Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Scheme of workflow to construct oligonucleotides for miRNAs (A) Workflow for construction of miRNA oligonucleotides using the BLOCK-iT ™ RNAi Designer (Invitrogen, https://rnaidesigner.thermofisher.com/rnaiexpress/ ) website. Several 21-mer-oligonucleotide sequences are automatically provided depending on the specificity for sequences of targeted genes (the sequences with “5 stars” are recommended as highly specific sequences). (B) Scheme of the contents in the annealed miRNAs for the targeted gene ( Setd1a as an example). The 64-mer oligonucleotides contain the linker sequence, the 21-mer sequence selected from the target gene, the loop sequence, and the 21-mer sequence excluding the 9th and 10th nucleotides. (C) Scheme of the process to insert the oligonucleotides into the pCAG-GFP empty vectors.

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Construct, Blocking Assay, Sequencing

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Summary of plasmids in this protocol

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Plasmid Preparation, Sequencing

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Summary for combinations of electroporated plasmids

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Plasmid Preparation, Imaging

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Summary for comparison of pairs of pyramidal neurons in paired-pulse stimulation

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Plasmid Preparation, Expressing

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet:

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Recombinant, Mutagenesis, Transfection, Blocking Assay, Expressing, Plasmid Preparation, DNA Purification, Ligation, Software, In Utero, Electroporation, Microscopy

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Summary of plasmids in this protocol

Article Snippet: Alternatives: We subcloned the mOrange sequence from pmOrange2-N1 Vector (Takara Bio USA) and replaced GFP with mOrange on the pCAG-GFP empty vector.

Techniques: Plasmid Preparation, Sequencing

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet:

Article Snippet: Alternatives: We subcloned the mOrange sequence from pmOrange2-N1 Vector (Takara Bio USA) and replaced GFP with mOrange on the pCAG-GFP empty vector.

Techniques: Recombinant, Mutagenesis, Transfection, Blocking Assay, Expressing, Plasmid Preparation, DNA Purification, Ligation, Software, In Utero, Electroporation, Microscopy

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Experimental images from patch-clamp recording using LED light (A) Patch-clamp setup for electrophysiological experiments. (B) Amplifier for the recording (upper) and patch pipettes made from glass capillaries using a puller machine (lower). (C) Blue light shining during the experiments to confirm the fluorescent expression of mOrange or GFP. (D) Images of GFP-negative control and GFP-positive knockdown neurons through DIC microscope during the experiment. The upper neuron that is surrounded by the black circle is the control neuron and white cells mean the GFP-positive neurons (lower black circle). Scale, 50 μm. (E) PC screen on which the PATCHMASTER program is running to record synaptic currents evoked by optogenetic stimulation. (F) Scheme of possible neuron pairs in the electroporated mPFC.

Article Snippet: Alternatives: We subcloned the mOrange sequence from pmOrange2-N1 Vector (Takara Bio USA) and replaced GFP with mOrange on the pCAG-GFP empty vector.

Techniques: Patch Clamp, Expressing, Negative Control, Knockdown, Microscopy, Control

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Summary of plasmids in this protocol

Article Snippet: Alternatives: We subcloned the mOrange sequence from pmOrange2-N1 Vector (Takara Bio USA) and replaced GFP with mOrange on the pCAG-GFP empty vector.

Techniques: Plasmid Preparation, Control, Sequencing

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Validation of efficiency of the KD miRNA in HEK 293T cells (A) Illustration of workflow to evaluate the efficiency of the KD-miRNA vector compared to the Scr-miRNA vector for targeted genes in HEK293T cells. The illustration was made by BioRender. WT: wildtype, Res: rescue. (B) Representative images of the GFP and mOrange from the transfected HEK293T cells in each condition. Scale, 100 μm.

Article Snippet: Alternatives: We subcloned the mOrange sequence from pmOrange2-N1 Vector (Takara Bio USA) and replaced GFP with mOrange on the pCAG-GFP empty vector.

Techniques: Biomarker Discovery, Plasmid Preparation, Transfection

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Sparsely-labeled opsin (Chronos with GFP) in the miRNA expressing neurons in the electroporated neurons (A–C) Representative Images of fluorescent expression of Chronos with GFP (A) and, KD miRNAs with mOrange (B), and the merge image showing the double-positive neurons (C). Scale, 500 μm. (D) Scheme of the patch-clamp recording using optogenetic stimulation. (E) Representative traces of optogenetically induced EPSCs from a non-fluorescent control neuron.

Article Snippet: Alternatives: We subcloned the mOrange sequence from pmOrange2-N1 Vector (Takara Bio USA) and replaced GFP with mOrange on the pCAG-GFP empty vector.

