Journal: Neuroscience Bulletin

Article Title: An Anterior Cingulate Cortex-to-Midbrain Projection Controls Chronic Itch in Mice

doi: 10.1007/s12264-022-00996-6

Figure Lengend Snippet: Contribution of ACC neurons to chronic itch-induced scratching behaviors. A Schematic showing the experimental timeline for establishing the chronic itch model. DCP, diphenylcyclopropenone; DW, distilled water. B Time course of DCP-induced scratching behavior. Numbers of scratching bouts within 60 min were counted. n = 5 mice per group; ** P <0.01, *** P <0.001; two-way ANOVA followed by Bonferroni post hoc analysis. C Representative immunostaining images of c-Fos expression in the ACC. Scale bars, 200 μm (left) and 30 μm (right). D , E Numbers of c-Fos + neurons in total ( D ) and different bregma planes ( E ) of the ACC in the DCP-treated group compared with the DW control group. n = 4 or 5 mice; *** P <0.001; unpaired Student’s t test for ( D ); two-way ANOVA followed by Bonferroni post hoc analysis for ( E ). F Schematic showing the experimental timeline for intrathecal ablation of itch-specific GRPR + neurons. Sap, saporin. G Scratching behavior evoked by DCP is significantly blocked in mice treated with bombesin-sap compared with blank-sap. *** P <0.001; unpaired Student’s t test. H , I Representative image ( H ) and quantification ( I ) showing a dramatic reduction of DCP-induced c-Fos expression in the ACC when the ascending itch signal is blocked by bombesin-sap. n = 3 mice per group; * P <0.05; unpaired Student’s t test. Scale bar, 200 μm (left), 50 μm (right). J Timeline for chemogenetic inhibition of ACC neuronal activity and behavioral tests. K Schematic of the sites of viral injection into the ACC. L A representative image showing the expression of hM4Di-mCherry in the ACC. Scale bars, 200 μm (left) and 50 μm (right). M , N Whole-cell patch clamp recordings reveal that bath application of CNO (5 μmol/L) blocks action potential firing of ACC pyramidal neurons. M Representative trace. N Quantification data. n = 5 neurons. *** P <0.001; paired Student’s t test. O Quantitative analyses of the number of scratching bouts within 60 min in mice treated with saline or CNO to chemogenetically inactivate ACC neurons. Sal: saline. n = 6 mice per group; *** P <0.001; unpaired Student’s t test. P , Q Total distance travelled ( P ) and average velocity ( Q ) in the open field. No significant difference was detected between the two groups. n = 6 mice per group. Data are presented as the mean ± SEM.

Article Snippet: AAV-hSyn-hM4D(Gi)-mCherry (AAV2/9, 3.18 × 10 12 genomic copies/mL, AG50475), AAV-CaMKIIα-hM4D(Gi)-mCherry (AAV2/9, 1.56 × 10 13 genomic copies/mL, H5778), AAV-EF1α-DIO-hM4D(Gi)-mCherry (AAV2/9, 1.38 × 10 13 genomic copies/mL, HYMBE1370), AAV-Retro-hSyn-mCherry (rAAV, 2.13 × 10 13 genomic copies/mL, AOV063), AAV-EF1α-DIO-mCherry (AAV2/9, 3.46 × 10 12 genomic copies/mL, and AG20299) and AAV-Retro-hSyn-Cre (rAAV, 8.18 × 10 12 genomic copies/mL, CN867) were all made by OBiO Technology, Shanghai, China.

Techniques: Immunostaining, Expressing, Inhibition, Activity Assay, Injection, Patch Clamp

Journal: Neuroscience Bulletin

Article Title: An Anterior Cingulate Cortex-to-Midbrain Projection Controls Chronic Itch in Mice

doi: 10.1007/s12264-022-00996-6

Figure Lengend Snippet: ACC modulates chronic itch in a cell type-dependent manner. A Schematic showing the experimental timeline for selective chemogenetic inhibition of excitatory neurons in the ACC. B Scheme for specific infection of excitatory neurons in the ACC with hM4Di. C A representative image illustrating the expression of hM4Di-mCherry in the ACC. Scale bars, 200 μm (left) and 50 μm (right). D Quantitative analyses of the number of scratching bouts within 60 min in mice treated with saline or CNO to chemogenetically inactivate excitatory ACC neurons. n = 8 or 9 mice; ** P <0.01; unpaired Student’s t test. E , F Total distance travelled ( E ) and average velocity ( F ) in the open field. No significant difference was found between the two groups. n = 8 mice per group. G Scheme for specific infection of inhibitory neurons in the ACC with hM4Di. H Histological verification of viral infection. Scale bars, 200 μm (left) and 50 μm (right). I Chemogenetic suppression of inhibitory neurons in the ACC enhances scratching behaviors in chronic itch. n = 5 mice; *** P <0.001; unpaired Student’s t test. Data are presented as the mean ± SEM.

