The overall goal of the following experiment is to rapidly activate or silence genetically defined neuronal populations on a physiologically relevant timescale. This is achieved by delivering a viral vector carrying transgenes that encode the blue light neuron activating channel opsin or green light neuron silencing hall opsin to genetically defined neurons. As a second step, a light emitting op road is constructed that consists of a fiber optic affixed to a recording electrode, which allows for simultaneous light delivery and electrical recording.
Next, the TRO is positioned over and lowered into the Opsin transduced brain region. In order to evoke channel opsin and ha opsin mediated responses, results are obtained that show light induced activation and inhibition of channel redsin and ha hallin transduced neurons based on the single unit recording technique that allows recording of individual neurons. This method addresses a fundamental need in neuroscience by allowing us to, to determine exactly how effective light can be at driving neuronal activity with millisecond time resolution and in specific neural subtypes and demonstrating this will be Chin Nakamura.
A postdoc in my laboratory Ensure that biosafety level one procedures are followed while preparing and handling virus, and that all local and government guidelines pertaining to animals are observed. Prior to stereotactic injection, allow an aliquot of virus to thaw on ice and then briefly spin down. In a benchtop centrifuge, slowly backfill a Hamilton glass syringe and needle with a small amount of silicone or mineral oil.
Make sure there are no visible air bubbles in the syringe barrel. Insert the syringe into a micro injector pump and attach the pump directly to the vertical stereotaxic arm. After setting the virus aliquot tube into a steady position, lower the stereotaxic arm until the tip of the needle touches the bottom of the virus aliquot tube.
Use the controller for the micro injector pump to withdraw the desired volume of virus. After anesthetizing a rat with ketamine and xylazine, check for the reflex response to a toe pinch to ensure an adequate depth of anesthesia using standard aseptic methods. Place the animal into a stereotaxic apparatus after making a midline incision through the skin on top of the animal skull with small surgical scissors or scalpel.
Gently separate the connective tissue and clean the top of the skull with a small bone scraper. Lower the needle and verify that the Z coordinates for bgma and lambda are equal to establish that the head is level. Then move the stereotaxic arm to the coordinates corresponding to the brain structure of interest.
Mark the intended site of injection with a surgical pen. Use a handheld drill to carefully thin the skull over the target area. Stop when the drill bit reaches the bottom of the skull and use a pair of extra fine forceps to remove the thinned bone.
After the dura is exposed, position the syringe needle over the target area and lower the needle until it touches the dura. And use this point to calculate the z. Coordinate now very slowly lower the injection needle into the brain until the proper Z position is reached.
In this protocol, the pre limbic region of the medial prefrontal cortex is targeted. Inject the virus at a rate of 0.1 microliters per minute. After the injection is completed, wait 10 minutes before withdrawing the needle to avoid backflow of the virus to the brain's surface.
After using a sewing suture with attached needle to seal the skin, apply antibiotics to the wound. First, attach a one to 1.5 mega ohm tungsten electrode to a glass capillary tube using superglue. Then use a fiber stripping tool to expose the bare end of a multi-mode optical fiber and clean the fiber tip with ethanol very lightly.
Score the fiber tip with a wedge-shaped diamond knife and use fine forceps to carefully remove the excess fiber. The fiber should cleave easily at the score. Use an optical power meter to measure the laser intensity at the tip of the optic fiber.
For in vivo recordings, light intensity as little as 20 to 100 milliwatts per square millimeter can reliably evoke channel redsin or hallin mediated activation or silencing respectively of neuronal activity. Next, insert the optical fiber into the capillary tube. Position the fiber tip approximately 500 microns above the tip of tungsten electrode and tie a thin suture thread twice at a few points near the tip so that the electrode and the fiber are straight.
Make sure that the distance between the tip of the electrode and the nearest knot is long enough for insertion to the target region as seen here. Prepare the animal for surgery as before. Then use the initial craniotomy as a guide to make a new, clean, small window in the skull.
For positioning the tro. Use a fine needle to make a small incision on the dura. Carefully lower the TRO through the skull window and into the brain to the transduced region using a microm manipulator.
