virus expressing channelrhodopsin aav9 flex rev chr2 h134r mcherry  (Addgene inc)

Journal: eLife

Article Title: Laminar-specific cortico-cortical loops in mouse visual cortex

doi: 10.7554/eLife.59551

Figure Lengend Snippet: ( A ) We probed the strength of CC inputs to looped and non-looped neurons in different cortical layers. ( B ) Example experiment configuration. Retrograde tracers are injected in two areas to label different projection neurons. One cortical area is also co-injected with adeno-associated virus (AAV)-channelrhodopsin-2 (ChR2) to express ChR2 in a specific CC projection. ( C ) Example of subcellular channelrhodopsin-2 (ChR2)-assisted circuit mapping (sCRACM) experiment. Pairs of neighboring retrogradely labeled neurons in the same cortical layer were sequentially recorded. During each recording, a laser beam was scanned over the dendrites of the cell at different locations in a grid pattern. ( D ) Brightfield image of an acute coronal cortical slice showing the recording pipette and photostimulation grid. ( E ) Excitatory postsynaptic currents (EPSCs) recorded from a pair of neighboring L5 neurons, evoked by photostimulating ChR2 + V2L→V1 FB terminals on a grid. ( F ) Left, dendritic morphology staining of the recorded pair. Right, identity of the recorded projection neuron was confirmed by fluorescence in the soma of both a retrograde tracer and a different-colored dye introduced from the internal patch pipette solution. ( G ) sCRACM maps of the recorded pair overlaid on their reconstructed dendrites. Responsive locations are color-coded to represent mean amplitude.

Article Snippet: Virus expressing ChR2 (AAV-2/1-CAG-Channelrhodopsin-2-Venus, Addgene #20071; 20–25 nl, titer ~5×10 12 vg/ml) was delivered intracortically either to V1 to label FF projections or V2L/V2M to label FB projections, and co-injected with red-fluorescent microspheres (Red Retrobeads IX, Lumafluor; 10–12.5 nl) to retrogradely label cells projecting to the source of FF/FB input.

Techniques: Injection, Labeling, Transferring, Staining, Fluorescence

Journal: eLife

Article Title: Laminar-specific cortico-cortical loops in mouse visual cortex

doi: 10.7554/eLife.59551

Figure Lengend Snippet: ( A ) Example of subcellular channelrhodopsin-2 (ChR2)-assisted circuit mapping (sCRACM) traces from individual neurons (data from L2/3 looped intratelencephalic [IT] neurons). Each trace corresponds to the average excitatory postsynaptic current (EPSC) in the location eliciting the largest amplitude. Blue tick, laser pulse. The arrowhead indicates a single neuron (trace in blue) in which the laser pulse evoked an early-onset EPSC, suggestive of a non-synaptic response. Ten neurons with early-onset EPSCs were detected in the entire dataset and removed from further analysis. ( B ) Anti-green fluorescent protein (GFP) immunostained section of primary visual cortex (V1) showing fluorescent medial visual area (V2M) axons in an animal injected with AAV2/1-CAG-ChR2-Venus. ( C ) Higher magnification image of a region in ( B ). The arrow indicates an example of a retrogradely infected neuron in V1. ( D ) Configuration of experiment comparing strength of V2M feedback (FB) input to pairs of L6 looped and non-looped IT neurons in V1 using AAV5-CaMKIIa-hChR2(H134R)-EYFP. ( E ) sCRACM traces from 11 looped IT neurons recorded in L6 from the experiment in ( D ). ( F ) Left, paired comparisons of total FB input to looped vs. non-looped IT neurons from the experiment in ( D ). Inset traces represent group averages for each projection class. Blue tick, light pulse. Right, sCRACM Response Index (SRI) of the same data. *, p=0.0116.

Article Snippet: Virus expressing ChR2 (AAV-2/1-CAG-Channelrhodopsin-2-Venus, Addgene #20071; 20–25 nl, titer ~5×10 12 vg/ml) was delivered intracortically either to V1 to label FF projections or V2L/V2M to label FB projections, and co-injected with red-fluorescent microspheres (Red Retrobeads IX, Lumafluor; 10–12.5 nl) to retrogradely label cells projecting to the source of FF/FB input.