Article Snippet: AAV-hSyn-hM4D(Gi)-mCherry (AAV2/9, 3.18 × 10 12 genomic copies/mL, AG50475), AAV-CaMKIIα-hM4D(Gi)-mCherry (AAV2/9, 1.56 × 10 13 genomic copies/mL, H5778), AAV-EF1α-DIO-hM4D(Gi)-mCherry (AAV2/9, 1.38 × 10 13 genomic copies/mL, HYMBE1370), AAV-Retro-hSyn-mCherry (rAAV, 2.13 × 10 13 genomic copies/mL, AOV063), AAV-EF1α-DIO-mCherry (AAV2/9, 3.46 × 10 12 genomic copies/mL, and AG20299) and AAV-Retro-hSyn-Cre (rAAV, 8.18 × 10 12 genomic copies/mL, CN867) were all made by OBiO Technology, Shanghai, China.

Techniques: Inhibition, Infection, Expressing

Journal: Neuroscience Bulletin

Article Title: An Anterior Cingulate Cortex-to-Midbrain Projection Controls Chronic Itch in Mice

doi: 10.1007/s12264-022-00996-6

Figure Lengend Snippet: Activation of the ACC→VTA excitatory projection by chronic itch. A Schematic showing the timeline of the immunostaining experiments. B Scheme for retrograde labelling of VTA-projecting ACC neurons. C Representative images of c-Fos co-staining with mCherry in the ACC for both DW- and DCP-treated groups. Arrows indicate c-Fos + /mCherry + neurons. Scale bars, 200 μm (left) and 50 μm (right). D , E Numbers of mCherry + neurons in total ( D ) and different sections ( E ) of the ACC. n = 5 or 4 mice per group. F , G Numbers of c-Fos + /mCherry + neurons in total ( F ) and different sections ( G ) of the ACC. n = 5 or 4 mice; * P <0.05, ** P <0.01; unpaired Student’s t test for ( F ); two-way ANOVA followed by Bonferroni post hoc analysis for ( G ). H Percentage of c-Fos + / mCherry + neurons in mCherry + neurons for different bregma planes of the ACC. n = 5 or 4 mice; *** P <0.001; two-way ANOVA followed by Bonferroni post hoc analysis. I Scheme for retrograde labelling of VTA-projecting ACC neurons in Vgat-Cre::H2B-GFP mice. J Representative images of GFP co-staining with mCherry in the ACC. Arrows indicate GFP + /mCherry + neurons. Scale bars, 200 μm (left) and 50 μm (right). K Summary of the numbers of mCherry + neurons in different sections of the ACC. n = 5 mice per group. L Numbers of GFP + /mCherry + neurons in different parts of the ACC. n = 5 mice per group. M Percentage of GFP + /mCherry + neurons in mCherry + neurons for different sections of the ACC. Few co-labeled neurons are detected. n = 5 mice per group. Data are presented as mean ± SEM.

Article Snippet: AAV-hSyn-hM4D(Gi)-mCherry (AAV2/9, 3.18 × 10 12 genomic copies/mL, AG50475), AAV-CaMKIIα-hM4D(Gi)-mCherry (AAV2/9, 1.56 × 10 13 genomic copies/mL, H5778), AAV-EF1α-DIO-hM4D(Gi)-mCherry (AAV2/9, 1.38 × 10 13 genomic copies/mL, HYMBE1370), AAV-Retro-hSyn-mCherry (rAAV, 2.13 × 10 13 genomic copies/mL, AOV063), AAV-EF1α-DIO-mCherry (AAV2/9, 3.46 × 10 12 genomic copies/mL, and AG20299) and AAV-Retro-hSyn-Cre (rAAV, 8.18 × 10 12 genomic copies/mL, CN867) were all made by OBiO Technology, Shanghai, China.

