Drug Microinjection with Electrophysiological Recording in a Rat Brain
Trascrizione
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 micropump 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.