Techniques: Injection, Infection

Journal: eLife

Article Title: Laminar-specific cortico-cortical loops in mouse visual cortex

doi: 10.7554/eLife.59551

Figure Lengend Snippet: ( A ) Total subcellular channelrhodopsin-2 (ChR2)-assisted circuit mapping (sCRACM) input per neuron as a function of cortical depth for both feedforward (FF) projections. Circles, individual cells. Triangles, mean values per projection class for each experiment. Averages from paired data are joined by a line. Color indicates projection class. ( B ) Total sCRACM input per neuron as a function of cortical depth for both feedback (FB) projections.

Article Snippet: Virus expressing ChR2 (AAV-2/1-CAG-Channelrhodopsin-2-Venus, Addgene #20071; 20–25 nl, titer ~5×10 12 vg/ml) was delivered intracortically either to V1 to label FF projections or V2L/V2M to label FB projections, and co-injected with red-fluorescent microspheres (Red Retrobeads IX, Lumafluor; 10–12.5 nl) to retrogradely label cells projecting to the source of FF/FB input.

Techniques:

Journal: eLife

Article Title: Laminar-specific cortico-cortical loops in mouse visual cortex

doi: 10.7554/eLife.59551

Figure Lengend Snippet: ( A ) Left, group averages of subcellular channelrhodopsin-2 (ChR2)-assisted circuit mapping (sCRACM) maps aligned by pia position showing primary visual cortex (V1) FF input to the different cell types (combining V1→V2L and V1→V2M inputs in the case of intratelencephalic [IT] neurons). Triangles, soma position. Right, vertical profiles of input strength. Error bars, s.e.m.; n, number of neurons; N, number of mice. ( B ) Group averages and vertical profiles of sCRACM maps showing FB input to the different cell types in V1 (combining V2L→V1 and V2M→V1 inputs in the case of IT neurons).

Article Snippet: Virus expressing ChR2 (AAV-2/1-CAG-Channelrhodopsin-2-Venus, Addgene #20071; 20–25 nl, titer ~5×10 12 vg/ml) was delivered intracortically either to V1 to label FF projections or V2L/V2M to label FB projections, and co-injected with red-fluorescent microspheres (Red Retrobeads IX, Lumafluor; 10–12.5 nl) to retrogradely label cells projecting to the source of FF/FB input.

Techniques:

Journal: eLife

Article Title: Laminar-specific cortico-cortical loops in mouse visual cortex

doi: 10.7554/eLife.59551

Figure Lengend Snippet: ( A ) Left, group averages of subcellular channelrhodopsin-2 (ChR2)-assisted circuit mapping (sCRACM) maps aligned by soma position showing primary visual cortex (V1) FF input to the different cell types (combining V1→V2L and V1→V2M inputs in the case of intratelencephalic [IT] neurons). Triangles, soma position. Right, vertical profiles of the mean distribution of inputs as a function of distance to soma. Error bars, s.e.m.; n, number of neurons; N, number of mice. ( B ) Group averages and vertical profiles of soma-aligned sCRACM maps showing FB input to the different cell types in V1 (combining V2L→V1 and V2M→V1 inputs in the case of IT neurons).

Article Snippet: Virus expressing ChR2 (AAV-2/1-CAG-Channelrhodopsin-2-Venus, Addgene #20071; 20–25 nl, titer ~5×10 12 vg/ml) was delivered intracortically either to V1 to label FF projections or V2L/V2M to label FB projections, and co-injected with red-fluorescent microspheres (Red Retrobeads IX, Lumafluor; 10–12.5 nl) to retrogradely label cells projecting to the source of FF/FB input.