Techniques: Activation Assay, Immunostaining, Staining, Labeling

Journal: Neuroscience Bulletin

Article Title: An Anterior Cingulate Cortex-to-Midbrain Projection Controls Chronic Itch in Mice

doi: 10.1007/s12264-022-00996-6

Figure Lengend Snippet: VTA neurons are significantly activated during chronic itch. A Timeline of the c-Fos immunostaining experiments. B Representative images of c-Fos expression in the VTA for both DW- and DCP-treated groups. Scale bars, 200 μm (left) and 50 μm (right). C , D Numbers of c-Fos + neurons in total ( C ) and different sections ( D ) of the VTA. n = 5 or 6 mice; *** P <0.001; unpaired Student’s t test for ( C ); two-way ANOVA followed by Bonferroni post hoc analysis for ( D ). E Schematic showing the timeline of the immunostaining experiments. F Scheme for specific labeling of ACC-innervated VTA neurons. G Representative images of c-Fos co-staining with mCherry in the VTA. Arrows indicate c-Fos + /mCherry + neurons. Scale bars, 200 μm (left) and 50 μm (right). H , I Numbers of mCherry + neurons in total ( H ) and different parts ( I ) of the VTA. n = 5 or 6 mice. J , K Numbers of c-Fos + / mCherry + neurons in total ( J ) and different sections ( K ) of the VTA. n = 5 or 6 mice; * P <0.05, ** P <0.01, *** P <0.001; unpaired Student’s t test for ( J ); two-way ANOVA followed by Bonferroni post hoc analysis for ( K ). L Percentage of c-Fos + /mCherry + neurons in mCherry + neurons for different parts of the VTA. n = 5 or 6 mice; * P <0.05, ** P <0.01; two-way ANOVA followed by Bonferroni post hoc analysis. Data are presented as the mean ± SEM.

Article Snippet: AAV-hSyn-hM4D(Gi)-mCherry (AAV2/9, 3.18 × 10 12 genomic copies/mL, AG50475), AAV-CaMKIIα-hM4D(Gi)-mCherry (AAV2/9, 1.56 × 10 13 genomic copies/mL, H5778), AAV-EF1α-DIO-hM4D(Gi)-mCherry (AAV2/9, 1.38 × 10 13 genomic copies/mL, HYMBE1370), AAV-Retro-hSyn-mCherry (rAAV, 2.13 × 10 13 genomic copies/mL, AOV063), AAV-EF1α-DIO-mCherry (AAV2/9, 3.46 × 10 12 genomic copies/mL, and AG20299) and AAV-Retro-hSyn-Cre (rAAV, 8.18 × 10 12 genomic copies/mL, CN867) were all made by OBiO Technology, Shanghai, China.

Techniques: Immunostaining, Expressing, Labeling, Staining

Journal: Neuroscience Bulletin

Article Title: An Anterior Cingulate Cortex-to-Midbrain Projection Controls Chronic Itch in Mice

doi: 10.1007/s12264-022-00996-6

Figure Lengend Snippet: ACC predominantly projects to non-dopaminergic neurons in the VTA. A Schematic showing the timeline for virus injection and TH co-staining experiments. B Scheme for specific labeling of ACC-innervated VTA neurons with mCherry. C Representative images of mCherry co-staining with TH in the VTA. Scale bars, 200 μm (upper) and 50 μm (lower). D , E Numbers of TH + ( D ) and mCherry + ( E ) neurons in different sections of the VTA. n = 10 mice. F , G Numbers ( F ) and percentage ( G ) of TH + /mCherry + neurons in different parts of the VTA. n = 10 mice. H Pie chart showing minimal expression of mCherry in TH + neurons in the VTA.

Article Snippet: AAV-hSyn-hM4D(Gi)-mCherry (AAV2/9, 3.18 × 10 12 genomic copies/mL, AG50475), AAV-CaMKIIα-hM4D(Gi)-mCherry (AAV2/9, 1.56 × 10 13 genomic copies/mL, H5778), AAV-EF1α-DIO-hM4D(Gi)-mCherry (AAV2/9, 1.38 × 10 13 genomic copies/mL, HYMBE1370), AAV-Retro-hSyn-mCherry (rAAV, 2.13 × 10 13 genomic copies/mL, AOV063), AAV-EF1α-DIO-mCherry (AAV2/9, 3.46 × 10 12 genomic copies/mL, and AG20299) and AAV-Retro-hSyn-Cre (rAAV, 8.18 × 10 12 genomic copies/mL, CN867) were all made by OBiO Technology, Shanghai, China.

Techniques: Injection, Staining, Labeling, Expressing