it towards the targeted terminal using a micro manipulator while moving the tip downward through the slice. Move the pipette in a lateral sweeping direction to ensure that the slice is not pulled along with the tip. Then move the pipette tip around the terminal to clean the membrane surface.
Push the tip downward against the edge of the terminal to create a dimple and release the positive pressure. If a giga ohm seal is not formed immediately apply slight negative pressure to the pipette. Once a giga ohm seal is formed, switch the E PC nine patch clamp amplifier to the onsell recording mode and change the holding potential to minus 60 or minus 70 millivolts.
Apply the fast capacitance correction and wait for the seal to stabilize between five to 10 giga ohms. Then rupture the membrane with sharp, gentle suction. To establish whole cell recording configuration.
You can also use mouth suction instead of a syringe. After the whole cell configuration has been established onto an atomized MB terminal, apply a 200 millisecond step depolarization from minus 70 to zero millivolts. Remember that a two kilohertz sine wave in a voltage clamp mode is applied before and after this depolarization In order to measure membrane capacitance, the depolarization allows the direct measurement of inward calcium currents activated by the opening of voltage gated L type calcium channels with a cesium based internal solution.
In parallel, one is able to monitor the capacitance jump caused by the calcium dependent exocytosis of synaptic vesicles, which increases the membrane surface area. If an intact MB bipolar cell is patched with the pipette on the synaptic terminal, no discernible calcium currents will be observed because of the large leak currents due to the gap junction coupling in the do rights of the MB bipolar cells. What one observes is a large outward leak like current.
However, one is still able to monitor the capacitance jump with a high frequency sinusoidal stimulus activated before and after the depolarizing step shown. Here are the examples of I-R-D-I-C, images of M bipolar cell terminals. The intact terminals are more likely to appear elliptical as shown in A and B while C shows the mized terminal, which appears round and circular.
The dye filling method can also be used to classify the cell terminals. Here are the examples of the fluorescence images of MBI bipolar cell terminals with an intact axon in D, a partially cut axon in e and a fully removed axon in F.Their teria are indicated by the blue arrows. Here shows the direct measurements of calcium imaging signals exocytosis and inhibitory feedback.
In an MB bipolar cell terminal, a transient rising calcium concentration in the teleria of an MB terminal was activated by a voltage clamp step depolarization in a the corresponding fluorescence image of the MB teleria can be seen in B.Examples of short-term synaptic depression of neurotransmitter release are shown in C.While attempting this procedure, it is important to remember to minimize the amount of solution around retina as it is placed into the kit agar. This will improve the attachment of Latinized from the agar block.
