MicroRNA-Mediated Inhibition of Excitatory Postsynaptic Currents in Mouse Hippocampal Slices

Take mouse hippocampal brain slices containing CA3 neurons infected with a recombinant viral vector.

The viral genome expresses a light-activated channel tagged with a fluorescent reporter and a microRNA that downregulates the expression of voltage-gated calcium channels.

Place a slice in a recording chamber under dark conditions. Using a microscope, identify the fluorescent CA3 neurons.

Patch a recording electrode onto a CA1 neuron that receives input from infected CA3 neurons. Rupture the membrane to record intracellular ionic currents.

Apply an inhibitor to block inhibitory neurotransmitter receptors, allowing only excitatory signals.

Using light pulses, stimulate the light-activated channels in the infected CA3 neurons, generating action potentials.

In uninfected mice, the action potential activates voltage-gated calcium channels, causing a calcium ion influx that triggers neurotransmitter release. The neurotransmitters trigger excitatory postsynaptic currents, or EPSCs, in the CA1 neurons.

In infected mice, the microRNA-mediated reduction of voltage-gated calcium channels results in EPSC inhibition.

For this protocol, use an animal that 15 days prior or longer, had rAAV1/2 injected into the brain, according to a previously published protocol by Cetin and company. Isolate the brain and make acute brain slices with a vibratome and gassed ice-cold aCSF.

Collect the slices of the region of interest, and minimize their exposure to light to avoid activation of the optogenetic probe. Let the slices recover for 30 minutes at 37 degrees Celsius in the same aCSF. Use a chamber specifically designed for holding brain slices. Then, return the slices to room temperature where they will remain healthy for six to eight hours.

To proceed, transfer a slice to the recording chamber, and super-fuse it at two milliliters per minute of aCSF. Next, briefly check the signal of the expressed fluorescent reporter to ascertain the localization and intensity of infection. Then, fill a patch electrode with the intracellular solution.

Now, under infrared illumination, obtain a tight-seal whole-cell configuration on a neuron that is receiving synaptic inputs from the infected neurons. The series resistance can be left uncompensated, but it should be constant and low. For example, if CA3 pyramidal neurons were infected, patch pyramidal neurons in the proximal to medial tract of the CA1 region.

When the cells start to look shrunk or swollen, when the patching becomes difficult or the patches are unstable, then the selected slices are not long healthy enough to record from.

Next, use pharmacology to isolate the synaptic currents under investigation. For example, if the aim is to investigate excitatory synaptic transmission, block inhibitory synaptic transmission by adding bicuculline to the bath.

Then, evoke synaptic currents such as excitatory postsynaptic currents using a 473-nanometer blue laser coupled to an optical fiber positioned on the somata of the infected neurons. Do not direct the laser onto the neuron's axons.

For example, avoid shining light on the Schaffer collaterals. Direct depolarization of the axons is not desirable. Then, adjust the stimulation length to a minimum to reduce the possibility of evoking more than one action potential per light pulse.

Next, refine the laser's intensity to evoke small but clearly detectable synaptic current. For example, for excitatory synaptic transmission between CA3 and CA1 pyramidal neurons, adjust the laser's intensity to evoke synaptic currents of 20 to 50 picoamps.