Bouw van een verbeterde multi Tetrode Hyperdrive voor grootschalige neurale opname in ratten gedragen

The overall goal of this procedure is to construct a recording device with multiple independently adjustable electrodes for the examination of brain activity in freely behaving rats. So this method is very exciting because it will help answer several questions related to special navigation, decision making, and other cognitive processes. The main advantage of this hyperdrive is that it provides reliable neuro recording in the brain using materials that are easy to obtain.

Although this method was designed to be used specifically with rats, it can be used with any species of similar or larger head sizes such as non-human primates, thereby allowing investigations of many species. Those new to this method might struggle with the nuance involved in the preparation of the steps cause there's a certain level of experience and accuracy that's required to develop these tricks that can make a successful drive. Begin by expanding the holes in the 3D printed core using different sized drill bits.

Use a diameter of 0.61 millimeter for the 12 ground wire through holes. For the 18 tetrode through holes, first use a 0.66 millimeter bit and then widen with a 0.71 millimeter bit. A 0.84 millimeter diameter bit is used for the 18 guide rod blind holes.

Next, thread the two through holes on top of the core and the remaining eight blind holes with a 0.80 tap. Use a bottoming tap for the blind holes. Create external threads at the base of the core using a 3/8-24 die.

Adjust the die properly so the hyperdrive nut will fit over the new threads. Depending on the desired number of ground wires, insert multiple six millimeter long segments of 23 gauge metal tubing into the ground wire holes in the core gluing them if necessary. File the outer ends of the ground wire cannulas until they are flushed with the core and then clean the cannulas with a 0.30 millimeter diameter steel wire.

Next, fully insert 18 08015.88 millimeter long flathead screws head down into the slots in the core. Do not bend the screws or damage the threads during this process. Using the rod positioning complex into the core station, position 18 17 millimeter segments of 0.89 millimeter diameter welding rod over the guide rod holes in the core and hammer them down about five millimeters until flushed with the screws.

Correct the positions of the welding rods and screws if necessary then tighten the central shoulder screw and the surrounding six screws in the rod positioning complex to secure the outward directions of the rods in the core. Screw the nut onto the core with the rod positioning complex and fit the core into the hyperdrive holder to allow easier positioning under a stereoscope. Fill the slots with dilute dental cement to secure the screws to the core.

Fill two to three slots at a time before the dental cement gets too thick. Scrape away any excess dental cement on the core to maintain a proper fit with the shield then allow to air dry for 15 minutes. Next, glue the screws and rods into the core with thin super glue and allow to dry for 15 minutes.

Clean and expand the two outer holes in the 3D printed shuttle with drill bits. Use a 0.61 millimeter diameter bit for the smaller hole and a 0.89 millimeter bit for the larger hole. Insert the shuttle bolt into the bolt holder base ensuring the proper orientation.

Then close the bolt holder lid. Hold the bolt holder tightly and thread slowly through the hole in the lid with a 0.80 tap. Tap two or three times until smooth.

Then insert the shuttle bolt into the shuttle from the side with the smaller opening. Place the shuttle-shuttle bolt complex upside down in the microdrive assembly station base. Next, smooth both ends of a 15 millimeter segment of 23 gauge metal tubing, then position the tubing over the 0.61 millimeter diameter hole guided by the slot in the station lid.

Hammer the cannula into the hole until the upper end is flushed with the station lid. Remove the outer half of the upper tip of the cannula with a sanding wheel. Clean the cannula with a 0.30 millimeter diameter metal wire.

Then glue the cannula onto the shuttle using thin super glue making sure not to glue the shuttle bolts to the shuttle. Prepare at least 18 microdrives. Test the microdrives on the microdrive rack.

Make sure that the shuttle bolt can rotate smoothly in the shuttle and that the entire microdrive moves freely along the length of the threaded rod. To insert guide cannulas into the hyperdrive core, begin by removing the heat shrink tubes and slide a four millimeter segment of silicone tubing along the bundle to the soldered-unsoldered border. Wedge the slit in the hyperdrive spacer to widen the central hole allowing the spacer to slip around the silicone tube.

Remove the wedge when the spacer sits at the center of the silicone tube. Next, organize the positions of the guide cannulas in the bundle by placing 10 centimeter long segments of 0.18 millimeter diameter metal wire through each cannula into a specific tetrode hole in the hyperdrive core preventing any crossover of the wires or cannulas in the process. Bend the end of the wires to hold them in place.

Push the cannulas through their respective holes in the core being careful to avoid bending or crossing between them until the free end of each cannula is at least two millimeters outside the upper end of the tetrode hole. Secure the spacer by screwing the nut onto the core being careful to prevent the spacer from rotating. Apply a drop of very dilute dental cement from the top of the core onto the junction of the cannulas to secure their relative positions.

Cut the guide wires from the soldered end of the bundle and remove them from the cannulas by retracting from the free end. Load the microdrives slowly and carefully onto each threaded rod of the core. Confirm that the 23 gauge microdrive cannula goes into the tetrode hole smoothly, the 30 gauge guide cannula goes into the 23 gauge microdrive cannula smoothly, and the shuttle bolt turn smoothly along the threaded rod.

Screw the microdrives down to one to 1.5 millimeters above the lower end of the threaded rods. Then cut 18 pieces of polyamide tubing into 43 millimeter segments which is the length of the guide cannula bundle plus seven millimeters. Clean each tube with a 0.8 millimeter diameter steel wire.

Invert the core, insert the polyamide tubes carefully into the guide cannulas from the soldered end and push them all the way in. Then flip the core upright and glue the upper end of the polyamide tube onto the microdrive cannula with thick super glue. Place the core upside down and let the glue dry for 15 minutes.

After drying, cut the extra polyamide tubing at the upper end leaving 0.5 to one millimeter outside the microdrive cannula. To assemble the ground wires, first cut lengths of 25 to 30 millimeters from coated steel wire. Then strip two millimeters of the plastic insulation from both tips of the wires and insert one end of each into both sides of a six to eight millimeter long 30 gauge cannula.

Flatten this end of each cannula to secure the connection to their respective wires. Finally, insert the round end of the 30 gauge cannula into the upper end of the ground wire cannula in the core and press to make the insertion tight. Then complete assembly of the multi-tetrode hyperdrive according to the instructions in the written protocol.

This scatter plot shows the relationship between peak to peak amplitudes of spikes recorded from two electrodes of a tetrode located in the post rhinal cortex. Each dot corresponds to one spike. Clusters of spikes are likely to originate from the same cell.

Four clusters are color coded as seen here. The mean spike waveforms from all four tetrode channels of the color coded cells shown in the previous scatter plot are seen here. Traces of local field potential in the theta frequency range recorded simultaneously from four different tetrodes located in the medial entorhinal cortex when the rat was freely foraging are seen here.

While attempting this procedure, it's important to work with precision throughout the entire procedure to ensure that all parts fit together properly. Also use care and wear proper PPE when working with the demo tool to avoid injury. After watching this video, you should have a good understanding of how to construct these improved hyperdrives carrying multiple tetrodes for chronic neuro recordings in behaving rats.