Techniques: Labeling, Expressing, Patch Clamp, Control

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Summary for comparison of pairs of pyramidal neurons in paired-pulse stimulation

Article Snippet: Alternatives: We subcloned the mOrange sequence from pmOrange2-N1 Vector (Takara Bio USA) and replaced GFP with mOrange on the pCAG-GFP empty vector.

Techniques: Comparison, Plasmid Preparation, Expressing

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: mOrange-sparse labeling for spine analysis (A) Images of the sparse labeling of GFP vector for the miRNA (left), mOrange vector (middle) as a reporter to label the individual neuron sparsely, and merge (right). Scale, 50 μm. (B) Representative images of dendrites from the mOrange-labeled neurons. Scale, 5 μm.

Article Snippet: Alternatives: We subcloned the mOrange sequence from pmOrange2-N1 Vector (Takara Bio USA) and replaced GFP with mOrange on the pCAG-GFP empty vector.

Techniques: Labeling, Plasmid Preparation

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet:

Article Snippet: Alternatives: We subcloned the mOrange sequence from pmOrange2-N1 Vector (Takara Bio USA) and replaced GFP with mOrange on the pCAG-GFP empty vector.

Techniques: Recombinant, Mutagenesis, Transfection, Blocking Assay, Expressing, Plasmid Preparation, DNA Purification, cDNA Synthesis, Ligation, Software, In Utero, Electroporation, Microscopy

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet:

Article Snippet: pmOrange2-N1 Vector , Takara Bio USA, Inc , CAT: # 632549.

Techniques: Recombinant, Mutagenesis, Transfection, Blocking Assay, Expressing, Plasmid Preparation, DNA Purification, Ligation, Software, In Utero, Electroporation, Microscopy

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Experimental images from patch-clamp recording using LED light (A) Patch-clamp setup for electrophysiological experiments. (B) Amplifier for the recording (upper) and patch pipettes made from glass capillaries using a puller machine (lower). (C) Blue light shining during the experiments to confirm the fluorescent expression of mOrange or GFP. (D) Images of GFP-negative control and GFP-positive knockdown neurons through DIC microscope during the experiment. The upper neuron that is surrounded by the black circle is the control neuron and white cells mean the GFP-positive neurons (lower black circle). Scale, 50 μm. (E) PC screen on which the PATCHMASTER program is running to record synaptic currents evoked by optogenetic stimulation. (F) Scheme of possible neuron pairs in the electroporated mPFC.

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Patch Clamp, Expressing, Negative Control, Knockdown, Microscopy, Control

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Summary of plasmids in this protocol

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Plasmid Preparation, Control, Sequencing

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Validation of efficiency of the KD miRNA in HEK 293T cells (A) Illustration of workflow to evaluate the efficiency of the KD-miRNA vector compared to the Scr-miRNA vector for targeted genes in HEK293T cells. The illustration was made by BioRender. WT: wildtype, Res: rescue. (B) Representative images of the GFP and mOrange from the transfected HEK293T cells in each condition. Scale, 100 μm.

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Biomarker Discovery, Plasmid Preparation, Transfection

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Summary for combinations of electroporated plasmids

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Plasmid Preparation, Imaging

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Sparsely-labeled opsin (Chronos with GFP) in the miRNA expressing neurons in the electroporated neurons (A–C) Representative Images of fluorescent expression of Chronos with GFP (A) and, KD miRNAs with mOrange (B), and the merge image showing the double-positive neurons (C). Scale, 500 μm. (D) Scheme of the patch-clamp recording using optogenetic stimulation. (E) Representative traces of optogenetically induced EPSCs from a non-fluorescent control neuron.

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Labeling, Expressing, Patch Clamp, Control

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: Summary for comparison of pairs of pyramidal neurons in paired-pulse stimulation

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Comparison, Plasmid Preparation, Expressing

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet: mOrange-sparse labeling for spine analysis (A) Images of the sparse labeling of GFP vector for the miRNA (left), mOrange vector (middle) as a reporter to label the individual neuron sparsely, and merge (right). Scale, 50 μm. (B) Representative images of dendrites from the mOrange-labeled neurons. Scale, 5 μm.

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Labeling, Plasmid Preparation

Journal: STAR Protocols

Article Title: Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

doi: 10.1016/j.xpro.2021.100469

Figure Lengend Snippet:

Article Snippet: pCAG-DIO-mOrange , CAG , mOrange , pCAG-GFP vector , DIO: subcloned from pAAV-EFa-DIO-mVenus mOrange: subcloned from pmOrange2-N1 Vector (Takara Bio USA).

Techniques: Recombinant, Mutagenesis, Transfection, Blocking Assay, Expressing, Plasmid Preparation, DNA Purification, cDNA Synthesis, Ligation, Software, In Utero, Electroporation, Microscopy