pcdna5 frt to ha Search Results


hm3dq  (Addgene inc)
93
Addgene inc hm3dq
(A and B) Intrathecal NA (A, 0.1 nmol) or Phe (B, 0.05 nmol)-induced mechanical hypersensitivity in control ( Adra1a flox/flox ) and Vgat + IN-selective α 1A R conditional knockout mice [ Vgat-Cre ; Adra1a flox/flox mice (Vgat + INs–α 1A R cKO mice)] (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; n.s., not significant). (C) Changes in PWT after acute exposure to restraint stress in control ( Adra1a flox/flox ; n = 6 mice) and Vgat + INs–α 1A R cKO mice ( n = 9 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test). (D) Representative trace of membrane potentials in tdTomato + (Vgat + ) SDH neurons after application of NA (20 μM) to spinal cord slices from Vgat-Cre ; Rosa-tdTomato mice. A 1 R agonist CPA (1 μM) was co-applied with NA. (E) Percentage of Vgat + SDH neurons whose NA-evoked response was inhibited by CPA ( n = 10 cells from 7 mice). (F) Representative images of Adora1 (A 1 R) mRNA expression (green) in Slc32a1 (Vgat) + INs (magenta). DAPI staining is shown in gray. Arrowheads indicate A 1 R-expressing Vgat + cells. Scale bar, 25 μm. (G) SaCas9 (yellow, detected by HA-tag) and mCherry (magenta) expression in the PAX2 + INs (cyan) at 3 weeks after intra-SDH injection of AAV-flex[SaCas9] and AAV-flex[mCherry]-U6-sgAdora1 in Vgat-Cre mice. Arrowheads indicate genome-editing Vgat + cells. Scale bar, 25 μm. (H) Representative traces of membrane potentials in Vgat + INs after application of NA and CPA to spinal cord slices from Vgat-Cre mice with conditional knockdown of A 1 Rs in Vgat + INs (SDH-Vgat + IN–A 1 R cKD) and their controls (Control; Vgat-Cre mice with intra-SDH injection of AAV-flex[mCherry]). (I) Percentage of mCherry + (Vgat + ) SDH neurons whose NA-evoked response was inhibited by CPA (Control, n = 10 cells from 8 mice; SDH-Vgat + IN–A 1 R cKD, n = 8 cells from 5 mice). (J) Expression of <t>hM3Dq</t> (green, detected by HA-tag) in the SDH at 3 weeks after intra-SDH injection of AAV-flex[hM3Dq] in Hes5-CreERT2 mice treated with TAM. Dashed line indicates an outline of the gray matter of SDH. GFAP (magenta, bottom left), SOX9 (magenta, bottom right). Arrowheads indicate HA-tag + astrocytes. Scale bars, 200 μm (top) and 25 μm (bottom). (K and L) Representative traces of sIPSCs (K) and quantitative analysis of their frequency (L) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-hM3Dq mice treated with TAM [Pre and CNO: before and after bath application of CNO (100 μM), respectively] ( n = 7 cells from 7 mice; Wilcoxon signed-rank test; * P < 0.05). (M and N) Representative traces of sIPSCs (M) and quantitative analysis of their frequency (N) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-flex[hM3Dq] mice treated with TAM [Pre and CNO: before and after bath application of CNO with CPT (1 μM), respectively] ( n = 13 cells from 13 mice; Wilcoxon signed-rank test; n.s., not significant). Data represent mean ± SEM.
