The current protocol analyzes an affordable and straightforward method for combined neuroanatomical and electrophysiological tracing of neurocircuits in vivo. The technique permits mapping of adjunct terminals from specific neuron population to a target site. Likewise, the anatomical map for these terminals can be verified by optogenetic stimulation during extracellular recording at the target site.
This protocol can be applied for studying multiple neural circuits associated with sensory, motor, and cognitive function. Performing this procedure requires some knowledge of rodent stereotaxic surgery and handling of delicate neural electrodes. The recording system connection to prevent noise in recordings.
Visual demonstration highlights basic steps in setting up an affordable LED system for in vivo optogenetics. Also, it shows how to connect the amplifier with the LED driver for synchronized recording and stimulation in an anesthetized mouse. Place a heating pad on the stereotaxic frame so that the body of the mouse is lying on it which will help maintain its body temperature throughout the procedure, then gently fix the head on the stereotaxic apparatus.
Prepare the surgical area by cleaning it first with iodine solution and then with alcohol to remove the iodine. Next, apply topical lidocaine to block sensation on the scalp. Then using a scalpel, make a midline incision extending from the frontal to the occipital region.
For ventral tegmental area or VTA injection, position an ultra-fine blunt point needle syringe at negative 3.08 millimeter anterior posterior and 0.5 millimeter medial lateral coordinates relative to the bregma. Use a drilling tool to bore a one millimeter hole in the skull at the marked coordinate. Follow the manufacturer's instructions to fix the syringe holder on a micromanipulator and fill the syringe with double distilled water to clean and test the flow of fluid.
Then dispense the water to test the flow of the syringe. Thaw aliquots of adeno-associated virus or AAV cocktail on ice. Fill the mounted syringe with 1, 000 nanoliters of AAV solution and dispense 10 nanoliters of the solution to confirm the flow of the liquid.
Use the micromanipulator to lower the needle to the injection site and inject 600 to 800 nanoliters of AAV into the VTA, delivering the solution at 60 nanoliters per minute. Three weeks after the AAV injection, affix the head of the animal on a stereotactic frame as previously described and perform a craniotomy to expose the dura. Use a drilling tool to remove part of the parietal bone.
Under a dissection microscope, use a bent 27 gauge needle tip to excise the exposed dura, taking care not to pull apart the delicate peel covering and cortical tissues in this area. Apply drops of artificial cerebrospinal fluid over the craniotomy area to prevent dryness. Bore a hole in the occipital bone to hold the ground screw and connect a stainless steel ground wire.
Before lowering the cannula, connect the fiber optic cable to a fiber coupled LED source. Lower the 400 micrometer diameter optic fiber into the VTA at coordinates of AP negative 3.08 millimeters and ML 0.5 millimeters. Using a micromanipulator, position the electrode contact sites in the pyramidal cell layer of the CA1.
To synchronize the light pulse with the neural recording, connect the LED driver and recording controller digital import to a transistor-transistor logic pulser with a BNC splitter. Adjust the knob to determine the effective intensity that can generate a response without producing photoelectric artifacts and connect the ground on the skull to the ground on the adapter. Connect the preamplifier head stage to the recording controller via a serial peripheral interface cable and check the LED color lights on the recording controller ports.
Green and yellow LEDs indicate proper voltage on the connected amplifier board and the red led indicates a working software head stage control. After launching the software, select the data file format and change the filename. Switch to the port for the connected head stage and click on disable all on port if the electrode has fewer channels than the head stage.
Select the appropriate number of channels to be displayed on the screen. To capture the TTL timestamp for synchronized neural recording and light pulse triggered timestamp, click on the digital imports and enable digital in zero one. This must correspond with the BNC connection on the amplifier digital imports.
Adjust the timescale and voltage scale for appropriate waveform display and select the sampling rate, keeping in mind that a higher sampling rate and number of channels will increase the file size. Next, set the amplifier bandwidth for single unit recording. A 300 to 5, 000 Hertz cutoff frequency was used here.
For spike sound, click on analog out audio and enable the analog port. Then adjust the gain and silencer for optimum sound. To synchronize the amplifier recording and light pulse train, click on the display tab and enable show marker.
Switch to the appropriate channel that corresponds with the BNC connection and reinspect the ports to make sure that the recording channels and digital in channel are enabled to capture neural spikes and light pulse timestamps. Click on spike scope to view waveforms that constitute the continuously recorded spike train. Use the mouse to set the threshold for the waveforms to be displayed.
Then inspect the RMS to determine the noise level in your recording. Adeno-associated virus expression was verified by immunofluorescence imaging of EYFP in the ventral tegmental area of mice 21 days post-injection. Fluorescence imaging was also used to detect pre-synaptic VTA glutamate projections in the hippocampus layers DG, CA3, and CA1.
Once extracellular voltage activity was detected, the baseline activity was recorded for approximately 10 minutes before triggering the light pulse at the desired frequency. This made it possible to compare the firing or burst rates of CA1 putative neurons before and after VTA glutamate photostimulation. To support this outcome, statistical comparison of the CA1 network firing rates before and after photostimulation revealed a significant increase for the post-stimulation period.
Subsequent analysis of the raster train to detect bursts also showed an increased burst rate for the CA1 putative pyramidal neurons after photostimulation. Response to photosimulation was identified by fluorescence of the AAV reporter expressions at the stimulus sites. Also, the description of should be correlated with the site of recording for responsive putative units.
This method can also be performed in awake mice undertaking behavioral tasks. In this case, chronic implantable probes and fiber optic cannula should be used. This technique will allow for robust tracing of neurocircuits through a combination of presynaptic terminal distribution in the target site and modulation of presynaptic terminals to detect target site response in vivo.
