Inhibition of Neuronal Activity and Its Detection Using Electrophysiology in a Rat

Begin with an anesthetized rat secured in a stereotaxic frame with its skull exposed.

Using the anatomical landmarks, the bregma and the lambda, locate the superior colliculus, a deep brain structure responsible for processing visual information.

Drill to create a bone flap, remove it, and apply a buffer to keep the exposed brain hydrated.

Position an injectrode assembly consisting of a micropipette with an electrode attached to a microsyringe.

The micropipette contains an inhibitory neurotransmitter solution to suppress neuronal excitability while the electrode simultaneously records neuronal activity.

Lower the injectrode into the superior colliculus and cover the exposed surface with molten agar to prevent dehydration.

Present light flashes to the eye to stimulate neurons and evoke synchronized action potentials.

Inject the neurotransmitter, which binds to its receptors and triggers chloride ion influx.

This influx hyperpolarizes the membrane potential, thereby suppressing action potentials.

As the neurotransmitter concentration declines over time, neuronal activity recovers.

To prepare the recording injection pipettes, first, pull a 7-centimeter capillary with a 1 millimeter outer diameter. For a cop vertical model 720 or similar unit, set the heater to 18 and the solenoid to one. Next, wearing gloves, manually break the tip to make an opening and verify under a microscope that the inner diameter is between 30 and 40 micrometers. Measure this with the viewfinder's ruler. It will produce a relatively low impedance of 0.3 to 0.6 megaohms.

Then from the non-tapered end, insert a silver wire into the pipette about 7 centimeters long, and leave 1 to 2 centimeters hanging out of the pipette. Bend this excess wire orthogonally to the capillary. Next, take a 30 gauge hypodermic needle and apply flexible plastic adhesive onto the shaft. Then slowly insert the needle into the pipette as far as possible.

Add more adhesive to create a seal at the junction between the pipette and the needle. With the pipette tip upwards, let the assembly dry for about 12 hours. After anesthetizing and putting the rat into a stereotaxic frame, apply ophthalmic ointment, shave its head clean.

After applying a local anesthetic and confirming a surgical plan of anesthesia, proceed with incising the scalp along the median using a number 10 scalpel blade. This will expose the coronal and sagittal sutures. Then, using a surgical spatula, identify the lambda and bregma anatomical references.

Adjust the position of the head so that these landmarks are level in the same horizontal plane. Then zero a fine rigid pointer like a glass tube on the bregma. Next, adjust the position of the tube to a location of interest using coordinates from the bregma.

Use a Sharpie to draw a square around the target area for the craniotomy. Now, with the surgical sterilized drill, slowly remove the bone along the marks. Begin with using as little pressure as possible and keep the drill moving to avoid head-induced tissue damage due to friction.

When the bone has become thin, carefully remove the square section with tweezers. The dura mater does not need to be removed as the inject rod will penetrate it. Going forward, keep the exposed cortex irrigated with cerebrospinal fluid. Prepare the injectrode by first filling a 5 to 10-microliter syringe with mineral oil. Next, prepare the drug of interest with a dye in physiological saline. Here, 0.5% Chicago Sky Blue is mixed with 300 micromolar GABA.

Now, fill the injection pipette by first loading a 1-milliliter syringe with the prepared solution, and then using the syringe to load the pipette. When removing the syringe, keep light pressure on the plunger, so a vacuum does not remove solution from the injectrode. Check for escaping fluid at possible leak points. Swab up any excess fluid and recheck for leakage.

Now attach the micro syringe with mineral oil to the injectrode. Then wipe away any excess solution with gauze. Check the injection tip. It should be possible to flow small drops of solution through it. Otherwise, it is blocked or leaking. Now, securely mount the injectrode to a micro pump system. Then move the position of the injectrode to the target coordinates, and lower the electrode to the superior colliculus, which can be identified through the recording of visual evoked potentials.

While the electrode is lowered, apply a small positive pressure by applying a low rate of injection to avoid clogging. Interrupt the injection when the structure is reached. After inserting the injectrode, cover the exposed cortex with warm agar to prevent desiccation.

After the final placement of the electrode, wait 30 minutes to allow the stabilization of the tissue surrounding the electrode. Then inject 400 to 800 nanoliters of solution at a rate of 40 nanoliters per minute, until inactivation of neuronal activity in the superior colliculus. The electrode should show a reduction in spikes during the injection of the inhibitory solution.