Hm3dq, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews
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pcdna5 frt ha hm4di  (Addgene inc)
93
Addgene inc pcdna5 frt ha hm4di
(A and B) Intrathecal NA (A, 0.1 nmol) or Phe (B, 0.05 nmol)-induced mechanical hypersensitivity in control ( Adra1a flox/flox ) and Vgat + IN-selective α 1A R conditional knockout mice [ Vgat-Cre ; Adra1a flox/flox mice (Vgat + INs–α 1A R cKO mice)] (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; n.s., not significant). (C) Changes in PWT after acute exposure to restraint stress in control ( Adra1a flox/flox ; n = 6 mice) and Vgat + INs–α 1A R cKO mice ( n = 9 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test). (D) Representative trace of membrane potentials in tdTomato + (Vgat + ) SDH neurons after application of NA (20 μM) to spinal cord slices from Vgat-Cre ; Rosa-tdTomato mice. A 1 R agonist CPA (1 μM) was co-applied with NA. (E) Percentage of Vgat + SDH neurons whose NA-evoked response was inhibited by CPA ( n = 10 cells from 7 mice). (F) Representative images of Adora1 (A 1 R) mRNA expression (green) in Slc32a1 (Vgat) + INs (magenta). DAPI staining is shown in gray. Arrowheads indicate A 1 R-expressing Vgat + cells. Scale bar, 25 μm. (G) SaCas9 (yellow, detected by HA-tag) and mCherry (magenta) expression in the PAX2 + INs (cyan) at 3 weeks after intra-SDH injection of AAV-flex[SaCas9] and AAV-flex[mCherry]-U6-sgAdora1 in Vgat-Cre mice. Arrowheads indicate genome-editing Vgat + cells. Scale bar, 25 μm. (H) Representative traces of membrane potentials in Vgat + INs after application of NA and CPA to spinal cord slices from Vgat-Cre mice with conditional knockdown of A 1 Rs in Vgat + INs (SDH-Vgat + IN–A 1 R cKD) and their controls (Control; Vgat-Cre mice with intra-SDH injection of AAV-flex[mCherry]). (I) Percentage of mCherry + (Vgat + ) SDH neurons whose NA-evoked response was inhibited by CPA (Control, n = 10 cells from 8 mice; SDH-Vgat + IN–A 1 R cKD, n = 8 cells from 5 mice). (J) Expression of <t>hM3Dq</t> (green, detected by HA-tag) in the SDH at 3 weeks after intra-SDH injection of AAV-flex[hM3Dq] in Hes5-CreERT2 mice treated with TAM. Dashed line indicates an outline of the gray matter of SDH. GFAP (magenta, bottom left), SOX9 (magenta, bottom right). Arrowheads indicate HA-tag + astrocytes. Scale bars, 200 μm (top) and 25 μm (bottom). (K and L) Representative traces of sIPSCs (K) and quantitative analysis of their frequency (L) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-hM3Dq mice treated with TAM [Pre and CNO: before and after bath application of CNO (100 μM), respectively] ( n = 7 cells from 7 mice; Wilcoxon signed-rank test; * P < 0.05). (M and N) Representative traces of sIPSCs (M) and quantitative analysis of their frequency (N) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-flex[hM3Dq] mice treated with TAM [Pre and CNO: before and after bath application of CNO with CPT (1 μM), respectively] ( n = 13 cells from 13 mice; Wilcoxon signed-rank test; n.s., not significant). Data represent mean ± SEM.
Pcdna5 Frt Ha Hm4di, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rm3d gs  (Addgene inc)
92
Addgene inc rm3d gs
(A and B) Intrathecal NA (A, 0.1 nmol) or Phe (B, 0.05 nmol)-induced mechanical hypersensitivity in control ( Adra1a flox/flox ) and Vgat + IN-selective α 1A R conditional knockout mice [ Vgat-Cre ; Adra1a flox/flox mice (Vgat + INs–α 1A R cKO mice)] (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; n.s., not significant). (C) Changes in PWT after acute exposure to restraint stress in control ( Adra1a flox/flox ; n = 6 mice) and Vgat + INs–α 1A R cKO mice ( n = 9 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test). (D) Representative trace of membrane potentials in tdTomato + (Vgat + ) SDH neurons after application of NA (20 μM) to spinal cord slices from Vgat-Cre ; Rosa-tdTomato mice. A 1 R agonist CPA (1 μM) was co-applied with NA. (E) Percentage of Vgat + SDH neurons whose NA-evoked response was inhibited by CPA ( n = 10 cells from 7 mice). (F) Representative images of Adora1 (A 1 R) mRNA expression (green) in Slc32a1 (Vgat) + INs (magenta). DAPI staining