A Guide<em> In vivo</emOptogenetically Belirlenen Kortikal inhibitör dan> Tek birim Kayıt

The overall goal of the following experiment is to obtain high quality extracellular single unit recordings from cortical inter neurons in mice expressing channel rootin two. This is achieved by advancing a high impedance recording electrode through the tissue to identify inter neurons from spiking responses to blue light pulses. The electrical signal is monitored for changes that indicate the appropriate rate of advance and likelihood of obtaining a stable well isolated recording.

If good single unit isolation cannot be achieved or conversely light responsive neurons are rarely encountered, replace the recording electrode. The results show the reliability of light evokes responses from optically identified into neurons and the typical signal to noise ratio obtained with this strategy. The photo stimulation approach is an accessible and inexpensive way to target genetically identified cell types using a standard extracellular amplifier and blue light.

This is our strategy for pimping specific inner neurons in anesthetize auditory cortex, but the more general points apply to other cortical areas than sparse cell types To begin this procedure. After the animal has been anesthetized with ketamine meine cocktail via intraperitoneal injection, place it in a stereotaxic or other head holding apparatus, ensure that the skull is well secured. This is essential for maintaining stable single cell recordings.

Next, apply ophthalmic ointment to the eyes to prevent dryness and maintain the animal’s body temperature at 37 degrees Celsius to perform a CI sternal drain for additional recording stability, remove tissue from the posterior skull with a scalpel blade to expose the CI sternum magna. Then make a small nick in the Dora with the tip of the blade to drain the cerebral spinal fluid. Now using the scalpel, perform a small craniotomy over the cortical area of interest.

After that, remove the dura, then cover the exposed cortex with the layer of warm aros. Keep the agros moist with saline. Prepare a tongues stand electrode.

Glue the electrode to a glass capillary tube with super glue and accelerator. Then add heat shrink tubing for grip. Afterward, mount the electrode on a motorized or hydraulic microm manipulator and set it to travel orthogonal to the cortical surface.

Slide a ground wire under the skin against the skull. Avoid contact with the muscles on the side of the head and the back of the neck as they can generate myo graphic artifacts. The electrical signal is amplified using an extracellular amplifier suitable for single unit recording, preferably equipped with an impedance check mode.

Ongoing spiking activity is monitored with two oscilloscopes and a set of powered speakers. Now advance the electrode through the agros and test its impedance to make sure it is within seven to 14 mega ohms. When the tip breaches the surface of the cortex zero this position on the microm manipulator.

To best estimate the depth of recording, visually confirm that the electrode tip exits the cortical surface at a depth of zero. When withdrawing the electrode at the end of a penetration in the auditory cortex to ensure that this zero set setpoint corresponds to the level of the cortex, monitors stimulus evoked field potentials. While advancing the electrode through the first several hundred micrometers of tissue, confirm that the polarity of the local field potential reverses around a depth of 100 micrometers.

Next, mount an optical fiber coupled to a blue light source onto a manual micro manipulator. Regulate the output of the light source with the control unit capable of delivering a TTL pulse train of specified width and duration. Then monitor the signal on both oscilloscopes.

Measure the total light power at the tip of the optical fiber using a power meter. Use this value to calculate the irradiance by dividing it by the cross-sectional area of the fiber core. Begin with the value in the range of 10 to 15 milliwatts per square millimeters.

Using the microscope position the tip of the fiber as close to the surface of the aros as possible. Center the beam where the electrode will enter the tissue. Now check for light artifacts with the electrode in the aros.

Deliver a pulse strain. Eliminate any transient light artifacts by repositioning the optical fiber relative to the electrode to change the angle of incident light. If artifacts persist, try decreasing the light power.

Next, advance the electrode slowly through brain at a rate of approximately one micrometer per second. Use one oscilloscope to monitor the ongoing activity of the electrode. Use the other for triggering off the laser pulses.

Listen for the faint hash of the light evoked spikes on the audio monitor, which indicates that the electrode is approaching a channel opsin positive cell. Then slow the rate of advance as soon as the light evokes. Spikes are large enough to be triggered off of individually on the oscilloscope.

Begin doing so. Adjust a scale on the horizontal axis to observe the exact shape of the spike waveform. At this point, stop advancing the electrode and wait for it to settle in the tissue.

This is critical for a stable recording. After several minutes, if the signal to noise ratio has not improved, advance five micrometers further and wait. Again, repeat this process until either the peak or the trough of an action potential can be reliably captured with a voltage threshold.

Set well above the noise floor while recording. Monitor the ongoing activity on the first oscilloscope. At the same time, pay attention to the size and shape of spikes using the second oscilloscope and note the quality of their sound on the speaker.

Listen attentively for abrupt changes, which indicate that the cell is either drawing too close to the electrode or drifting away. If the spikes become large and distorted, withdraw the electrode. If they get smaller, advance the electrode.

Move slowly in two micrometer steps to confirm the quality of the spikes. Superimpose all the spike waveform across a wide range of threshold values. Only one consistent spike shape of uniform height should be seen.

Most PV positive cells can sustain firing for a full one second. If the signal becomes contaminated with spikes from a neighboring neuron, advanced the electrode and look for a new cell in each penetration, lower the electrode through the entire depth of cortex. If no light responsive neurons are encountered after many penetrations or conversely, recordings are routinely contaminated with spikes from neighboring channel, opsin negative cells, try a new electrode.

This figure shows example recordings from three types of optically identified inter neurons. Cells respond to the search pulse train with a reliable burst of spikes. Shown here are 100 tr line spikes and the average interpolated waveform.

These roster plots show the consistent timing of light evoked spikes. The first spike latencies for a sample of power. Volin positive inter neurons are presented here.

The photo stimulation approach is conceptually very simple, but it can be surprisingly challenging and low yield. In practice. Glass patch electrodes are strongly biased towards parametal neurons, so you don’t get inner neurons very often.

On the other hand, with tetros, you get plenty of light responsive inter neurons, but the spikes are small and there’s a lot of overlapping wave forms, so it’s very hard to reliably sort into single units. We try to accomplish this many different ways, and for inner neurons, we found that these sharp 10 to 14 mega electrodes struck the right balance in terms of listening radius, which must be large enough to detect light of oak spikes from a distance, but restricted enough to achieve good single unit isolation. There’s some voodoo to it though.

If you can’t achieve that, you’ll save yourself a lot of time just by ditching that electrode and moving on to the next until you find one that works.