Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

The overall goal of this procedure is to introduce the process of unfolding rodent hippocampus and placing the flat tissue preparation onto the penetrating micro electrode array. For recording. This is accomplished by first isolating a mouse hippocampus from half of its brain.

The second step is to unfold the curved structure of the hippocampus with custom made tools. Next, the unfolded hippocampus is transferred to the recording chamber and placed onto the recording array. The final step is to remove the unfolded hippocampus from the array after the experiment.

Ultimately, individual normalization mapping method is used in data analysis to show the neural activity propagation recorded by the penetrating micro electrode array. Well, the main advantage of this technique is that it allows us to record neural activity in two direction transverse and longitudinal. In order to see the actual propagation of neural activity, you need an intact hippocampus, unroll the dent jars and lay down flat into a recording chamber, and the propagation then will take place both transverse and longitudinal directions.

And we were able to then figure out the speed of propagation of the tissue. We were of the activity in the two directions, and we were able to study the propagation mechanisms, in particular, the nons synaptic mechanisms. We were able to see activity propagating with that synaptic transmission with that gap junctions, and because of the speed, we know it's not diffusion.

So now we're trying to work out the actual mechanisms of this propagation. Generally, individuals new to this technique will struggle because of the challenging procedures of unfolding the hippocampus and place the flat tissue preparation onto this penetrating micro electrode array without damaging either the tissue or the array. To begin this procedure, place the mouse head in ice cold oxygenated sucrose.

A CSF use a pair of fine scissors to remove the skin of the skull. Next, cut the skull along the midline and the two ends near the temporal lobe. Then peel the cut skull towards the sides of the head.

In order to expose the brain carefully remove the brain from the skull with the spatula. After that, place the brain on an ice cold surgical stage covered with wet filter paper. Remove the cerebellum with an ice chilled blade.

Subsequently separate the two hemispheres by cutting the midline of the brain. Then place the two separated hemispheres into a beaker filled with ice cold sucrose. A CSF bubbled with 95%oxygen, 5%carbon dioxide.

Transfer one hemisphere to the ice cold stage covered with filter paper. Place regular paper towels or filter papers around the brain to absorb the extra solution. Afterward, use two fire polished glass pipettes to separate the cortex from the central part of the brain in order to expose the hippocampus.

Next, apply two to three drops of ice cold suse A CSF on the tissue, and remove extra solution. Cut the connections with the cortex at the two ends of the hippocampus. Then dissect the hippocampus out of the brain with the fire polished glass tools.

Subsequently separate the whole hippocampus with its ous side facing up and the hippocampal sulcus facing down Quickly apply two to three drops of ice cold sase CSF on the tissue again, and remove the extra solution around it. Then use the fire polished glass tool to turn the whole hippocampus over. To expose the sulcus, use an ice cold blade to trim the septal and temporal ends of the hippocampus.

If necessary, insert a custom made glass needle into the sulcus and cut the fiber track from DG to ca three. Then use a metal wire loop to pull the DG over and unfold the hippocampus. The whole surgery process usually lasts for about two minutes.

Apply another two to three drops of ice cold sucrose A CSF onto the tissue and remove the extra solution around it. Then trim the edges of the unfolded hippocampus with an ice cold blade. Transfer the preparation to the recovering chamber filled with normal A CSF and bubbled with 95%oxygen, 5%carbon dioxide at room temperature for one hour before transferring it to the recording chamber.

In this procedure, fill one bottle with normal A CSF and another bottle with four AP A CSF bubble. The solutions with 95%oxygen, 5%carbon dioxide from the beginning of the experiments. Use a trivalve connector to control which solution will be selected during an experiment.

Next, connect a vacuum tube to the outlet of the chamber. To remove the solution into a dust container, heat the pipeline before delivering the solution into the recording chamber and keep it at 35 degrees Celsius controlled temperature. After closing the inlet and outlet of the recording chamber, use a custom made glass pipette dropper to transfer the unfolded hippocampus to the recording chamber.

Under the microscope position the unfolded hippocampus with its alvia side facing down ca a three area pointing away and ca a one field pointing towards the researcher carefully aspirate away the solution in the chamber using a vacuum pipette from the edge of the recording chamber until the chamber is dried and the tissue lies on the array. Then carefully place a custom made tissue anchor on top of the tissue to hold the unfolded hippocampus onto the array. Refill the recording chamber with a few drops of solution.

Gradually open the inlet and outlet to adjust the flow rate to about two drops per second in the IV trip chamber. Incubate the tissue in the recording chamber with normal A CSF for about one minute. Then switch the solutions.

Apply to four AP dissolved A CSF and adjust the flow rate properly. Incubate the tissue in four AP dissolved A CSF for about five to 10 minutes before recording. To remove the tissue from the PMEA, control the tissue anchor by a micro manipulator and gradually lift the anchor from the recording chamber.

Shut down both the inlet and outlet in order to stop the flow. In the recording chamber, use a small paintbrush to lift the corner of the tissue. If the tissue is not floating in the solution, employ the vacuum tube to dry the chamber carefully with the tissue still sitting on the array.

Then carefully open the inlet to gradually refill the chamber and shut down the inlet to stop the flow. When the recording chamber is full, use the small paintbrush to lift the corner of the tissue.Again. If the tissue is floating in the solution, use the vacuum tube to suck the tissue away.

Open the flow at the inlet and the vacuum at the outlet. Wash the system with distilled water and dry it out. This is an example of the spontaneous activity induced by 100 Micromolar four AP A CSF recorded from one of the micro electrodes located in the basal dendritic region with a signal to noise ratio of 34.9 decibels.

And here are the enlarged activities in the red rectangle. This is the raw data from another micro electrode located closer to the somata with a signal to noise ratio of 27.2 decibels shown. Here is another example of a recording obtained from an electrode positioned within the somatic layer.

In this example, the signal to noise ratio is 18.53 decibels, and here is the baseline noise recorded from a micro electrode. The baseline usually has a peak to peak value from 150 to 200 microvolts, and the impedance of a single electrode is around one to two mega ohms. This video shows the neural propagation recorded with the array and then processed using individual normalization method.

In data analysis, the whole propagation across the entire tissue lasts about 100 milliseconds Following this procedure. Other methods like neuroimaging in the unfolded, hippocampus in vitu can also be performed in order to answer additional questions like the movement of neuro foci. After watching this video, you should have a good understanding of how to unfold hippocampus at place, the flat tissue preparation onto the micro lecture array.