is shown in gray. Arrowheads indicate A 1 R-expressing Vgat + cells. Scale bar, 25 μm. (G) SaCas9 (yellow, detected by HA-tag) and mCherry (magenta) expression in the PAX2 + INs (cyan) at 3 weeks after intra-SDH injection of AAV-flex[SaCas9] and AAV-flex[mCherry]-U6-sgAdora1 in Vgat-Cre mice. Arrowheads indicate genome-editing Vgat + cells. Scale bar, 25 μm. (H) Representative traces of membrane potentials in Vgat + INs after application of NA and CPA to spinal cord slices from Vgat-Cre mice with conditional knockdown of A 1 Rs in Vgat + INs (SDH-Vgat + IN–A 1 R cKD) and their controls (Control; Vgat-Cre mice with intra-SDH injection of AAV-flex[mCherry]). (I) Percentage of mCherry + (Vgat + ) SDH neurons whose NA-evoked response was inhibited by CPA (Control, n = 10 cells from 8 mice; SDH-Vgat + IN–A 1 R cKD, n = 8 cells from 5 mice). (J) Expression of <t>hM3Dq</t> (green, detected by HA-tag) in the SDH at 3 weeks after intra-SDH injection of AAV-flex[hM3Dq] in Hes5-CreERT2 mice treated with TAM. Dashed line indicates an outline of the gray matter of SDH. GFAP (magenta, bottom left), SOX9 (magenta, bottom right). Arrowheads indicate HA-tag + astrocytes. Scale bars, 200 μm (top) and 25 μm (bottom). (K and L) Representative traces of sIPSCs (K) and quantitative analysis of their frequency (L) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-hM3Dq mice treated with TAM [Pre and CNO: before and after bath application of CNO (100 μM), respectively] ( n = 7 cells from 7 mice; Wilcoxon signed-rank test; * P < 0.05). (M and N) Representative traces of sIPSCs (M) and quantitative analysis of their frequency (N) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-flex[hM3Dq] mice treated with TAM [Pre and CNO: before and after bath application of CNO with CPT (1 μM), respectively] ( n = 13 cells from 13 mice; Wilcoxon signed-rank test; n.s., not significant). Data represent mean ± SEM.
Rm3d Gs, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pcdna5+frt+to+ha/pcDNA5%2FFRT-HA-rM3D(Gs)+(Plasmid+%2345549)/pmc10842385-148-0-2
Average 92 stars, based on 1 article reviews
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pcdna5 frt to sbp tnrc6a  (Addgene inc)
92
Addgene inc pcdna5 frt to sbp tnrc6a
(A and B) Intrathecal NA (A, 0.1 nmol) or Phe (B, 0.05 nmol)-induced mechanical hypersensitivity in control ( Adra1a flox/flox ) and Vgat + IN-selective α 1A R conditional knockout mice [ Vgat-Cre ; Adra1a flox/flox mice (Vgat + INs–α 1A R cKO mice)] (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; n.s., not significant). (C) Changes in PWT after acute exposure to restraint stress in control ( Adra1a flox/flox ; n = 6 mice) and Vgat + INs–α 1A R cKO mice ( n = 9 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test). (D) Representative trace of membrane potentials in tdTomato + (Vgat + ) SDH neurons after application of NA (20 μM) to spinal cord slices from Vgat-Cre ; Rosa-tdTomato mice. A 1 R agonist CPA (1 μM) was co-applied with NA. (E) Percentage of Vgat + SDH neurons whose NA-evoked response was inhibited by CPA ( n = 10 cells from 7 mice). (F) Representative images of Adora1 (A 1 R) mRNA expression (green) in Slc32a1 (Vgat) + INs (magenta). DAPI staining is shown in gray. Arrowheads indicate A 1 R-expressing Vgat + cells. Scale bar, 25 μm. (G) SaCas9 (yellow, detected by HA-tag) and mCherry (magenta) expression in the PAX2 + INs (cyan) at 3 weeks after intra-SDH injection of AAV-flex[SaCas9] and AAV-flex[mCherry]-U6-sgAdora1 in Vgat-Cre mice. Arrowheads indicate genome-editing Vgat + cells. Scale bar, 25 μm. (H) Representative traces of membrane potentials in Vgat + INs after application of NA and CPA to spinal cord slices from Vgat-Cre mice with conditional knockdown of A 1 Rs in Vgat + INs (SDH-Vgat + IN–A 1 R cKD) and their controls (Control; Vgat-Cre mice with intra-SDH injection of AAV-flex[mCherry]). (I) Percentage of mCherry + (Vgat + ) SDH neurons whose NA-evoked response was inhibited by CPA (Control, n = 10 cells from 8 mice; SDH-Vgat + IN–A 1 R cKD, n = 8 cells from 5 mice). (J) Expression of <t>hM3Dq</t> (green, detected by HA-tag) in the SDH at 3 weeks after intra-SDH injection of