Journal: bioRxiv

Article Title: Mapping of dI3 neuron sensorimotor circuits across the cervical and lumbar spinal cord

doi: 10.1101/2024.11.17.624039

Figure Lengend Snippet: A. Diagram of genetic strategy for generating Isl1 Cre +/- ; Slc17a6 FlpO +/+ ; Ai80 (RCFL-CatCh)-D and Isl1 Cre +/- ; Slc17a6 FlpO +/+ ; Ai65 (RCFL-tdT)-D mice. Isl1 Cre +/- mice were crossed with Slc17a6 FlpO +/+ mice to drive the expression of Cre and FlpO recombinase in dI3 neurons and Vglut2 + nociceptive afferents, forming dI3-driver mice. For optogenetic experiments, Isl1 Cre +/- ; Slc17a6 FlpO +/+ (dI3-driver) mice were crossed with Ai80 (RCFL-CatCh)-D (CatCh) mice containing a frt -flanked STOP cassette and a loxP -flanked STOP cassette upstream of the CatCh/EYFP fluorescent protein, resulting in Isl1 Cre +/- ; Slc17a6 FlpO +/+ ; Ai80 (RCFL-CatCh)-D (dI3-driver-CatCh) mice. To visualize the dI3 neurons and Vglut2 + afferents, dI3-driver mice were crossed with Ai65 (RCFL-tdT)-D (TdTomato) mice containing a frt -flanked STOP cassette and a loxP -flanked STOP cassette upstream of the TdTomato fluorescent protein, resulting in Isl1 Cre +/- ; Slc17a6 FlpO +/+ ; Ai65 (RCFL-tdT)-D (dI3-driver-TdTomato) mice. B. Representative image of TdTomato (red) labelled dI3 neurons and Vglut2 + afferents in dI3-driver-TdTomato mice. To avoid activation of these afferents in dI3-driver-CatCh mice, a dorsal hemisection was performed at the indicated level. C. Diagram of the ex vivo spinal cord preparation. The spinal cord was isolated in neonatal P1-P7 dI3-driver-CatCh mice, and a dorsal hemisection was performed at the cervical (C1-C8) and lumbar (L1-L5) segments to remove Vglut2-CatCh + afferents. Three suction electrodes were simultaneously applied to various ventral roots to record from C4-C8 and L1-L5 segments, and blue light was applied to ipsilateral segments. Within the same animals, blue light was also independently applied to contralateral segments from C4-C8 and L1-L5.

Article Snippet: To visualize the dI3 neurons and Vglut2 + afferents, dI3-driver mice were crossed with Ai65 (RCFL-tdT)-D (TdTomato) mice (The Jackson Laboratory, Strain No.: 021875) containing a frt -flanked STOP cassette and a loxP -flanked STOP cassette upstream of the TdTomato fluorescent protein, resulting in Isl1 Cre +/- ; Slc17a6 FlpO +/+ ; Ai65 (RCFL-tdT)-D (dI3-driver-TdTomato) mice.

Techniques: Expressing, Activation Assay, Ex Vivo, Isolation

Journal: Cell reports

Article Title: Generation of a DAT-P2A-Flpo mouse line for intersectional genetic targeting of dopamine neuron subpopulations

