視床下部スライスに緑色蛍光タンパク質（GFP）によって識別されるゴナドトロピン放出ホルモン（GnRH）ニューロンから二重の体細胞の録音

This video describes a procedure for performing dual cell attached recordings from gonadotropin releasing hormone neurons. Hypothalamic brain slices are prepared in either coronal or sagittal orientation. The slices are then visualized using an immersion objective and pipettes are positioned over two neurons.

Low resistant seals are formed by releasing positive pressure, then applying negative pressure to the somatic membrane cell. Attached recordings can then be made. Hi, I'm Kelly Suiter.

Welcome to my laboratory in the Department of Biology at the University of Texas San Antonio. I'm Peter Heman from the Suitor Lab. Today we're gonna show you a procedure for obtaining dual somatic recordings from gonadotropin releasing hormone neurons and hypothalamic slices from mice.

We use this procedure in my laboratory to study coordinated firing in GNH neurons. So let's get started. Begin the procedure by removing the brain from a mouse euthanized by decapitation under isof fluorine anesthesia.

The mouse used in today's procedure expresses green fluorescent protein under the regulation of the GNRH peptide promoter, which is expressed exclusively in GNRH neurons. After removal, place the brain in cold, artificial cerebral spinal fluid or A CSF. Place the brain on its dorsal surface to expose the hypothalamus throughout the blocking procedure.

Continuously moisten the brain with cold A CSF using a glass pipette. To prepare a sagittal slice, use a razor blade to remove the cerebellum. Then remove the lateral regions of the brain.

The rostral portions of the cortex provide the support for brain slicing and for placement of silver wires to secure the brain. In the recording, well next place a small amount of superglue on the cutting platform. Using a spatula, lift the brain, bring a Kim wipe to the surface of the spatula and draw excess A CSF away from the brain.

Then using a razor blade, gently slide the brain forward onto the glue. It is important to avoid touching or pushing down on the brain during the transfer. The weight of the brain itself should be sufficient to secure it.

Secure the brain and the cutting platform to a vibrating microtome and fill the cutting well with cold A CSF. The cold solution firms the brain protecting it from damage During the cutting procedure, slowly cut slices. Each slice should float freely away from the brain.

Withdraw slices from the cutting chamber as they're cut, and place them in a slice incubation chamber continuously supplied with oxygen, using a mixture of 95%oxygen and 5%carbon dioxide in a warm water bath, approximately 32 degrees Celsius for one to two hours. Continue cutting slices and placing them in the incubation chamber. If the slices begin to curl during cutting, slow down the speed at which the blade advances.

Using a vertical puller, prepare glass pipettes. A uniformly tapered tip with an opening of 0.1 millimeters is recommended. Next, using a second pipette coat, the pipettes with Sard 180 4 to reduce capacitance.

Then using a heat gun cure the sill guarded tips. Add a small amount of Lucifer yellow to a thaw 0.5 milliliter aliquot of intracellular solution and sonicate. The solution should appear yellow.

Fill the pipette with the yellow intracellular solution. This will allow visualization of the tips using a GFP filter during the clamp recording procedure. After slices of incubated for one to two hours, remove one from the incubation chamber and move it to the recording chamber.

Load the Lucifer yellow intracellular filled pipettes into the pipette holders for each head stage. Then attach an empty three milliliter syringe to each pipette holder with a piece of tubing. These will be used to apply positive pressure to prevent clogging of the pipettes during the procedure.

Using the GFP filter, visualize the pipette tips. Ensure that the slice has greater than four recording quality neurons. A good quality neuron will be somewhat pear shaped.

Very round neurons, as well as shriveled neurons are generally in the process of dying. Select two neurons for recording selected neurons are often at different levels of the slice. Using the computer software, set the Axo clamp to be amplifier to pass current injection pulses.

Then using a microm manipulator position, the first pipette over the largest surface of the neuron, that is deepest in the slice position, the second pipette at the more superficial neuron. This will prevent the positioning of the second pipette from shifting the positioning of the first pipette. As the slice will move slightly in the vertical plane as pipettes, enter the slice next, lower the first pipette onto the first neuron, and immediately apply positive pressure using the syringe.

The positive pressure will induce a small dimple on the surface of the cell. Once the first pipette has been placed, return to the top of the recording chamber and lower the second recording pipette into position again, applying positive pressure using the syringe. Once both pipettes have been placed, seal the first neuron by removing the syringe to release positive pressure.

Membranes of healthy neurons will adhere to the pipette. Then using a small amount of mouth suction, apply negative to form a seal with the appropriate resistance. If a seal is not successfully generated between the pipette and neuron, raise the microscope objective and pipette away from the surface of the slice to the top of the perfusion.

Well replace the used pipette with a clean pipette. Once the pipette has been changed, return to the slice and generate a seal using the new pipette. Once a loose seal has been established based on resistance, terminate the current injection pulses from the Axo clamp to be amplifier.

Place the Axo clamp to be amplifier in series with the AM systems 3000 amplifier so that the output of the OC clamp two B amplifier serves as the input for the AM systems 3000 amplifier. The latter amplifier provides the signal for data acquisition. Next, establish a seal for the second neuron and reconfigure the amplifiers.

Once seals have been established with both pipettes begin directly measuring action potentials by acquisition of data. Lucifer yellow was used solely to increase visualization for demonstrating the dual recording technique. However, Lucifer yellow is toxic to neurons and alters the inactivation of sodium currents, which underlie the action potential if one ruptures the seals to fill neurons.

Thus in our method, Alexa 5 68 is used in experiments following recording, the seal can be ruptured, allowing neurons to be filled with Alexa 5 68 for subsequent histology. The cell attached recording method presented in this video was validated by simultaneous traditional whole cell recording and cell attached recording of gonadotropin releasing hormone neurons. As shown here, there's complete agreement between action potentials in the cell attached recording and the whole cell recording during both slow spontaneous firing and repetitive firing.

In the left hand panel, the responses in both the cell attached and whole cell recording configuration to application of a low frequency of simulated AMPA type receptor activation are shown. Note that each action potential in the cell attached recording configuration is associated with a corresponding action potential in the whole cell record. The right hand panel shows the response of the neuron to a high frequency burst of simulated AMPA type receptor activation indicated by the dashed line.

Note the increase in action potential frequency detected in both the cell attached recording configuration in response to the stimulated burst of input and in the whole cell recording configuration, trace G expands the timescale of the portion of the recording shown in D beneath that dashed line when the frequency of activation of simulated AMPA type receptors is increased. Finally, there is agreement between the cell attached and whole cell recording. When a depolarizing current injection pulse is applied to cause the neuron to discharge action potentials.

This panel shows the response of A-G-N-R-H neuron to a current injection pulse in the cell attached recording configuration and in the whole cell recording. The inset in the middle trace expands the waveform of the detected action potential. Note that each detected action potential in the cell attached configuration has a corresponding action potential detected in the whole cell recording configuration.

We've just shown you how to make dual somatic recordings in hypothalamic slices when doing this procedure. It's important to remember that this takes practice. So that's it.

Thanks for watching and good luck with your experiments.