Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

The overall goal of this procedure is to monitor and localize the activity of single neurons within the subcortical brain structures of alert head restrained rats. This is accomplished by first acclimating the rat to body restraint in a cloth sock. The second step is to surgically implant a head restraint apparatus and recording chamber such that the rat can be maintained in the stereotaxic plane during multiple subsequent recording sessions.

Next, the rat is placed in an appropriate jig for conducting the behavioral and electrophysiological experiments and juxta cellular neuronal recordings are performed. This process enables unambiguous isolation of single units. The final step is to mark the anatomical location of the recording site with Chicago Sky blue dye so that it can be visualized in post hoc is totology.

The main advantage of this technique over commonly used methods like extracellular recording with metal micro electrodes are that spikes from single units can be isolated unambiguously and that the recording site can be revealed in post hoc histology. To begin this procedure, anesthetize the animal with a ketamine xylazine injection. Check the toe withdrawal reflex to determine the plane of anesthesia and maintain anesthesia by further administration of anesthetic.

If necessary, shave the head and disinfect the surgical site with povidone iodine. Afterward, place the rat in a stereotaxic holding frame and secure its head with ear bars. Make an incision with a scalpel along the midline of the cranium from the midpoint between the eyes to the back of the ears.

Then remove a two to three millimeter strip of skin on both sides of the incision. Scrape away the periosteum to expose the cranium extending to the lateral ridges. Subsequently cover the exposed cranium with a thin layer of super glue.

Next drill a small hole immediately posterior to where the bgma suture meets the lateral ridge at a 30 to 45 degree angle into the cranium. Then screw in a zero 80 flat bottom machine. Screw to the hole at a 30 to 45 degree angle, and be careful not to screw in too deeply to avoid damaging the underlying brain tissue.

Repeat this procedure for six additional screws in this configuration. After that, apply super glue to the base of all the screws. Now places syringe needle in the stereo tax manipulator.

Make a dent in the cranium with the needle to mark an appropriate reference location. Then mark the dent with a permanent marker. Subsequently, make a craniotomy centered on the desired coordinate and leave the dura matter intact.

Cover the craniotomy with modified artificial cerebral spinal fluid. Next, cut a 0.2 milliliter centrifuge tube into four to five millimeters in length and cut off the cap. Place the tube on the cranium and center it over the craniotomy.

Then apply dental cement around the bottom of the tube to seal the base of the tube to the cranium. And be careful not to leak any cement into the exposed craniotomy. Now drill another small hole in the contralateral cranial plate.

Carefully insert the reference wire that has been soldered to a pin connector. After that, apply super glue to the hole in which the wire was inserted to keep the pin and wire in place. Then mix the two parts of the silicone gel kit in equal portions.

Wait two minutes and fill the centrifuge tube with gel. At approximately one third full, attach the head plate to the head restraint bar. Here the bar has been attached to a right angle post clamp at a 45 degree pitch angle.

Subsequently, attach the holding bar to the stereo tax manipulator arm using the post clamp so that the bar is parallel to the ear bars, lower the bar and plate so that the plate is posterior of the lambda suture and anterior to the coddle mos screw. Then grasp the front head bolt at an approximately 45 degree angle with a clamp so the head of the screw faces down and towards the tail of the animal. After that, lower the screw to touch the cranium.

Next, secure the bolt and plate in place with dental acrylic. Apply a layer of dental cement around the edges of the dental acrylic to cement the skin to the implant. After the cement has dried, poke several holes in the cap of the centrifuge tube and place the cap on the tube.

Now remove the clamp. Carefully remove the head holding bar from the stereo tac. Then remove the head plate from the bar.

At the end, remove the animal from the stereo attacks and administer postoperative care and monitoring. In this procedure, polo pipette with quartz capillary tubing on a carbon dioxide laser micro pipette polar. Then secure the pipette in place under a differential interference contrast microscope equipped with a long working distance objective.

Slowly move a glass block into the field of view with a pipette tip. Using the stage micro manipulator, break the tip of the pipette by gently touching it to the glass. Repeat the procedure until the pipette tip.

Outer diameter is between one and three micrometers. Next, prepare the extracellular saline and add 2%Chicago sky blue. Using a syringe with a 30 gauge needle, fill the back end of the pipette with a solution.

Then place the rat in the cloth sock and transfer the cloth sock into a rigid tube on an appropriate experimental jig. Attach the head restraining plate on the rat to the corresponding piece on the jig. Next place an 8 32 nut on the head restraining bolt that is implanted on the rat.

Subsequently, screw in a threaded stainless steel rod to the head bolt. After that, open the recording chamber and remove the silicone gel. Clean the craniotomy using fine forceps.

If tissue has regrown in the craniotomy, use iso fluorine anesthesia during this process if necessary. Next, attach the pipette to the motorized micro manipulator. Move the pipette to the desired recording location in the anterior, posterior, and medial lateral axis.

Then advance the pipette ventrally through the dura until it is approximately 500 micrometers dorsal to the intended recording location. Slowly advance the pipette while listening for spiking events on an audio monitor of the amplified voltage recorded. Once spiking events are identified, continue to move the pipette about zero to 100 micrometers until the positive voltage deflections are greater than approximately 500 microvolts.

To label the recording site, set the current source to negative four microamps with two second pulses at 50%duty cycle and pass the current for four minutes to ionophore, inject Chicago sky blue through the pipette. After the experiment, perfuse the animal according to standard practice. Then section the brain and counterstain as necessary to identify the anatomical location of the recording site shown.

Here is the sample spiking activity of a VPM thalamic unit as a rat is actively whisking. Here is a histogram of spike times aligned to the instantaneous phase of Vibra motion. There are more spikes during the retraction phase of whisking.

After the recording, the location of this unit was labeled via aphoresis of Chicago sky blue dye. As shown here, the tissue is counter staind for cytochrome oxidase activity to reveal the neuro anatomical borders of VPM thalamus us. After watching this video, you should have a good understanding of how to perform surgical and electrophysiological techniques that maintain the animal's head in the stereotaxic plane, and that unambiguously isolate the spiking activity of single neurons.