The overall goal of this procedure is to describe the methodology that will allow us to examine how individual neurons and local field potentials in different cortical layers of the primary visual cortex. In code sensory information. The procedure begins with describing the construction of the computer controlled micro drive system and the use of a multi contact laminar electrode for the recording in the primary visual cortex.
The next step is to perform an evoked response potential paradigm after the electrode has been advanced to the target brain region. Following this current source density analysis is used to identify the cortical layers according to the polarity inversion, accompanied by the sync source configuration. The final step of the procedure is to perform receptive field mapping and analyze the differences in neural activity in response to visual stimulation.
Ultimately, results can be obtained that show layer specific changes in encoding sensory information. Hi, my name is Sarah Eagleman and I'm a graduate student at the University of Texas Medical School in Houston. The main advantage of this technique over existing methods like multi electrode arrays, is that the U probe can record neural activity across many millimeters of cortex simultaneously in a single penetration.
Hi, my name is Brian Hansen. I'm a graduate student working at the University of Texas Medical School in Houston. This method can answer key questions in the neuroscience field, exploring whether and how information is processed in a laminar specific manner.
To construct the name electrode drive assembly first, assemble the necessary tools and pieces, including the guide tubes, guide wire, complete Dremel, set name tools and parts, and the U probe. Measure the guide tubes so that when attached to the recording device, they're long enough to rest on top of the dura without damaging it. Next, after measuring the depth of the recording chamber, cut the guide tubes to the measured length of about five to seven centimeters while cutting the guide tubes.
Try to ensure that no metal fragments enter inside the tube. Use a stiff wire smaller than the inside diameter of the guide tube to remove any metal fragments inside the tube. Next, place the name grid into the name base.
Tighten the clamp screw and the grid screw. Then identify the recording region of interest and position the micro drive towers over that region. After the region of interest has been identified, advance the guide tube through the bottom of the grid until it is about one to two millimeters outside the name chamber.
Next, assemble two clamps on each NA micro drive tower. A motor drives the top clamp while the bottom clamp can be either fixed in place or loose. The top clamp is attached to the reinforcement tube of the U probe.
Attach the bottom clamp to the guide tube and apply a small amount of super glue to secure the guide tube in place. The two clamps provide stability and precision to the system. Carefully align the tip of the U probe with the top of the guide tube and pass the U probe through the guide tube until you can secure the tower to the main base.
Adjust the tower position with the thumbscrew so that there's no added tension on the U probe or guide tube. Place the name system on the cylinder base and connect the motor cables to the corresponding towers. If using multiple towers, color coded zip ties are used to help distinguish between motor cables and towers, use the name software program to begin advancing the U probe, either by setting a target position that automatically advances the U probe to that location or by clicking down on the name software interface, advance the U probe so that at minimum 10 millimeters of the tip is through the guide tube past the end of the name chamber.
Sterilize the U probe by placing it in metro side activated aldehyde solution for 20 to 30 minutes prior to attaching the name base to the implanted recording chamber. After that, rinse the U probe and name base with sterile water zero. The name software locations by retracting the U probe so that the tip is just inside the guide tube in the name software.
Click zero all positions. Attach the name base to the implanted recording chamber and tighten all four screws. Then align the base according to a pin that is on the side of the recording chamber.
Tighten all four screws again and make sure that the name base is securely attached to the recording chamber. To prepare for advancing recording, the U probe is grounded and considered floating according to the grounding and referencing instructions. This is accomplished by placing the wire attached jumper.
On the bottom connectors head stages are secured to the U probe connector, and then the amplifier cables are connected and grounded. The U probe is initially advanced about one to two millimeters in a fast and strong manner. Set the velocity parameter in the range of 0.1 to 0.2 millimeters per second, and the depth step to 0.2 to 0.3 millimeters.
These values ensure that the U probe is able to puncture the dura cleanly and is an important first step in the recording. Once through the dura, reduce the velocity to 0.50 to 0.1 millimeters per second and reduce the depth step to 0.5 to 0.1 millimeters. The goal is to advance the U probe as smoothly and slowly as possible such that no tissue is damaged.
One of the indications that the probe has entered the brain is a change in the amplitude of the LFP accompanied by a reduction in the noise level to verify that the electrode is spanning all cortical layers measure the change in amplitude in response to the full field white flash stimulus. The changes in the LFP amplitude across time underlie the evoked response potential analysis. This analysis provides the basis for identifying cortical layers to identify the cortical layers.