Techniques:

Journal: eLife

Article Title: Laminar-specific cortico-cortical loops in mouse visual cortex

doi: 10.7554/eLife.59551

Figure Lengend Snippet: ( A ) Configuration of experiments comparing strength of primary visual cortex (V1) FF input to pairs of L6 looped and non-looped IT neurons in lateral visual area (V2L) or medial visual area (V2M). ( B ) Example pair of subcellular channelrhodopsin-2 (ChR2)-assisted circuit mapping (sCRACM) maps overlaid on reconstructed dendrites showing monosynaptic V1 FF inputs to a looped IT neuron (left) and an adjacent non-looped IT neuron (right) recorded in V2L. ( C ) Left, paired comparisons of perisomatic FF input to looped vs. non-looped IT neurons (n, number of cell pairs; N, number of mice); black dots, V1→V2L inputs; gray dots, V1→V2M inputs. Traces were generated by averaging the mean perisomatic excitatory postsynaptic current (EPSC) of each neuron across all neurons in the same projection class. Colors correspond to ( A ). Blue tick, laser pulse. Scale bars in all panels, 2 pA and 20 ms. Right, sCRACM Response Index (SRI) of the same data. Number of cell pairs and animals are the same as in the left plot unless otherwise specified. Horizontal line, mean. *, p<0.05, see text for exact value. ( D ) Same as C for apical inputs (SRI: V1→V2L, n = 7, N = 6; V1→V2M, n = 7, N = 6). ( E ) Configuration of experiment comparing strength of V1 FF input to pairs of L6 looped IT and CT neurons in V2L. ( F ) Example pair of sCRACM maps overlaid on reconstructed dendrites showing monosynaptic V1 FF inputs to a looped IT neuron (left) and an adjacent CT neuron (right) recorded in V2L. ( G ) Paired comparisons and SRI of perisomatic FF input to looped IT vs. CT neurons. ( H ) Paired comparisons and SRI (n = 5, N = 5) of apical FF input to looped IT vs. CT neurons. ( I ) Configuration of experiments comparing strength of V2L or V2M FB input to pairs of L6 looped and non-looped IT neurons in V1. ( J ) Example pair of sCRACM maps overlaid on reconstructed dendrites showing monosynaptic V2L FB inputs to a looped IT neuron (left) and an adjacent non-looped IT neuron (right) recorded in V1. ( K ) Paired comparisons and SRI of perisomatic FB input to looped vs. non-looped IT neurons. Dark green dots, V2L→V1 inputs; light green dots, V2M→V1 inputs. ( L ) Paired comparisons and SRI (V2L→V1, n = 5, N = 5; V2M→V1, n = 4, N = 4) of apical FB input to looped vs. non-looped IT neurons. ( M ) Configuration of experiment comparing strength of V2L FB input to pairs of L6 looped IT and CT neurons in V1. ( N ) Example pair of sCRACM maps overlaid on reconstructed dendrites showing monosynaptic V2L FB inputs to a looped IT neuron (left) and an adjacent CT neuron (right) recorded in V1. ( O ) Paired comparisons and SRI of perisomatic FB input to looped IT vs. CT neurons. ( P ) Paired comparisons and SRI (n = 8, N = 7) of apical FB input to looped IT vs. CT neurons.

Article Snippet: Virus expressing ChR2 (AAV-2/1-CAG-Channelrhodopsin-2-Venus, Addgene #20071; 20–25 nl, titer ~5×10 12 vg/ml) was delivered intracortically either to V1 to label FF projections or V2L/V2M to label FB projections, and co-injected with red-fluorescent microspheres (Red Retrobeads IX, Lumafluor; 10–12.5 nl) to retrogradely label cells projecting to the source of FF/FB input.