AAV-flex[hM3Dq] in Hes5-CreERT2 mice treated with TAM. Dashed line indicates an outline of the gray matter of SDH. GFAP (magenta, bottom left), SOX9 (magenta, bottom right). Arrowheads indicate HA-tag + astrocytes. Scale bars, 200 μm (top) and 25 μm (bottom). (K and L) Representative traces of sIPSCs (K) and quantitative analysis of their frequency (L) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-hM3Dq mice treated with TAM [Pre and CNO: before and after bath application of CNO (100 μM), respectively] ( n = 7 cells from 7 mice; Wilcoxon signed-rank test; * P < 0.05). (M and N) Representative traces of sIPSCs (M) and quantitative analysis of their frequency (N) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-flex[hM3Dq] mice treated with TAM [Pre and CNO: before and after bath application of CNO with CPT (1 μM), respectively] ( n = 13 cells from 13 mice; Wilcoxon signed-rank test; n.s., not significant). Data represent mean ± SEM.
Pcdna5 Frt To Sbp Tnrc6a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pcdna5+frt+to+ha/pcDNA5%2FFRT%2FTO%2FFLAG-HA-SBP-TNRC6A+(Plasmid+%2342044)/pmc07216507-153-69-75
Average 92 stars, based on 1 article reviews
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maria vartiainen  (Addgene inc)
91
Addgene inc maria vartiainen
(A and B) Intrathecal NA (A, 0.1 nmol) or Phe (B, 0.05 nmol)-induced mechanical hypersensitivity in control ( Adra1a flox/flox ) and Vgat + IN-selective α 1A R conditional knockout mice [ Vgat-Cre ; Adra1a flox/flox mice (Vgat + INs–α 1A R cKO mice)] (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; n.s., not significant). (C) Changes in PWT after acute exposure to restraint stress in control ( Adra1a flox/flox ; n = 6 mice) and Vgat + INs–α 1A R cKO mice ( n = 9 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test). (D) Representative trace of membrane potentials in tdTomato + (Vgat + ) SDH neurons after application of NA (20 μM) to spinal cord slices from Vgat-Cre ; Rosa-tdTomato mice. A 1 R agonist CPA (1 μM) was co-applied with NA. (E) Percentage of Vgat + SDH neurons whose NA-evoked response was inhibited by CPA ( n = 10 cells from 7 mice). (F) Representative images of Adora1 (A 1 R) mRNA expression (green) in Slc32a1 (Vgat) + INs (magenta). DAPI staining is shown in gray. Arrowheads indicate A 1 R-expressing Vgat + cells. Scale bar, 25 μm. (G) SaCas9 (yellow, detected by HA-tag) and mCherry (magenta) expression in the PAX2 + INs (cyan) at 3 weeks after intra-SDH injection of AAV-flex[SaCas9] and AAV-flex[mCherry]-U6-sgAdora1 in Vgat-Cre mice. Arrowheads indicate genome-editing Vgat + cells. Scale bar, 25 μm. (H) Representative traces of membrane potentials in Vgat + INs after application of NA and CPA to spinal cord slices from Vgat-Cre mice with conditional knockdown of A 1 Rs in Vgat + INs (SDH-Vgat + IN–A 1 R cKD) and their controls (Control; Vgat-Cre mice with intra-SDH injection of AAV-flex[mCherry]). (I) Percentage of mCherry + (Vgat + ) SDH neurons whose NA-evoked response was inhibited by CPA (Control, n = 10 cells from 8 mice; SDH-Vgat + IN–A 1 R cKD, n = 8 cells from 5 mice). (J) Expression of <t>hM3Dq</t> (green, detected by HA-tag) in the SDH at 3 weeks after intra-SDH injection of AAV-flex[hM3Dq] in Hes5-CreERT2 mice treated with TAM. Dashed line indicates an outline of the gray matter of SDH. GFAP (magenta, bottom left), SOX9 (magenta, bottom right). Arrowheads indicate HA-tag + astrocytes. Scale bars, 200 μm (top) and 25 μm (bottom). (K and L) Representative traces of sIPSCs (K) and quantitative analysis of their frequency (L) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-hM3Dq mice treated with TAM [Pre and CNO: before and after bath application of CNO (100 μM), respectively] ( n = 7 cells from 7 mice; Wilcoxon signed-rank test; * P < 0.05). (M and N) Representative traces of sIPSCs (M) and quantitative analysis of their frequency (N) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-flex[hM3Dq] mice treated with TAM [Pre and CNO: before and after bath application of CNO with CPT (1 μM), respectively] ( n = 13 cells from 13 mice; Wilcoxon signed-rank test; n.s., not significant). Data represent mean ± SEM.