Measure the evoked response potential during a passive fixation task while exposing the subject to a full field black screen that flashes white for 100 milliseconds and then returns to black. This sequence constitutes one trial, which is repeated 200 times. The plex on multichannel acquisition processor saves all the continuous data signals directly to the recording computer through a national Instruments PCI board.
After the data is saved, begin processing the signals for current source density analysis. Use the software correction FP align provided by Plex on to correct the time delays in the LFP signals induced by the filters in the head stages and the pre amplification boards. At this point, data is transferred to MATLAB with neuro explorer.
Each LFP channel is filtered using standard high and lowpass filters with cutoff frequencies of 0.5 hertz and 100 hertz. After each electrode contact has been filtered, identify each trial and average across trials to obtain the mean LFP time series for each electrode contact, then organize each contact into a matrix with LFP amplitude as a function of time, run the ICSD toolbox in MATLAB by typing CSD plotter in the workspace. Given that the sampling frequency of the continuous data is one kilohertz, set the DT parameter to one millisecond.
Next, set the cortical conductivity value to 0.4 Siemens per meter, which approximates the current source density in units of nano peers per cubic millimeter, and change the electrodes position as a vector of 0.1 by a step of 0.1 to 1.6, which is the total number of contacts. When all the parameters have been inserted, click run this. View the CSD profile in the CSD plotter interface and paste it to a new figure.
Common functions in MATLAB such as image SC can be used to plot the layer profile and various smoothing algorithms and normalization routines can be applied for representing the CSD data and comparing the layer identification across hours and sessions. To identify the polarity inversion accompanied by the sink source configuration at the base of layer four first, verify the presence of a primary sink in the granular layer. Using the laminar CSD profile, locate the sink driven negative polarity in the CSD plot.
Then compute the center of mass of the granular sink. A OID is obtained from the analysis consisting of the contact number and time when the sync was largest. The contact with the syn Centro serves as the granular layer reference at zero micrometers.
Analyze all the contacts above and below the reference and group them into one of three possible layers. Supra, granular, granular, and infra granular validate the granular sink by shuffling the electrode positions, leaving the temporal domain unchanged. After shuffling, the CSD matrix compute the OID analysis.
Again, shuffling electrode contact as a function of cortical depth should destroy any laminar specificity. To find receptive fields begin by presenting a reverse correlation stimulus on the monitor where receptive fields are potentially located. The stimulus is comprised of four orientation gradings at 45 0 90, and 135 degrees.
Perform cluster analysis on the firing rate maps to locate the receptive field. First, calculate maximum firing rate locations and their centroid for each time delay. Then calculate the distances between the Centro and these maximum firing rate locations.
Compute maps of firing rates at each spatial location for conduction delays between 40 and 120 milliseconds at five millisecond intervals for each neuron independently. Find the total distance between the OID and the surrounding maximum firing rate points at all time delays. The receptive field is at the time delay that minimizes that distance.
Once a receptive field is found for each cell present a reverse correlation stimulus Larger than all the receptive field locations overlapping all receptive fields in the recorded population. A real-time firing rate plot can be used to determine whether the correct receptive field locations have been identified. Lastly, sort spike wave forms using plex on's offline sorter program that implements waveform clustering based on parameters such as the principle components spike, width valley and peak properties.
Be sure to remove signal units that abruptly change responses and only keep units with stable firing rates for further analysis shown here. An example to illustrate the CSD analysis in localizing cortical layers across cortical depth as a function of time, the position of super granular, granular and infra granular layers remain stable even four hours after the recording session began. The CSD traces represent the average of those contacts assigned to a given layer.
In this example, the granular layer undergoes a clear decrease in CSD amplitude at about 50 milliseconds. Another critical analysis while using the laminar electrode is to accurately identify and localize the neurons receptive field. The origin of these plots is the fixation point, which is a small white circle displayed centrally on a black computer screen.
The color in these plots represents the firing rate of each neuron in response to a dynamic reverse correlation stimulus. This figure represents two examples of spike wave forms isolated on the same channel. Cluster analysis was performed by using principle component analysis and spike waveform characteristics.
The average spike wave forms are shown in solid line. Standard deviations are shown in dash line. While attempting these procedures, it is important to remember to carefully advance and to allow enough time for the brain to sufficiently settle after advancement.
We typically start recording about 30 to 45 minutes after the last advance Following this procedure. Other spectral techniques such as LFP Power and Spike Field synchronization can be used to study network structure within and between cortical layers.