Techniques: Generated

Journal: eLife

Article Title: Laminar-specific cortico-cortical loops in mouse visual cortex

doi: 10.7554/eLife.59551

Figure Lengend Snippet: ( A ) Configuration of experiments comparing strength of primary visual cortex (V1) FF input to pairs of L5 looped and non-looped IT neurons in lateral visual (V2L) or medial visual (V2M) areas. ( B ) Example pair of subcellular channelrhodopsin-2 (ChR2)-assisted circuit mapping (sCRACM) maps overlaid on reconstructed dendrites showing monosynaptic V1 FF inputs to a looped IT neuron (left) and an adjacent non-looped IT neuron (right) recorded in V2L. ( C ) Left, paired comparisons of perisomatic FF input to looped vs. non-looped IT neurons; black dots, V1→V2L inputs; gray dots, V1→V2M inputs. Traces were generated by averaging the mean perisomatic excitatory postsynaptic current (EPSC) of each neuron across all neurons in the same projection class. Blue tick, laser pulse. Scale bars in all panels, 2 pA and 20 ms. Right, sCRACM Response Index (SRI) of the same data. Number of cell pairs and animals are the same as in the left plot unless otherwise specified. Horizontal line, mean. *, p<0.05, see text for exact value. ( D ) Same as C for apical inputs (SRI: V1→V2L, n = 12, N = 8; V1→V2M, n = 11, N = 7). ( E ) Configuration of experiment comparing strength of V1 FF input to pairs of L5 looped IT and PT neurons in V2L. ( F ) Example pair of sCRACM maps overlaid on reconstructed dendrites showing monosynaptic V1 FF inputs to a looped IT neuron (left) and an adjacent PT neuron (right) recorded in V2L. ( G ) Paired comparisons and SRI of perisomatic FF input to looped IT vs. PT neurons. ( H ) Paired comparisons and SRI (n = 11, N = 7) of apical FF input to looped IT vs. PT neurons. ( I ) Configuration of experiments comparing strength of V2L or V2M FB input to pairs of L5 looped and non-looped IT neurons in V1. ( J ) Example pair of sCRACM maps overlaid on reconstructed dendrites showing monosynaptic V2L FB inputs to a looped IT neuron (left) and an adjacent non-looped IT neuron (right) recorded in V1. ( K ) Paired comparisons and SRI of perisomatic FB input to looped vs. non-looped IT neurons. Dark green dots, V2L→V1 inputs; light green dots, V2M→V1 inputs. ( L ) Paired comparisons and SRI (V2L→V1, n = 11, N = 10; V2M→V1, n = 11, N = 10) of FB input in L1 to looped vs. non-looped IT neurons. ( M ) Configuration of experiment comparing strength of V2L FB input to pairs of L5 looped IT and PT neurons in V1. ( N ) Example pair of sCRACM maps overlaid on reconstructed dendrites showing monosynaptic V2L FB inputs to a looped IT neuron (left) and an adjacent PT neuron (right) recorded in V1. ( O ) Paired comparisons and SRI of perisomatic FB input to looped IT vs. PT neurons. ( P ) Paired comparisons and SRI (n = 12, N = 9) of FB input in L1 to looped IT vs. PT neurons.

Article Snippet: Virus expressing ChR2 (AAV-2/1-CAG-Channelrhodopsin-2-Venus, Addgene #20071; 20–25 nl, titer ~5×10 12 vg/ml) was delivered intracortically either to V1 to label FF projections or V2L/V2M to label FB projections, and co-injected with red-fluorescent microspheres (Red Retrobeads IX, Lumafluor; 10–12.5 nl) to retrogradely label cells projecting to the source of FF/FB input.

Techniques: Generated

Journal: eLife

Article Title: Laminar-specific cortico-cortical loops in mouse visual cortex

doi: 10.7554/eLife.59551

Figure Lengend Snippet: ( A ) Configuration of experiments comparing strength of primary visual cortex (V1) FF input to pairs of L2/3 looped and non-looped intratelencephalic (IT) neurons in lateral visual area (V2L) or medial visual area (V2M). ( B ) Example pair of subcellular channelrhodopsin-2 (ChR2)-assisted circuit mapping (sCRACM) maps overlaid on reconstructed dendrites showing monosynaptic V1 FF inputs to a looped IT neuron (left) and an adjacent non-looped IT neuron (right) recorded in V2L. ( C ) Left, paired comparisons of perisomatic FF input to looped vs. non-looped IT neurons; black dots, V1→V2L inputs; gray dots, V1→V2M inputs. Traces were generated by averaging the mean perisomatic excitatory postsynaptic current (EPSC) of each neuron across all neurons in the same projection class. Blue tick, laser pulse. Scale bars in all panels, 2 pA and 20 ms. Right, sCRACM Response Index (SRI) of the same data. Number of cell pairs and animals are the same as in the left plot unless otherwise specified. Horizontal line, mean. *, p<0.05, see text for exact value. ( D ) Same as C for inputs in L1 (SRI: V1→V2L, n = 11, N = 7; V1→V2M, n = 7, N = 5). ( E ) Configuration of experiments comparing strength of V2L or V2M FB input to pairs of L2/3 looped and non-looped IT neurons in V1. ( F ) Example pair of sCRACM maps overlaid on reconstructed dendrites showing monosynaptic V2L FB inputs to a looped IT neuron (left) and an adjacent non-looped IT neuron (right) recorded in V1. ( G ) Paired comparisons and SRI of perisomatic FB input to looped vs. non-looped IT neurons. Dark green dots, V2L→V1 inputs; light green dots, V2M→V1 inputs. ( H ) Same as G for inputs in L1 (SRI: V2L→V1, n = 11, N = 10; V2M→V1, n = 12, N = 11).