doi: 10.1016/j.celrep.2021.109123

Figure Lengend Snippet: (A–C) Confocal images from a P28 male NEX-Cre;Ai9 mouse showing tdTomato Cre-reporter expression (representative of 3 mice). (A) Whole brain coronal section showing tdTomato expression throughout the cortex and hippocampus. Boxed region denotes the VTA and ventral SNc (B) TdTomato expression in the midbrain including the SNc, VTA, and IPN. (C) TdTomato expression in the dorsal striatum (dStr) and NAc. (D–F) Confocal images from a P50 male DAT-P2A-Flpo;RCE-FRT mouse showing EGFP Flp reporter expression (representative of 7 mice). (D) Whole brain coronal section showing restricted EGFP expression in the midbrain (boxed region). (E) EGFP expression in the midbrain. (F) EGFP expression in the dStr and NAc. (G–I) Confocal images from a P50 female DAT-P2A-Flpo;NEX-Cre;Ai65 mouse showing tdTomato expression (representative of 5 mice). (G) Whole brain coronal section showing restricted tdTomato expression in the VTA (boxed region). (H) TdTomato expression in the midbrain. (I) TdTomato expression in the dStr and NAc. (J) Confocal images of a VTA section from a P50 DAT-P2A-Flpo;NEX-Cre;Ai65 mouse. Top panel shows tdTomato fluorescence, bottom panel shows a merged image with TH immunostaining in green and tdTomato in magenta. (K) Confocal images of the projection targets of Neurod6 + DA neurons in the striatum and NAc (representative of 5 mice). Left panel shows TH immunostaining. Middle panel shows tdTomato + projections. Right panel shows merged image with TH in green and tdTomato + projections in magenta. DMS, dorsomedial striatum; DLS, dorsolateral striatum; C, nucleus accumbens core; ac, anterior commissure; LSh, lateral shell (of NAc); MSh, medial shell (of NAc); OT, olfactory tubercle.

Article Snippet: DAT-P2A-Flpo mice were generated using Ai65 mouse embryonic stem cells (mESCs) from the Allen Institute for Brain Science (a gift from Tanya Daigle, Ph.D.).

Techniques: Expressing, Fluorescence, Immunostaining

Journal: Cell reports

Article Title: Generation of a DAT-P2A-Flpo mouse line for intersectional genetic targeting of dopamine neuron subpopulations

doi: 10.1016/j.celrep.2021.109123

Figure Lengend Snippet: (A) Whole brains from 2 male and 1 female P120 DAT-P2A-Flpo;NEX-Cre;Ai65 mice were optically cleared and imaged without sectioning using a light sheet microscope. Shown is a representative max projection of 600 μm from a horizontal plane z stack (representative of 3 mice). Right panels show zoomed-in images of Neurod6 + DA neurons in the midbrain (I), axonal tracts from the midbrain to the NAc MSh (II), Neurod6 + DA neuron axon terminals in the MSh (III), and fibers crossing the midline (IV). Ctx, cortex. (B) Coronal view (XZ projection) comprising a 1.5 mm anterior/posterior (A/P) cross section of the VTA in optically cleared DAT-P2A-Flpo;NEX-Cre;Ai65 mice. Three separate brains were aligned and merged. Image is overlaid with brain region outlines from the middle position of the 1.5 mm cross section from the Allen Brain Atlas. (C) Coronal z stack image of a 1.5 mm cross section of the VTA from a single brain color coded by depth. Depth scale is shown in bottom panel. (D) Coronal z stack image of TrailMap-extracted axons from a 500 μm section of the striatum and NAc. Three separate brains were aligned and merged. Extracted axons are overlaid onto a reference slice from the middle position of the 500 μm section from the Allen Brain Atlas. (E) Projections and processes of DAT-P2A-Flpo;NEX-Cre;Ai65-positive neurons visualized in a 3D view of TrailMap-extracted processes from three aligned brains. See also , , and , , and .

Article Snippet: DAT-P2A-Flpo mice were generated using Ai65 mouse embryonic stem cells (mESCs) from the Allen Institute for Brain Science (a gift from Tanya Daigle, Ph.D.).

Techniques: Microscopy

Journal: Cell reports

Article Title: Generation of a DAT-P2A-Flpo mouse line for intersectional genetic targeting of dopamine neuron subpopulations

doi: 10.1016/j.celrep.2021.109123

Figure Lengend Snippet:

Article Snippet: DAT-P2A-Flpo mice were generated using Ai65 mouse embryonic stem cells (mESCs) from the Allen Institute for Brain Science (a gift from Tanya Daigle, Ph.D.).