Then advance the offroad slowly in 10 to 50 micron steps until the emergence of a light responsive cell. Here, a custom software user interface written in lab view is used to control the single diode laser and to visualize and record ongoing neural activity. Recorded signals are captured with an ex AMP 20 K amplifier band pass filtered at 0.3 to eight kilohertz and a national instrument's analog to digital board.
The neuro lux Pro user interface allows the choice of analog inputs and digital output. Choose two analog channels for the neuronal and transistor transistor logic or TTL signal. Set the sampling rate at 20 kilohertz, the baseline pre light period to two seconds and post light period to two seconds.
Then set the TTL laser stimulation pulse width, frequency, and stimulation period. Here the frequency is 20 hertz and the stimulation period is 500 milliseconds, which means that 10 laser pulses with defined pulse width are delivered. The user can choose either continuous light delivery or for repeated recordings.
They can choose the interval between the pulses and the number of pulse strains. Data can be acquired with or without recording to disc Following recording. The animal is sacrificed according to approved procedures and the brain tissue is processed to verify erode placement and opsin expression as shown here.
This screenshot of the Neuro LX Pro software interface set up for simultaneous light delivery and electrophysiological recording shows Hall Rod Dobson induced silencing of spontaneous activity of rap pre limbic parametal cell in response to 10 seconds of continuous 532 nanometer light delivery. This photograph shows representative hallo redsin expression in the dorsal ulu. The schematic on the right represents the location where the photograph was taken.
The arrowhead indicates the position of the tungsten electrode and the arrow indicates the position of the optical fiber. This photograph shows representative channel opsin expression in pre limbic cortex. Again, the schematic on the right represents the location where the photograph was taken.
The arrowhead indicates the position of the tungsten electrode and the arrow indicates the position of the optical fiber. This example trace recorded from rat pre limbic cortex shows channel redsin two triggered action potentials in response to 20 hertz delivery of blue 473 nanometer, light pulses for 10 milliseconds each as indicated by the blue bar.Below. The raster plot shows channel redsin induced spiking in six representative neurons.
Each unit activity is plotted as a dot. This example trace shows hall opsin induced suppression of spontaneous activity during continuous green 532 nanometer light illumination for 10 seconds as indicated by the green bar in the same brain region. The raster plot shows haller dosin induced silencing in six representative neurons.
Each unit activity is plotted as a dot. This image shows repetitive 20 hertz channel opsin driven spiking of a rat pre limpic parametal neuron in vivo voltage traces of the light evoked, spiking, acquired at the time points 0 30, 60 90, and 120 minutes after the beginning of the recordings are displayed on the left hand side. On the right hand side, the raster plot shows all 61 repetitions of the light induced activation.
Each unit activity is plotted as a dot. Here in vivo, electrophysiological recordings from Haller Dossin transduced rat dorsal sicm are shown at the top. The example trace demonstrates that 10 seconds continuous 532 nanometer illumination indicated by the green bar of the dorsal subm expressing Hall Dobson eliminates spontaneous activity.
Below are the average waveforms of the two recorded units from the above trace. The amplitude threshold was used for identifying two distinct neurons. Finally, the raster plot shows five repetitions of Haller dossin induced silencing of these units.
Each unit activity is plotted as a dot. Finally, this image shows repetitive 20 hertz channel opsin driven spiking of a rat dorsal subular neuron in vivo voltage traces of the light evoked, spiking, acquired at the time points 0, 30, 60, 90, and 120 minutes. After the beginning of the recordings are shown on the left, the inset below shows the typical bursting activity of this cell.
The raster plot on the right shows all 61 repetitions of the light induced activation. Each unit activity is plotted as a dot. This technique is important because it demonstrates that specific neural subtypes can be activated or silenced for long periods of time.
In fact, we've demonstrated that we can activate neurons for over two hours and applied to. Ultimately the goal would be to apply this to the behaving animal in animals that are performing attentional or memory tasks so that we can activate specific circuits and see how that controls different memory functions.