Article Snippet: Virus expressing ChR2 (AAV-2/1-CAG-Channelrhodopsin-2-Venus, Addgene #20071; 20–25 nl, titer ~5×10 12 vg/ml) was delivered intracortically either to V1 to label FF projections or V2L/V2M to label FB projections, and co-injected with red-fluorescent microspheres (Red Retrobeads IX, Lumafluor; 10–12.5 nl) to retrogradely label cells projecting to the source of FF/FB input.

Techniques: Generated

Journal: eLife

Article Title: Laminar-specific cortico-cortical loops in mouse visual cortex

doi: 10.7554/eLife.59551

Figure Lengend Snippet:

Article Snippet: Virus expressing ChR2 (AAV-2/1-CAG-Channelrhodopsin-2-Venus, Addgene #20071; 20–25 nl, titer ~5×10 12 vg/ml) was delivered intracortically either to V1 to label FF projections or V2L/V2M to label FB projections, and co-injected with red-fluorescent microspheres (Red Retrobeads IX, Lumafluor; 10–12.5 nl) to retrogradely label cells projecting to the source of FF/FB input.

Techniques: Recombinant, Plasmid Preparation, Software

Journal: eLife

Article Title: Hippocampal inputs engage CCK+ interneurons to mediate endocannabinoid-modulated feed-forward inhibition in the prefrontal cortex

doi: 10.7554/elife.55267

Figure Lengend Snippet: Figure 1. vHPC-evoked feed-forward inhibition and CCK+ interneurons. (A) Schematic for injections of AAV-ChR2-EYFP into vHPC and CTB-647 into cPFC. (B) Left, Confocal image of vHPC axons (blue) in IL PFC. Scale bar = 100 mm. Right, Confocal image of biocytin-filled L5 IT cell in IL PFC. Scale bar = 100 mm. (C) Left, Average vHPC-evoked EPSCs at 65 mV (black) and IPSCs at +15 mV (gray). Blue arrows = 5 pulses at 20 Hz. Right, Average response amplitudes as a function of pulse number (n = 7 cells, 3 animals). (D) Td-tomato labeling of PV+ (blue) and SOM+ (green) interneurons in PV-Cre Ai14 and SOM-Cre Ai14 animals, respectively. Scale bar = 100 mm. (E) Left, Schematic for injections of viruses into PFC of CCK-Cre mice. Middle, Injection of AAV-DIO-GFP labels CCK+ interneurons and pyramidal cells (n = 3 animals). Right, Injection of AAV-Dlx-Flex-GFP labels CCK+ interneurons (n = 3 animals). Scale bars = 100 mm. (F) Quantification of co-labeling of CCK+ interneurons with PV (top) and SOM (bottom) (PV staining, n = 308 cells, 17 slices, 6 animals; SOM staining, n = 105 cells, 8 slices, 3 animals). (G) Left, Confocal image of a biocytin-filled CCK+ interneuron in L5 Figure 1 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.55267 15 of 22 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Other AAV1-DIO-ChR2-eYFP UPenn Cat #: AV-1–20298P AAV virus expressing Cre-dependent ChR2 Other AAV1-ChR2-eYFP UPenn Cat #: AV-26973P AAV virus expressing ChR2 Other AAV1-DIO-eYFP UPenn Cat #: AV-1–27056 AAV virus expressing Cre-dependent eYFP Other AAVrg-GFP Addgene Cat #: 37825-AAVrg RRID:Addgene_37825 Retrograde virus expressing GFP Other AAVrg-TdTomato Addgene Cat #: 59462-AAVrg RRID:Addgene_59462 Retrograde virus expressing TdTomato Other AAV-Dlx-Flex-GFP Addgene Cat #: 83900 RRID:Addgene_83900 AAV virus expressing Cre-dependent GFP in interneurons Other AAV-Dlx-FlexChR2-mCherry This paper AAV virus expressing Cre-dependent ChR2 in interneurons Antibody Anti-PV (mouse, monoclonal) Millipore Cat#: MAB1572 RRID:AB_2174013 (1:2000) Antibody Anti-CB1R (guinea pig, polyclonal) Frontier Institute Cat#: Af530 RRID:AB_2314113 (1:500) Antibody Anti-SOM (rat, monoclonal) Millipore Cat#: MAB354 RRID:AB_2255365 (1:200) Chemical compound, drug WIN 55,212–2 Tocris Cat#: 1038 1 mM Chemical compound, drug AM-251 Tocris Cat#: 1117 10 mM

