The overall goal of this procedure is to construct an affordable inject road to micro inject neurotransmitters into the rat superior colliculus while monitoring multi-unit neuronal activity. This is accomplished by first creating a glass pipette using a glass capillary and a vertical micro pipette puller. The second step is to insert a silver wire into the capillary with a section of wire protruding out from the non tapered end.
Next glue is used to create a tight seal between the glass pipette and a hypodermic needle. The final steps are to fill the pipette with a Chicago sky blue colored GABA solution attached to a micro syringe filled with mineral oil and fixed to a micro injector. Ultimately, this construction of an inject with accessible and affordable parts can be used to inactivate deep brain structures while recording neuronal activity of the inactivated area before, during, and after injection of a drug.
There are a few advantages of this technique over other existing methods such as gas-based pressure pulses and photic injection systems. One is that it is a cost efficient method that allows for function-based control over the drug delivery, which limits the tissue damage that is caused from over injection. Another advantage that this system offers is a combined recording capability, which allows for efficient control over the temporal component of neural deactivation.
To prepare the recording injection pipettes, first pull a seven centimeter capillary with a one millimeter outer diameter for a cop vertical model. Seven 20 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 viewfinders ruler. It will produce a relatively low impedance of 0.3 to 0.6 mega ohms. Then from the non tapered end, insert a silver wire into the pipette about seven centimeters long, and leave one to two 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 plane 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 bgma 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 bgma.
Next, adjust the position of the tube to a location of interest using coordinates from the bgma. Use the 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 Dora MA does not need to be removed as the inject will penetrate it going forward. Keep the exposed cortex irrigated with cerebral spinal fluid.
Prepare the inject by first filling of five 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 one milliliter syringe with a 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 inject road. Check for escaping fluid at possible leak points.
Swab up any excess fluid and recheck for leakage. Now, attach the micros syringe with mineral oil to the inject road. 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 now securely mount the injector to a micro pump system.
Then move the position of the inject road to the target coordinates and lower the electrode to the superior colus, 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 inject, 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 an activation of neuronal activity in the superior colus, the electrode should show a reduction in spikes during the injection of the inhibitory solution.
Visually evoked potentials were obtained in the superior colus. Following a 300 millisecond flash to the contralateral eye upon the injection of gaba, the spiking activity in response to a flash stimulus was suppressed for 45 to 60 minutes. While attending this procedure, it is important to check for leaks during loading and to verify that the drug solution is flowing freely through the inject tip.
