The overall goal of this experiment is to investigate functional and synaptic properties of neural projections and micro-circuits using ex-vivo optogenetics. This includes expression of channel rhodopsin and its activation in specific projections combined with patch-clamp recordings. This method can help answer key questions about synaptic properties of local and long-range connectivity within and between brain areas.

This is illustrated here investigating the neural circuits that support fear behavior. The main advantage of this technique is that it allows to study circuits and synapses that are not amenable with conventional electrical stimulation techniques. Generally, people new to this method will have to overcome the challenge of performing all the required steps in an optimal way.

This includes the stereotactic targeting of viral injections, identifying the optimal expression time for channel rhodpsin, the preparation of good quality brain slices, and stable light activation and recordings. Before beginning this part of the procedure, a recombinant adeno-associated viral vector engineered to express channel rhodopsin fused to a fluorescent protein was introduced into the medial pre-frontal cortex by stereotactic surgery. Prepare the vibratome by fitting a sapphire blade and setting it to cut at 320 micron thickness.

After sacrifice according to approved methods and removal of the brain, use a scalpel to cut off the cerebellum, then isolate the anterior portion of the brain containing the medial pre-frontal cortex. Place the anterior portion of the brain in ice-cold cutting solution until slicing. Fill the cutting chamber with ice-cold cutting solution maintained at 4 degrees Celsius using a cooling unit.

Oxygenate the solution. Blot the brain dry with filter paper. Glue the four per cent agar block that that has been cut at a 35 degree angle to the vibratome stage, and glue the posterior part of the brain tissue onto this block.

Glue two additional agar blocks in front of and behind the brain block for stability while slicing. Place the stage with tissue attached in the cutting chamber and ensure it is submerged. Begin cutting the acute slices.

Cut each slice in half and transfer to an interface chamber supplied with oxygenated ACSF at room temperature. After cutting the acute amygdala slices, place the interface chamber in a water bath at 36 degrees Celsius and allow the slices to recover for 35 to 45 minutes. Fix a subset of the slices containing the injection site for post-hoc analysis by sandwiching them between two pieces of filter paper and submerging them in four per cent paraformaldehyde in PBS overnight.

Here, a recombinant adeno-associated viral vector engineered to express channel rhodopsin fused to a fluorescent protein was introduced into the medial division of the medial geniculate nucleus and adjacent posterior intra-laminar nucleus by stereotactic surgery. After obtaining the brain, use a scalpel to remove the cerebellum and anterior part of the brain and then cool the middle portion of the brain in ice-cold cutting solution as before. Blot the brain with filter paper and then glue it to the vibratome stage.

Again, fix an additional agar block behind the brain for stability while slicing. After fitting the stage into the cutting chamber filled with four degree Celsius cutting solution, cut 320 micron coronal sections through the amygdala, cut them in half, and collect the sections in the interface chamber as before. Under a stereomicroscope using fluorescent illumination check the injection site.

To prepare the patch microscope for optogenetic activation of fibers and cells, center the mounted light-emitting diode or LED onto the light delivery pathway. Use a power meter to measure the LED light intensity at the back focal plane and at the output of each objective at a wavelength of 470 nanometers. Use a spreadsheet to calculate the light intesity in milliwatts per millimeter squared and create a calibration curve for each objective.

Next, retrieve an acute amygdala slice from the interface chamber and place it in the slice chamber mounted onto the microscope. Position the slice so that the slice surface facing upward in the interface chamber is also facing upward in the recording chamber. Perfuse the slice with fresh, oxygenated ACSF at a rate of one to two milliliters per minute.

The temperature should be approximately 31 degrees Celsius. Turn on the fluorescent lamp and select the appropriate filter set for the specific fluoresent protein expressed. Use the 5x objective to obtain an overview.

And the 60x objective for assessment of fiber density within the target area. Next, open or restrict the aperture in the microscope light pathway as necessary for the experiment. To obtain a patch recording, fill a patch pipette with internal solution and mount it in the electrode holder.

Apply positive pressure to the patch pipette and slowly lower it into the bath solution. Then, under visual control use the micromanipulator to lower the patch pippette into the slice. Approach the neuron of interest with the patch pipette from the side and top.

Release the positive pressure when the pipette reaches the surface of the cell as indicated by a dimple visible on the cell surface. Apply negative pressure to obtain a gigaseal. Apply further suction to rupture the membrane patch and obtain the whole cell recording.

Next, stimulate the labeled fibers with a connected LED by activating channel rhodopsin with 470 nanometer wavelength light while recording electrical responses from the cell. For synaptic stimulation, use the digital outputs of the data-acquisition software to trigger the LED. Adjust the LED stimulation intensity manually.

Repeat the stimulation with an opened or restricted aperture as necessary for the next recorded cell or in the presence of specific test substances. After recording, fix slices for post-hoc analysis by sandwiching them between two filter papers and submerging them in four percent paraformaldehyde overnight. Fibers from the medial pre-frontal cortex were stimulated using optogenetic paired pulse stimulation while excitatory post-synaptic currents were recorded from basolateral amygdala principal neuron and an interneuron.

These representative traces show the paired pulse facilitation and paired pulse depression elicited from the stimulation. The following two images demonstrate feed-forward inhibition elicited by optogenetic activation of fibers from the medial prefrontal cortex. This first image shows a representative excitatory postsynaptic current at 70 millivolts and inhibitory postsynaptic current at zero millivolts in a basolateral amygdala principal neuron.

The inhibitory postsynaptic current has a longer synaptic latency compared to the excitatory postsynaptic current indicating disynaptic and monosynaptic input, respectively. This image shows that the light evoked by phasic excitatory and inhibitory postsynaptic current sequence at 50 millivolt is blocked by the AMPA kainate antagonist, CNQX, further supporting the disynaptic nature of the inhibitory postsynaptic current. This image shows the effects of a subsequent block of the excitatory and inhibitory postsynaptic current sequence at 50 millivolts by the chloride channel-blocker, picrotoxin, and picrotoxin plus CNQX.

The inhibitory postsynaptic current is blocked by picrotoxin and the remaining excitatory postsynaptic current by CNQX. Once mastered, the preparation of brain slices and the recording of light-evoked responses can be done within several hours to a full day. Prerequisites are good injection sites and sufficient expression of channel rhodopsin in cells and projections to be studied.

Following this procedure, other methods like anatomical studies of labeled axons and synapses can be performed at the light and electron microscopic level. This allows to correlate functional with anatomical and molecular properties of activated synapses. After watching this video, you should have good understanding of all the required steps for analysis of light-activated fibers and brain slices.

This includes channel rhodopsin expression, evaluation and activation of labeled fibers, as well as recording and analysis of the synaptic responses.