Techniques: Virus, Plasmid Preparation, Recombinant, Electron Microscopy, Blocking Assay, Protease Inhibitor, Cloning, BIA-KA, Derivative Assay, Generated, Software

Journal: bioRxiv

Article Title: Generation of a DAT-Flp mouse line for intersectional genetic targeting of dopamine neuron subpopulations

doi: 10.1101/2020.06.24.167908

Figure Lengend Snippet: A-C) Confocal images from a P28 male NEX-Cre;Ai9 mouse showing tdTomato expression. A) Whole coronal section at A/P −3.4 from Bregma, showing high expression throughout the cortex and hippocampus. B) TdTomato expression in midbrain including the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). Note the fiber tracts in the medial lemniscus and cell bodies in the VTA and adjacent interpeduncular nucleus (IPN). C) TdTomato expression in the dorsal striatum (dStr) and nucleus accumbens (NAc) at A/P +1.34 from Bregma. D-F) Confocal images from a P50 male DAT-Flp;RCE:FRT mouse showing EGFP expression. D) Whole coronal section at A/P −3.4 from Bregma, showing restricted expression in the midbrain. E) TdTomato expression in the midbrain. F) TdTomato expression in the striatum and NAc at A/P +1.34 from Bregma. G-I) Confocal images from a P50 female DAT-Flp;NEX-Cre;Ai65 mouse showing tdTomato expression. G) Whole coronal section at A/P −3.4 from Bregma, showing restricted expression in the VTA. H) TdTomato expression in the midbrain. I) TdTomato expression in the striatum and NAc at A/P +1.34 from Bregma. J) Confocal images of a VTA section from a P50 DAT-Flp;NEX-Cre;Ai65 mouse showing tdTomato (upper panel) co-localization with TH immunostaining (lower panel, merged view). K) Confocal images of the projection targets of DAT-Flp;NEX-Cre;Ai65 neurons in the striatum and NAc. Left panel shows TH immunostaining. Middle panel shows tdTomato+ projections from DAT-Flp;NEX-Cre;Ai65 neurons. Right panel shows merged image with TH in green and DAT-Flp;NEX-Cre;Ai65 projections in magenta. [DMS = dorsomedial striatum, DLS = dorsolateral striatum, C = nucleus accumbens core, ac = anterior commissure, LSh = nucleus accumbens lateral shell, MSh = nucleus accumbens medial shell, OT = olfactory tubercle] L) Confocal images of the SNc/VTA of a P90 female DAT-Flp;NEX-Cre mouse injected with AAV-Fon/Coff-EYFP (top panel) with TH immunostaining (bottom panel, merged view). M) Confocal images of the projection targets of DAT-Flp;NEX-Cre neurons expressing Fon/Coff-EYFP in the striatum and NAc. Left panel shows TH immunostaining. Middle panel shows EYFP+ projections from DAT-Flp-positive/NEX-Cre negative neurons. Right panel shows merged image with TH in green and DAT-Flp-positive/NEX-Cre-negative projections in magenta. See also .

Article Snippet: Ai65 mESCs were karyotyped (Cell Line Genetics, Inc. Madison, WI) to ensure chromosomal health both before and after gene editing.

Techniques: Expressing, Immunostaining, Injection

Journal: bioRxiv

Article Title: Generation of a DAT-Flp mouse line for intersectional genetic targeting of dopamine neuron subpopulations

doi: 10.1101/2020.06.24.167908

Figure Lengend Snippet: A) Schematic of the triple transgenic cross to generate DAT-Flp;NEX-Cre;Ai65 mice. In Ai65 mice, tdTomato is expressed in cells that express both Flp- and Cre-recombinase . B) Confocal images of projections from DAT-Flp;NEX-Cre;Ai65-expressing neurons to the lateral septum (LS). Left panel shows TH immunostaining. Middle panel shows tdTomato+ projections in the LS. Right panel shows a merged image of TdTomato (magenta) and TH (green) overlap in the LS. DMS = dorsomedial striatum C) Schematic of the strategy to label DA neurons that do not express Neurod6 (see experiment in ). A Flp-on, Cre-off EYFP AAV construct was generated by Fenno et al with artificially engineered introns to allow expression of full length EYFP only when Flp is expressed in the absence of Cre.

Article Snippet: Ai65 mESCs were karyotyped (Cell Line Genetics, Inc. Madison, WI) to ensure chromosomal health both before and after gene editing.

