The overall goal of this procedure is to optically control a neuronal protein's function using a genetically encoded, unnatural amino acid in neurons. This method can help answer key questions about the contribution of a specific neuronal protein to neuronal signalling in brain function with spatial temporal resolution. The main advantage of this technique is it has a potential to be applied to various neuronal proteins to achieve optical regulation of various neuronal processes.
Begin this procedure by plating hippocampal neurons onto cover slips in a 24-well plate with 500 microlitres of growth media, after filtering through a 40 micrometre nylon mesh. After that, incubate the neuronal culture at 35 degrees Celcius in a five percent carbon dioxide, 95 percent air humidified incubator for 2 to 3 weeks. On the day of transfection, make fresh 2.5 molar calcium chloride solution in double distilled water, and sterile filter it with a 0.22 micrometre filter.
Next, make fresh 2X BBS at pH 7, and sterile filter it with a 0.22 micrometre filter. After that, replace the culture growth media with 500 microlitres of fresh, pre-warmed transfection media, and do not discard the old growth media. Prepare the transfection solution immediately before adding it to the culture by combining calcium chloride and double-distilled water, while slowly agitating the tube.
Continue agitating the tube and slowly add DNA into the solution. Next, add 2X BBS buffer dropwise. Preparation of 2X BBS buffer is a critical step in calcium phosophate transfection, and it is a good idea to calibrate optimal pH for 2X BBS buffer between 6.90 to 7.15 for new DNA constructs or preps.
Then, immediately add 30 microlitres of the transfection solution to each cover slip of the neuronal culture. Rock the culture dish a few times to mix the solution, and incubate it at 35 degrees Celcius for 45 minutes to one hour. After about an hour, a layer of fine calcium phosphate precipitates should be observed covering the neurons.
Next, replace the transfecting media with 500 microlitres of pre-warmed washing buffer, and incubate it at 35 degrees Celcius for 15 to 20 minutes. The calcium phosphate precipitates should disappear after the wash. Now, replace the washing buffer with 500 microlitres of fresh growth media.
Afterward, replace the growth media with the saved original media. Then, add pre-mixed, unnatural amino acid Cmn in 50 microlitres of warm growth media to the culture, that results in a one millimolar final concentration. Incubate the transfected culture at 35 degrees Celcius for 12 to 48 hours before recording.
In this procedure, prepare the extra-and intracellular solutions. In our setup, the LED with emission of 385 nanometres is installed next to the microscope to deliver light to one centimetre away from the focal point at a 45 degree angle. Then, pull some patch pipettes with a glass electrode using a micro pipette puller.
Afterward, take a neuron cultures coverslip from the incubator and rinse it once in the extracellular solution. Profuse the rig with extracellular solution. Subsequently, place the coverslip on the electrophysiology microscope platform, using standard patch clamping techniques.
Patch a neuron transfected with M-citrine fluorescents. Next, record the neuronal activity using the current clamp method. First, adjust the resting potential to around negative 72 millivolts.
Then, inject a step current to induce continuous firing of action potentials at five to 15 Hertz. While recording, deliver single LED pulses to the neuron, and monitor the changes of its action potentials. Then, add 0.5 millimolar barium chloride to the bath, to recover the action potentials.
In the setting of acute brain slice, patch a neuron with M-cherry GFP fluorescents from the neocortical region. Then, record PIRK activity using the voltage ramp method. Specifically, monitor the Kir2.1 specific inward currents at negative 100 millivolts.
Then, deliver single LED pulses to the neuron while recording, and monitor if PIRK proteins are activated. Once PIRK is activated, inward currents at negative 100 millivolts would increase significantly. At the end, add 0.5 millimolar barium chloride to the bath, and verify if PIRK is inactivated.
Shown here are exemplary pictures of healthy rat hippocampal neuronal cultures. And two healthy neurons exhibiting plump cell bodies and pronounced dendrites and axons. These are the DIC and fluorescence images of rat hippocampal neurons cultures in vitro, and transfected for PIRK expression without CMN.
In the presence of one millimolar CMN, transfected neurons are capable of expressing full-length PIRK M-citrine proteins, thus showing green fluorescence. And here is an example of a single light pulse, suppressing the activity of a PIRK expressed hippocampal neuron. These fluroescence images of mouse embryonic cortical neurons show the successful incorporation of CMN into GFP and Kir2.1 proteins in vivo.
In this graph, the light-dependent activation of PIRK is indicated in the currents recorded from the mouse neocortical neurons. The implications of this technique extends to therapy of brain diseases caused by malfunction of specific neuronal proteins, for example, mutated Kir2.1 in Andersens syndrome, because this technique helps uncover which brain circuitry is affected by Kir2.1 deficits. Once mastered, this technique can be used to control neuronal proteins function with light in high specificity in live neurons.
After watching this video, you should have a good understanding of how to incorporate unnatural amino acids into proteins in neurons to achieve photo control over neuronal proteins of interest.
