We thank Dr. S. Szobota for helpful discussion on neuron culture and Ca-P transfection. L.W. acknowledges support from The Salk Innovation Grant, the California Institute for Regenerative Medicine (RN1-00577-1), and the National Institutes of Health (1DP2OD004744).

All procedures in the current study were performed using Institutional Animal Care and Use Committee (IACUC) approved protocols for animal handling at The Salk Institute for Biological Studies, La Jolla, CA.
1. Uaa Incorporation in Kir2.1 and Expression of the Resultant PIRK in the Primary Neuronal Culture
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	<li>DNA Construction
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		<li>Select a target site of Kir2.1 for Uaa incorporation. Exploit prior knowledge and information about structure and function of Kir2.1, so that the cho...

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To achieve effective photo-modulation, the important initial step is to decide where to incorporate the photoresponsive Uaa in the target protein. Structural and functional information of the target protein is very helpful to guide the selection of candidate sites. At the same time, the purpose of light regulation would determine which site is most suitable. After choosing candidate sites, we recommend to test the sites in mammalian cell lines such as the Human Embryonic Kidney (HEK) cells for easier culture and manipula...

Compared to conventional electric stimulation, photostimulation offers greater temporal and spatial resolution with minimum interference to the physiological system of specimens. Since the demonstration of using lasers to stimulate neurons in 19711, many creative ways have been invented to exogenously control neuronal activity with light. Optical release of photocaged agonists has long been used to study physiological response of the neuronal network to ligands2,3,4. This technique has limited specificity due to diffusion of caged agonists. Genetic specificity is achieved by ectopically expressing light-sensitive opsin channels and pumps5,6,7, and it has been successfully applied to modulate selected neuronal networks in diverse model organisms. However, it would be difficult to apply this method to optically control various other neuronal proteins, since grafting photoresponsiveness from the opsin proteins to other proteins would require intense engineering that may alter the natural characteristics of the protein under study. Chemically tethering an exogenous photosensitive ligand to a protein has demonstrated another way to control the functionality of channel proteins8,9,10. The ligand is presented or withdrawn from the binding site of the protein through the photoisomerization of the azobenzene moiety. Tethering chemistry limits the application mainly to the extracellular side of membrane proteins, excluding the intracellular side and intracellular proteins.
Photoresponsive Uaas, after being incorporated into proteins, provide a general strategy to manipulate proteins with light. In early efforts, tRNAs chemically acylated with photocaged Uaas were microinjected into Xenopus oocytes to incorporate the Uaas into membrane receptors and ion channels11, which have advanced the understanding of their structure-function relationships12,13,14. This microinjection approach is mainly limited to large oocytes. Genetic incorporation of a Uaa bypasses the technically challenging tRNA acylation and microinjection by using an orthogonal tRNA/synthetase pair, which incorporates the Uaa through endogenous protein translation in live cells15,16,17,18. Uaa incorporation into neuronal proteins has been demonstrated in primary neurons and neural stem cells19,20. More recently, photoresponsive Uaa has been genetically incorporated into a neuronal protein in the mammalian brain in vivo for the first time21. These advancements make it possible to study neuronal proteins with Uaas in their native cellular environment.
Inwardly rectifying potassium channel Kir2.1 is a strong rectifier that passes K+ currents more readily into than out of the cell, and it is essential in regulating physiological processes including cell excitability, vascular tone, heart rate, renal salt flow and insulin release22. Overexpression of Kir2.1 hyperpolarizes the membrane potential of the target neuron, which becomes less excitable23,24. To optically control Kir2.1 in its native cells, Kang et al. genetically incorporated a photo-responsive Uaa into Kir2.1 expressed in mammalian cells, neurons and embryonic mouse brains21. A brief pulse of light was able to convert the Uaa into a natural amino acid Cys, thus activating the target Kir2.1 protein. When this photo-inducible inwardly rectifying potassium (PIRK) channel protein was expressed in rat hippocampal primary neurons, it suppressed neuronal firing in response to light activation. In addition, the PIRK channel was expressed in the embryonic mouse neocortex, and the light-activated PIRK current in cortical neurons was measured. The successful implementation of the Uaa technology in vivo in the mammalian brain opens the door to optically control neuronal proteins in their native environment, which will enable optical dissection of neuronal processes and mechanisms at the molecular level.
In this protocol we describe procedures for genetic incorporation of Uaas into primary neurons in culture and in the embryonic mouse brain in vivo. The photoresponsive Uaa Cmn and Kir2.1 are used to illustrate the process. Methods to assess successful Uaa incorporation and optical control of neuronal protein activity are provided. This protocol provides a clear guide to genetically encoding Uaas in neurons and in vivo, and to optically regulating neuronal protein function via a photoresponsive Uaa. We expect this protocol to facilitate the adoption of the in vivo Uaa technology for neuroscience and optogenetic biological studies.

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optical control of a neuronal protein using a genetically encoded unnatural amino acid in neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

To genetically incorporate a Uaa into a protein in neurons, the first important step is to design appropriate gene constructs to deliver and express genes efficiently in neurons. There are three genetic components for Uaa incorporation: (1) the target gene with the TAG amber stop codon introduced at the chosen site for Uaa incorporation (2) an orthogonal tRNA to recognize the mutated TAG stop codon, and (3) an orthogonal aminoacyl-tRNA synthetase to charge the Uaa onto the orthogonal tRNA...

Watch this Scientific Journal Video about Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons at JoVE.com

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

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.

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Research

JoVE Journal

Neuroscience

7.9K 조회수.  Tufts University. 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 ...