Combinação de Optogenetics com microRNAs Artificial para caracterizar os efeitos do Gene Knockdown na função pré-sináptica dentro circuitos neuronais intacta

The overall goal of combining artificial microRNA mediated RNA interference with optogenetics is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. This method can help answer key questions in the field of synaptic physiology, such as addressing the physiological role of presynaptic proteins in intact brain circuits. The main advantage of this technique is that it makes it possible to monitor synaptic transmission selectively at neurons with reduced expression of the presynaptic protein under investigation.

Also, we have used this method to investigate presynaptic function in acute brain slices. It can also be applied to manipulate and probe neuronal security in vivo. Demonstrating the procedure will be Carmela Vitale, a grad student from our laboratory.

After designing the microRNAs and constructing a recombinant vector to express the microRNAs and the optogenetic probe, prepare primary neuronal cultures from the brain region of interest. To do this, follow an established corticoculturing protocol by L.A.Cingolani and Company with the following modifications. First, use six-well plates, plate half a million neurons per well, and use 2.5 milliliters of attachment medium per well.

After incubating the plate for four hours, switch to 3.3 milliliters of maintenance medium per well, and if astrocyte overgrowth is observed add 0.5 milliliters of maintenance medium supplemented with 7.5 micromolar of AraC at three to four DIVs. At five to six DIVs, infect three wells for each microRNA to test. Use the lowest infectious dose that will infect at least 99%of the neurons.

Add the virus directly to the neurons, mix gently, and place the plates back in the incubator at 37 degrees Celsius. At 17 to 18 DIVs, lyse the neurons for an RNA extraction. Tilt the plates, and use glass pipettes to remove all of the medium from each well.

Then, immediately add 700 microliters of lysis reagent to each well. After a brief mixing, transfer the cell lysate out of each well and into separate 1.5-milliliter tubes. Then, add 140 microliters of chloroform to each lysate.

Quickly cap and shake the tubes vigorously for 15 seconds to emulsify the contents. Next, centrifuge the tubes at 12, 000 times G for 15 minutes at four degrees Celsius. Then, carefully transfer the upper aqueous phase containing RNA to a new tube.

Do not let the pipette tip touch the organic phase. To the aqueous phase add 1.5 volumes of 100%ethanol, and pipette the solution slowly three times to mix. Then, purify the RNA using a commercially-available kit, and quantify the yields.

Expect at least 3.5 micrograms per sample. The ratios for purity over proteins and organic compounds should both be at least 1.9. Next, use a kit to retrotranscribe 250, 500, or 1, 000 nanograms of RNA.

Then, quantify the knockdown efficiency for the endogenous gene of interest by quantitative RTPCR. Aim for a knockdown efficiency of at least 60%For this protocol use an animal that 15 days prior or longer had RAAV12 injected into the brain according to a previously published protocol by A.Cetin and Company. Isolate the brain, and make acute brain slices with a vibratome and gassed ice-cold ACFF.

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 ACFF. 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 superfuse it at two milliliters per minute of ACFF. 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 wholesale 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 batches are unstable, then the selected slices are not longer 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 neurons'axons.

For example, avoid shining light on the Shaffer 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. At the end of the experiment, apply tetrodotoxin at 0.5 micromolar to block the sodium channels.

The sensitivity of the optically-evoked currents to tetrodotoxin indicates that they are action-potential driven. Continue collecting data until at least eight neurons have been patched for a given condition from at least three different animals. This is the minimum data needed to analyze results.

Using the described method, alternative splice isoforms of the presynaptic P/Q-type voltage-gated calcium channels were knocked down. These channels are thought to regulate short-term synaptic plasticity at CA3 to CA1 excitatory synapses. Four different constructs expressing isoform-specific microRNAs were made;each was expressed together with an ultra-fast channel rhodopsin along with the fluorescent protein Td-Tomato.

First, each construct was confirmed to have sufficient knockdown efficiency and selectivity in primary rat neuronal cultures using isoform-specific quantitative RTPCR. Next, each construct was injected into the hippocampus of P18 rats. Ultimately, the effect of the constructs on short-term synaptic plasticity at CA3 to CA1 synapses was investigated by stimulating infected CA3 pyramidal neurons with brief pulses of 473-nanometer light and by recording the resulting EPSCs in wholesale configuration from pyramidal neurons of the CA1 region.

Knockdown of different splice isoforms affected the responses to paired pulse stimulation in opposite directions. Knockdown of the A isoform boosted paired pulse facilitation;whereas knockdown of the B isoform abolished it. Combining RNA interferences optogenetics allows research in the field of synaptic physiology to explore how presynaptic proteins regulate short-term synaptic plasticity in intact circuits.

After watching this video you should have a good understanding of how to validate microRNAs for RNA interference and how to combine them with optogenetics to investigate synaptic transmission in acute brain slices.