Maria Vartiainen, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pcdna5+frt+to+ha/HA-BirA*-pDEST-N-pcDNA5%2FFRT%2FTO+(Plasmid+%23118375)/pm37235702-237-6-8
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ddx54 flag ha  (Addgene inc)
86
Addgene inc ddx54 flag ha
(A and B) Intrathecal NA (A, 0.1 nmol) or Phe (B, 0.05 nmol)-induced mechanical hypersensitivity in control ( Adra1a flox/flox ) and Vgat + IN-selective α 1A R conditional knockout mice [ Vgat-Cre ; Adra1a flox/flox mice (Vgat + INs–α 1A R cKO mice)] (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; n.s., not significant). (C) Changes in PWT after acute exposure to restraint stress in control ( Adra1a flox/flox ; n = 6 mice) and Vgat + INs–α 1A R cKO mice ( n = 9 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test). (D) Representative trace of membrane potentials in tdTomato + (Vgat + ) SDH neurons after application of NA (20 μM) to spinal cord slices from Vgat-Cre ; Rosa-tdTomato mice. A 1 R agonist CPA (1 μM) was co-applied with NA. (E) Percentage of Vgat + SDH neurons whose NA-evoked response was inhibited by CPA ( n = 10 cells from 7 mice). (F) Representative images of Adora1 (A 1 R) mRNA expression (green) in Slc32a1 (Vgat) + INs (magenta). DAPI staining is shown in gray. Arrowheads indicate A 1 R-expressing Vgat + cells. Scale bar, 25 μm. (G) SaCas9 (yellow, detected by HA-tag) and mCherry (magenta) expression in the PAX2 + INs (cyan) at 3 weeks after intra-SDH injection of AAV-flex[SaCas9] and AAV-flex[mCherry]-U6-sgAdora1 in Vgat-Cre mice. Arrowheads indicate genome-editing Vgat + cells. Scale bar, 25 μm. (H) Representative traces of membrane potentials in Vgat + INs after application of NA and CPA to spinal cord slices from Vgat-Cre mice with conditional knockdown of A 1 Rs in Vgat + INs (SDH-Vgat + IN–A 1 R cKD) and their controls (Control; Vgat-Cre mice with intra-SDH injection of AAV-flex[mCherry]). (I) Percentage of mCherry + (Vgat + ) SDH neurons whose NA-evoked response was inhibited by CPA (Control, n = 10 cells from 8 mice; SDH-Vgat + IN–A 1 R cKD, n = 8 cells from 5 mice). (J) Expression of <t>hM3Dq</t> (green, detected by HA-tag) in the SDH at 3 weeks after intra-SDH injection of AAV-flex[hM3Dq] in Hes5-CreERT2 mice treated with TAM. Dashed line indicates an outline of the gray matter of SDH. GFAP (magenta, bottom left), SOX9 (magenta, bottom right). Arrowheads indicate HA-tag + astrocytes. Scale bars, 200 μm (top) and 25 μm (bottom). (K and L) Representative traces of sIPSCs (K) and quantitative analysis of their frequency (L) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-hM3Dq mice treated with TAM [Pre and CNO: before and after bath application of CNO (100 μM), respectively] ( n = 7 cells from 7 mice; Wilcoxon signed-rank test; * P < 0.05). (M and N) Representative traces of sIPSCs (M) and quantitative analysis of their frequency (N) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-flex[hM3Dq] mice treated with TAM [Pre and CNO: before and after bath application of CNO with CPT (1 μM), respectively] ( n = 13 cells from 13 mice; Wilcoxon signed-rank test; n.s., not significant). Data represent mean ± SEM.