Techniques: Transgenic Assay, Expressing, Immunostaining, Construct, Generated

Journal: bioRxiv

Article Title: Generation of a DAT-Flp mouse line for intersectional genetic targeting of dopamine neuron subpopulations

doi: 10.1101/2020.06.24.167908

Figure Lengend Snippet: A) Whole brains from 2 male and 1 female P120 DAT-Flp;NEX-Cre;Ai65 mice were optically cleared and processed using the Adipo-Clear pipeline. TdTomato fluorescence was amplified with an anti-RFP antibody and whole brains were imaged without sectioning using a light-sheet microscope. Shown is a max projection of 600 μm from a horizontal plane Z-stack. Right panels show zoomed-in images of Neurod6 + DA neurons in the midbrain (I), axonal tracts from the midbrain to the nucleus accumbens medial shell (NAc MSh) through the medial forebrain bundle (II), Neurod6 + DA neuron axon terminals in the MSh (III), and contralateral fibers crossing the midline (IV). Ctx = cortex. B) Coronal view (XZ projection) comprising a 1.5 mm A/P cross section of the VTA in optically cleared DAT-Flp;NEX-Cre;Ai65 mice. 3 separate brains were aligned and merged. Image is overlaid with brain region outlines from the middle position of the 1.5 mm cross-section from the Allen Brain Institute reference atlas. C) Coronal Z-stack image of a 1.5 mm cross section of the VTA from a single brain color coded by depth. Depth scale is shown in lower panel. D) Coronal Z-stack image of TrailMap-extracted axons from a 500 μm section of the striatum and NAc. 3 separate brains were aligned and merged. Extracted axons are overlaid onto a reference slice from the middle position of the 500 μm section from the Allen Brain Atlas. E) Projections and processes of DAT-Flp;NEX-Cre;Ai65-positive neurons visualized in a 3D view of TrailMap-extracted processes from three aligned brains. See also , Supplemental Table 1, and Supplemental Videos 1-3

Article Snippet: Ai65 mESCs were karyotyped (Cell Line Genetics, Inc. Madison, WI) to ensure chromosomal health both before and after gene editing.

Techniques: Fluorescence, Amplification, Microscopy

Journal: bioRxiv

Article Title: Generation of a DAT-Flp mouse line for intersectional genetic targeting of dopamine neuron subpopulations

doi: 10.1101/2020.06.24.167908

Figure Lengend Snippet: A) Brains from 2 male and 1 female P120 DAT-Flp;NEX-Cre;Ai65 mice were processed with Adipo-Clear and imaged on a light sheet microscope. Projections and processes were quantified using TrailMap. Heatmap shows the total axonal/dendrite content of 277 brain regions using boundaries from the Allen Mouse Common Coordinate Framework (CCF). Values are normalized to both the region volume and total projection content per brain (taken from Supplemental Table 1 with ‘root’, ‘fiber tracts’, ‘ventricular systems’, and ‘Nucleus accumbens’ removed). The rows represent 3 independent brains. AON = Anterior optic nucleus; OT = Olfactory tubercle; SI = Substantia innominata; LPO = Lateral preoptic area; PST = Preparasubthalamic nucleus; PSTN = Parasubthalamic nucleus; PeF = Perifornical nucleus; PN = Paranigral nucleus; RR = Retrorubral area; IPN = Interpeduncular nucleus; CLi = Central linear nucleus raphe; DR = Dorsal nucleus Raphe. B) DAT-Flp;Nex-Cre;Ai65 mice exhibit tdTomato expression (magenta) in a small number of cortical neurons that do not co-express TH by immunostaining (green). C-D) Horizontal 450 μm Z-stack projections of light-sheet microscope whole brain images. DAT-Flp;NEX-Cre;Ai65 mice show consistent tdTomato expression in subpopulations of neurons in the anterior olfactory area (C) and cerebellum (D). See also Supplemental Table 1 and Supplemental Videos 1-3.

Article Snippet: Ai65 mESCs were karyotyped (Cell Line Genetics, Inc. Madison, WI) to ensure chromosomal health both before and after gene editing.

Techniques: Microscopy, Expressing, Immunostaining