The overall goal of this procedure is to manipulate the impact of synaptic inputs of neuronal processing using microiontophoretic injections of neuroactive substances in awake animals. This method can help disentangle key functionalities using sensory systems such as the role of neurotransmitters and their interaction on sensory stimuli and coding. The main advantage of this technique is that it allows the manipulation of local synatic inputs to the tab and the study of neuronal modulatory systems without being affected by anesthesia.
Though this prodigal was here in restrained mice, it can also be used in other mammals such as bats, rats, or guinea pigs. To begin this procedure, place the tungsten wires in a custom built alignment tool for cutting. Carefully attach the loose ends of the wires with adhesive tape and cut them to the desired length.
Next, pull the tape gently in order to remove the tungsten wires from the alignment tool. Making sure that all the wires remain attached to the tape. Then, roll the tape onto a polished brass spindle and hold the wires firmly against the spindle so there's a good electrical connection.
After that, connect the spindle to a 5 RPM motor in the workstation. The spindle access is angled 45 degrees relative to the surface of the etching solution. Subsequently, immerse one third of the wires in the solution.
Next, immerse a carbon rod electrode into the etching bath to close the circuit. Turn on the workstation so the spindle starts to rotate then adjust the current that passes through the electrodes and the etching solution to 250 milliamps. Stop the tip etching when the current drops to 200 milliamps in order to etch the wire tips into very fine points.
Now, place a wire into a borosilicate glass capillary with it's lower end blocked with modeling clay. Hold the pipette using a heating coil and a suspended plunger. Secure the glass covered wire to a slide before placing it under the microscope.
Set the tip and touch you boric bead in focus. Then heat the bead until it melts slightly. After that, insert about 10 to 15 microns of the wire tip and turn the heating potentiometers off.
As the bead cools down, remove the glass from the tip of the electrode to expose the tungsten. To obtain a tip of about 15 millimeters, set the polar to the appropriate parameters. Pull the capillary to obtain two pipettes with the tip secluded.
Mount a pole pipette on a slide using modeling clay and break its tip using fine scissors, forceps or the flat side of a scalpel. Adjust the angle between the electrode and the multi-barrel slightly to obtain a better bonding and thinner ensemble. Under the microscope, carefully align the tungsten electrode over the multi-barrel tip.
Once aligned, lower the electrode until it contacts the multi-barrel. Fitting in the groove between the two upper barrels then slide the tip of the electrode until it protrudes 15 to 20 micrometers away from the tip of the multi-barrel. Now apply a small drop of light curable adhesive on the tungsten and the multi-barrel ensemble about 5 millimeters away from their tips.
Keep the glue from reaching the tips to avoid blocking the multi-barrel channels and cure the glue using a blue light LED lamp Now, place the anesthetized animal on a heating blanket to maintain it's temperature at 38 degrees celsius and stabilize the animals head in a stereotaxic frame with two ear bars and a bite bar Then apply ophthalmic gel to protect the eyes. Next, shave the scalp and apply povidone iodine to disinfect the skin. Using a scalpel make an incision along the midline to expose the skull and retract the perastium covering the peridal and the more rostral part of the occipital bones.
Next, apply a thin layer of light curable adhesive on the skull and the base of the head post. Wait 10 seconds and place the head post over the rostral area of the parietal bones along the midline. Use a blue light source to cure the adhesive for 20 seconds for an effective bonding strength.
Subsequently, drill two holes around and behind the base of the head post using a small drill bit. Place a watch screw in a drilled hole to serve as an additional contact point between the skull and the head stage. Make one and a half turns to snuggley fit the screws and test with forceps to make sure they are not loose.
Then, insert the tip of the silver ground wire into the third hole and make sure it contacts the dura. Apply a small drop of water resistant cyanoacrylate glue to bind the wire to the skull. Afterward, apply light curing composite to reinforce the bond of the head post with the skull by covering the base of the head post, the lower part of the silver wire and the screws.
Be careful not to extend beyond lambda in order to allow access during recording and cure the composite using a blue light source. Then, retract the muscle at the back of the neck near it's insertion on the skull. Using a small trephine, drill a round window just below the lambda suture and lateral to the midline to expose the inferior colliculus.
In this procedure, place the animal's body into a custom made foam cushioned restrainer. Secure the head post to a holder attached to the stereotaxic frame. This is a good time to give the animal a liquid reward.
Next, cover the animal's body with a cotton blanket and loosely fasten it using a plastic hemi-cylinder and adhesive tape. Use a syringe with a flexible tip to fill the barrels with the chosen drugs. Using the micro-manipulator, place the multi-barrel electrode on its holder.
Next, place an un-coded silver wire inside each barrel so one end contacts the solution and the other end is accessible. Then connect those ends to the microiontophoresis device. After that, connect the positive terminal to the end of the tungsten electrode and the ground terminal to the silver wire that contacts the dura.
Subsequently, move the multi-barrel electrode until the tip contacts the surface of the brain. Apply warm sterile saline to prevent the desiccation of the brain tissue. At that point, apply retention current to avoid drug leakage from the barrel tip while looking for single unit activity.
Record neural activity and iontophoretic application of gabazine. Shown here is the frequency response area of four recorded neurons in the inferior colliculus before and during the micro iontophoretic injection of gabazine. The black crosses indicate that frequencies chosen to be presented as rare and repetitive sounds.
Here, are the accumulated peristimulus time histograms of the neural response to all the frequencies and intensities presented to construct the response area before and during the injection of gabazine. The black bars indicate the sound duration. Here are the dot rasters of the spiking response to a pair of frequencies presented as rare in red and repetitive stimulus in blue before and during the application of gabazine.
The shaded background indicates the duration of the stimulus. These dot rasters show the responses when the relative probabilities of the parafrequencies were switched. This graph, shows the single neuron responses to seven pairs of frequencies presented as the rare and the repetitive sound before and during gabazine application.
And this graph, shows the SSA indexes of the neural response to rare and repetitive sounds measured before and during the application of gabazine. Once mastered, the described Procedure allow the adjustment of the specific parameters according to the neural structure and type of neuron of interest. While attempting head restraint animal recordings it's critical to remember that the recording times is determined and limited by the level of habituation of the animal but an important advantage is that multiple record incisions can be performed on the same animal several days minimizing the number of animals used.
By performing microiontophoretic using piggy back electrodes other procedures like injections of neural transistors can be combined in order to answer additional questions like the source of inputs and projection target of neurons within the recording site. A critical issue when working with the awake animals is their well-being so never exceed the amount of recalling time approved in your protocol and please constantly monitor to check any sign of discomfort. If so, you must stop recording immediately.