Ddx54 Flag Ha, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pcdna5/frt/to/flag-ha-sbp-tnrc6a-nls-mut (plasmid #42045)  (Addgene inc)
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pcdna5/frt/to/flag-ha-sbp-tnrc6a-nes-mut (plasmid #42046)  (Addgene inc)
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pcdna5/frt/to-fntb-ha (plasmid #86869)  (Addgene inc)
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(A and B) Intrathecal NA (A, 0.1 nmol) or Phe (B, 0.05 nmol)-induced mechanical hypersensitivity in control ( Adra1a flox/flox ) and Vgat + IN-selective α 1A R conditional knockout mice [ Vgat-Cre ; Adra1a flox/flox mice (Vgat + INs–α 1A R cKO mice)] (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; n.s., not significant). (C) Changes in PWT after acute exposure to restraint stress in control ( Adra1a flox/flox ; n = 6 mice) and Vgat + INs–α 1A R cKO mice ( n = 9 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test). (D) Representative trace of membrane potentials in tdTomato + (Vgat + ) SDH neurons after application of NA (20 μM) to spinal cord slices from Vgat-Cre ; Rosa-tdTomato mice. A 1 R agonist CPA (1 μM) was co-applied with NA. (E) Percentage of Vgat + SDH neurons whose NA-evoked response was inhibited by CPA ( n = 10 cells from 7 mice). (F) Representative images of Adora1 (A 1 R) mRNA expression (green) in Slc32a1 (Vgat) + INs (magenta). DAPI staining is shown in gray. Arrowheads indicate A 1 R-expressing Vgat + cells. Scale bar, 25 μm. (G) SaCas9 (yellow, detected by HA-tag) and mCherry (magenta) expression in the PAX2 + INs (cyan) at 3 weeks after intra-SDH injection of AAV-flex[SaCas9] and AAV-flex[mCherry]-U6-sgAdora1 in Vgat-Cre mice. Arrowheads indicate genome-editing Vgat + cells. Scale bar, 25 μm. (H) Representative traces of membrane potentials in Vgat + INs after application of NA and CPA to spinal cord slices from Vgat-Cre mice with conditional knockdown of A 1 Rs in Vgat + INs (SDH-Vgat + IN–A 1 R cKD) and their controls (Control; Vgat-Cre mice with intra-SDH injection of AAV-flex[mCherry]). (I) Percentage of mCherry + (Vgat + ) SDH neurons whose NA-evoked response was inhibited by CPA (Control, n = 10 cells from 8 mice; SDH-Vgat + IN–A 1 R cKD, n = 8 cells from 5 mice). (J) Expression of hM3Dq (green, detected by HA-tag) in the SDH at 3 weeks after intra-SDH injection of AAV-flex[hM3Dq] in Hes5-CreERT2 mice treated with TAM. Dashed line indicates an outline of the gray matter of SDH. GFAP (magenta, bottom left), SOX9 (magenta, bottom right). Arrowheads indicate HA-tag + astrocytes. Scale bars, 200 μm (top) and 25 μm (bottom). (K and L) Representative traces of sIPSCs (K) and quantitative analysis of their frequency (L) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-hM3Dq mice treated with TAM [Pre and CNO: before and after bath application of CNO (100 μM), respectively] ( n = 7 cells from 7 mice; Wilcoxon signed-rank test; * P < 0.05). (M and N) Representative traces of sIPSCs (M) and quantitative analysis of their frequency (N) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-flex[hM3Dq] mice treated with TAM [Pre and CNO: before and after bath application of CNO with CPT (1 μM), respectively] ( n = 13 cells from 13 mice; Wilcoxon signed-rank test; n.s., not significant). Data represent mean ± SEM.

