バイオミメティック化学ニューロモデュレーション神経伝達物質グルタミン酸の in Vitroラット網膜のための方法論

The overall goal of this methodology is to enable a novel chemical-based neuromodulation of retinal neurons using neurotransmitters, which could potentially replace the functionality of defective retinal cells in patients with neurodegenerative blindness to restore vision. This method can help answer key questions in the field of artificial vision. Such as can glutamate, a primary retinal neurotransmitter, be used to therapeutically stimulate a photoreceptor degenerated retina?

The main advantage of our technique is it is biomimetic, stimulating the retina this way offers the potential for higher visual acuity and more naturalistic vision than is possible with electrical stimulation. The implications of this technique extend toward the treatment of blindness, due to photoreceptor degenerative diseases because early therapeutic intervention of a degenerated retina could restore glutamate sensitivity in retinal neurons. To begin this procedure, place both enucleated eyes in a 60 milimeter diameter Petri dish with fresh oxygenated Ames'medium solution.

Using a dissection stereo microscope, make a small incision in the corneal face using a scalpel or a pair of sharp scissors. Next, make a cut from this small incision to the edge of the cornea. And extend it in a circumferential section around the entire edge of the cornea.

Then, remove the detached cornea along with the lens, translucent aqueous, and vitreous humours. While gently holding the eye cup with one pair of forceps, carefully make two small incisions on opposite sides of the eye cup. Next, use two pairs of forceps to gently pull apart the eye cup at each of the incisions.

And separate the retina from the sclera. Slowly lift the entire retina from the sclera and the eye cup and cut the optic nerve if it is still attached. After that, make longitudinal cuts in the retina to obtain half or quarter sections.

And then gently spread one retinal section onto a nylon mesh with the ganglion cells facing away from the mesh. Place the mesh and retina onto a perforated MultiElectrode Array with the ganglion cells in contact with a PMEA surface. And then place a weight on top of the mesh to keep the retina in place.

In this procedure, under dim red illumination, place PMEA in the MEA amplifier and close the amplifier latches. Position the top perfusion outlet inside the PMEA chamber and turn on the top perfusion valve to achieve a perfusion rate of approximately three milliliters per minute. Next, position the top suction inlet at the desired perfuse eight level.

Ensure that it is working. Afterward, open the data acquisition software and click the play button to start receiving data. Ensure that all the PMEA channels are noise free and recording neuro-signals.

If not, reposition the PMEA within the amplifier to obtain better contact between the amplifier pins and the PMEA contacts. Then, ensure that the bottom perfusion line is clear of any air bubbles. Turn on the bottom perfusion valve to ensure the retina is supplied with oxygen and nutrients.

Subsequently, turn on the high speed camera attached to the inverted optical microscope and open the imaging software. Once the bottom perfusion is confirmed to be flowing, slowly ramp up the bottom suction by manually turning the vacuum pressure knob on the vacuum waste kit while observing the retina through the inverted microscope. Once a suction force is observed to act on the retina, cease increasing the suction.

After ensuring the retina has been held in place by the bottom suction, take the weight off the retina using forceps and gently remove the nylon mesh by peeling one corner carefully from the retina. Keep the perfusion running for approximately 30 minutes to allow the retina to stabilize from the surgical trauma. In this step, carefully insert a pre-pulled 10 micrometer diameter micropipette into a standard pipette holder containing a 50 micrometer diameter silver-silver chloride wire electrode.

If using a multiport microfluidic device instead, insert the stainless steel rod connected to the device into a standard pipette holder without a wire electrode. If utilizing a glass micropipette, interface the pipette holder with a patch clamp headstage and connect the pressure port lower connection of the pipette holder to channel one of the pressure injection system. Or, if using the multiport device, connect the pressure port luer connections of each of the eight injection ports, with channel one through eight of the pressure injection system.

Next, manually turn on the pressure injection system and turn on channel one. Ensure that the system is vented to atmosphere and set the injection pressure to 0.1 pounds per square inch. Afterward, turn on the micromanipulator and calibrate it by pressing the calibrate button on the manipulator controller.

Position the micromanipulator so that the micropipette tip is approximately 30 millimeters above the MEA amplifier. Then, fill a small Petri dish with glutamate solution and place it underneath the micromanipulator. Lower the micropipette tip into the solution and fill the glass micropipette or the multiport device tubing with approximately 20 millimeters of solution.

Subsequently, lift the micropipette tip, or device, out of solution. Remove the Petri dish and position the micromanipulator above the PMEA chamber. Using a boom stand mounted stereo microscope, align the micropipette tip, or the corners of the device, with a reference mark etched into the PMEA chamber ring.

Then, store the manipulator positions into the control surface using the store reference A and store reference B buttons to map the coordinate system of the manipulator of the PMEA electrodes. Following that, use the manipulator control software. Select a target PMEA electrode with robust spontaneous activity and click the move to channel button to align the glass micropipette with the target electrode.

If using the multiport device, align the device microports with target PMEA electrodes with robust spontaneous activity using the same process. If impedance measurement is available, turn on patch clamp amplifier and initiate the impedance visualization software by clicking the start button to visualize the impedance of the silver-silver chloride electrode inside the pipette holder. While observing the realtime impedance signals, slowly lower the micropipette, or device, until it contacts the retinal surface as indicated by a rapid increase in the impedance signal.

For subsurface stimulation, lower the pipette 40 micrometers further for S334 tera three retinas, or 70 micrometers for wild-type retinas. Perform a few short duration injections using the pressure injection system. To determine if the cells near the micropipette tip, or device microports, are receptive to glutamate stimulation by observing the neurosignals.

Now, orient the green LED towards the top surface of the retina. Begin recording using the data acquisition software by typing the file name and clicking the record button. Once recording has started, open the stimulus control program and load the default stimulus file by clicking the read stimulus file button.

Next, click on the run stimulus file button to initiate the default stimulus file. After the stimulus file has been completed, stop the recording to save the file for future spike sorting and data analysis. Here are the representative recordings from nine PMEA electrodes, showing the high-pass filtered electrode data during visual light stimulation with a green LED, where each rectangle shows the neural data from a unique electrode.

Each electrode recording illustrates data collected in the first second after turning on the green LED. Spikes were identified using a threshold voltage of negative 18 microvolts. Visual stimulation caused a burst of spikes in all electrodes except the top center one, which possessed an inhibitory response to light.

A similar plot for the same electrodes, showing spontaneous neural activity without visual or injection stimulation. Although smaller bursts were present, the patterns of spikes were very different from those recorded in response to visual stimulation. Here are the representative recordings from the same subset of electrodes recorded immediately after a glutamate injection at the central electrode.

The injected glutamate elicited a burst of spikes in the central electrode that was very similar to the visually evoked spike bursts. All other electrodes were unaffected by the glutamate injection which demonstrates the fine spatial resolution of the chemical stimulation technique. Once mastered, this technique can be done in four to six hours if it is performed properly.

While attempting this procedure, it's important to remember to monitor the perfusion to ensure the retina is continuously supplied with oxygen and nutrients throughout the entire experiment. This technique could pave the way for researchers in the field of artificial vision and visual prosthesis development to explore chemical-based neuromodulation for treating blindness from photoreceptor degenerative diseases. After watching this video, you should have a good understanding of how to accomplish neuromodulation of retinal neurons using neurotransmitters.