Techniques: Inhibition, Labeling, Injection, Staining

Journal: eLife

Article Title: Hippocampal inputs engage CCK+ interneurons to mediate endocannabinoid-modulated feed-forward inhibition in the prefrontal cortex

doi: 10.7554/elife.55267

Figure Lengend Snippet: Figure 4. CCK+ interneurons make connections onto pyramidal cells. (A) Left, Injections of AAV-Dlx-Flex-ChR2 into the PFC of CCK-Cre animals. Middle, Example AP traces from a ChR2-expressing CCK+ interneuron, stimulated with 2 ms light pulses at 20 Hz, showing 5 offset trials. Purple arrow = light pulse. Right, Average AP numbers across stimulation pulses (n = 6 cells, 3 animals). (B) Left, Average CCK+-evoked IPSCs at L5 pyramidal cells in IL PFC. When recording at 50 mV with a low Cl- internal solution, only outward IPSCs were observed (black). IPSCs were unchanged after wash-in of NBQX + CPP (blue) but abolished by wash-in of gabazine (red). Right, Summary of IPSC amplitudes in the different conditions. Purple arrow = light pulse. (C) Schematic of triple injections, with CTB-647 into cPFC, red retrobeads into PAG, and AAV-Dlx-Flex-ChR2 into PFC. (D) Left, Recording schematic of CCK+ inputs onto IT and PT cells. Middle, CCK+-evoked IPSCs at PT and IT cells. Right, Summary of IPSC amplitudes at PT and IT cells (7 Figure 4 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.55267 15 of 22 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Other AAV1-DIO-ChR2-eYFP UPenn Cat #: AV-1–20298P AAV virus expressing Cre-dependent ChR2 Other AAV1-ChR2-eYFP UPenn Cat #: AV-26973P AAV virus expressing ChR2 Other AAV1-DIO-eYFP UPenn Cat #: AV-1–27056 AAV virus expressing Cre-dependent eYFP Other AAVrg-GFP Addgene Cat #: 37825-AAVrg RRID:Addgene_37825 Retrograde virus expressing GFP Other AAVrg-TdTomato Addgene Cat #: 59462-AAVrg RRID:Addgene_59462 Retrograde virus expressing TdTomato Other AAV-Dlx-Flex-GFP Addgene Cat #: 83900 RRID:Addgene_83900 AAV virus expressing Cre-dependent GFP in interneurons Other AAV-Dlx-FlexChR2-mCherry This paper AAV virus expressing Cre-dependent ChR2 in interneurons Antibody Anti-PV (mouse, monoclonal) Millipore Cat#: MAB1572 RRID:AB_2174013 (1:2000) Antibody Anti-CB1R (guinea pig, polyclonal) Frontier Institute Cat#: Af530 RRID:AB_2314113 (1:500) Antibody Anti-SOM (rat, monoclonal) Millipore Cat#: MAB354 RRID:AB_2255365 (1:200) Chemical compound, drug WIN 55,212–2 Tocris Cat#: 1038 1 mM Chemical compound, drug AM-251 Tocris Cat#: 1117 10 mM

Techniques: Expressing