Journal: bioRxiv

Article Title: Descending locus coeruleus noradrenergic signaling to spinal astrocyte subset is required for stress-induced pain facilitation

doi: 10.1101/2024.11.14.623627

Figure Lengend Snippet: (A and B) Intrathecal NA (A, 0.1 nmol) or Phe (B, 0.05 nmol)-induced mechanical hypersensitivity in control ( Adra1a flox/flox ) and Vgat + IN-selective α 1A R conditional knockout mice [ Vgat-Cre ; Adra1a flox/flox mice (Vgat + INs–α 1A R cKO mice)] (n = 6 mice per group; two-way ANOVA with post hoc Bonferroni’s multiple comparisons test; n.s., not significant). (C) Changes in PWT after acute exposure to restraint stress in control ( Adra1a flox/flox ; n = 6 mice) and Vgat + INs–α 1A R cKO mice ( n = 9 mice) (two-way ANOVA with post hoc Bonferroni’s multiple comparisons test). (D) Representative trace of membrane potentials in tdTomato + (Vgat + ) SDH neurons after application of NA (20 μM) to spinal cord slices from Vgat-Cre ; Rosa-tdTomato mice. A 1 R agonist CPA (1 μM) was co-applied with NA. (E) Percentage of Vgat + SDH neurons whose NA-evoked response was inhibited by CPA ( n = 10 cells from 7 mice). (F) Representative images of Adora1 (A 1 R) mRNA expression (green) in Slc32a1 (Vgat) + INs (magenta). DAPI staining is shown in gray. Arrowheads indicate A 1 R-expressing Vgat + cells. Scale bar, 25 μm. (G) SaCas9 (yellow, detected by HA-tag) and mCherry (magenta) expression in the PAX2 + INs (cyan) at 3 weeks after intra-SDH injection of AAV-flex[SaCas9] and AAV-flex[mCherry]-U6-sgAdora1 in Vgat-Cre mice. Arrowheads indicate genome-editing Vgat + cells. Scale bar, 25 μm. (H) Representative traces of membrane potentials in Vgat + INs after application of NA and CPA to spinal cord slices from Vgat-Cre mice with conditional knockdown of A 1 Rs in Vgat + INs (SDH-Vgat + IN–A 1 R cKD) and their controls (Control; Vgat-Cre mice with intra-SDH injection of AAV-flex[mCherry]). (I) Percentage of mCherry + (Vgat + ) SDH neurons whose NA-evoked response was inhibited by CPA (Control, n = 10 cells from 8 mice; SDH-Vgat + IN–A 1 R cKD, n = 8 cells from 5 mice). (J) Expression of hM3Dq (green, detected by HA-tag) in the SDH at 3 weeks after intra-SDH injection of AAV-flex[hM3Dq] in Hes5-CreERT2 mice treated with TAM. Dashed line indicates an outline of the gray matter of SDH. GFAP (magenta, bottom left), SOX9 (magenta, bottom right). Arrowheads indicate HA-tag + astrocytes. Scale bars, 200 μm (top) and 25 μm (bottom). (K and L) Representative traces of sIPSCs (K) and quantitative analysis of their frequency (L) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-hM3Dq mice treated with TAM [Pre and CNO: before and after bath application of CNO (100 μM), respectively] ( n = 7 cells from 7 mice; Wilcoxon signed-rank test; * P < 0.05). (M and N) Representative traces of sIPSCs (M) and quantitative analysis of their frequency (N) in SG neurons in spinal cord slices from Hes5-CreERT2 ;AAV-flex[hM3Dq] mice treated with TAM [Pre and CNO: before and after bath application of CNO with CPT (1 μM), respectively] ( n = 13 cells from 13 mice; Wilcoxon signed-rank test; n.s., not significant). Data represent mean ± SEM.

Article Snippet: We then inserted AcGFP, DTR-EGFP (kindly provided by Prof. Kenji Kohno, Nara Institute of Science and Technology), mCherry and hM3Dq (#45547; Addgene) into pAAV-EF1α-FLEX.

Techniques: Control, Knock-Out, Membrane, Expressing, Staining, Injection, Knockdown