d1 receptor  (Advanced Cell Diagnostics Inc)

Journal: bioRxiv

Article Title: Abnormal hyperactivity of specific striatal ensembles encodes distinct dyskinetic behaviors revealed by high-resolution clustering

doi: 10.1101/2024.09.06.611664

Figure Lengend Snippet: (A) Cartoon of a mouse head showing the micro-endoscope on top of the mouse head connected to a 1 mm GRIN lens placed in the dorso-lateral striatum and the wireless IMU on the back of the micro-endoscope. On the right, coronal sections of two example mouse brains at the level of the striatum. Photomicrographs were acquired from A2a-Cre transgenic intact (A2a Int, top row) and 6-OHDA lesioned (A2a Les, bottom row) mice injected intrastriatally with the AAV5-GCamP6f viral vector. GFP expression (revealed with GFP antibody in green) in the dorso-lateral striatum shows the region of the striatum transduced with AAV5-GCamP6f viral vector, which is expressed below the 1 mm lens. TH (tyrosine hydroxylase, in red) shows the dopamine terminals in the striatum (note that A2a Les has a complete dopamine depletion shown by the lack of TH immunostaining in the right striatum). Merged photograph shows colocalization of GFP and TH showing the expression of GCamP6f in an intact (top) and a lesioned striatum (bottom) (scale bar: 1 mm). (B) Field of view of the striatum (striatal F.O.V.) through the lens of a D1-Cre lesioned mouse treated with LD, corresponding to a maximum projection of 3000 frames of the video recording. Fluorescent calcium signal shows increased fluorescence in neuronal somas (example neurons depicted by the arrows) and lack of fluorescence of a blood vessel (shown by an asterisk). Right picture shows the same F.O.V. with the total number of neurons (283 neurons) detected with the CNMFe algorithm during both BL and LD sessions, shown as footprints or ROIs (regions of interest) colored in green (scales: 60 µm). (C) Event rate of D1-SPNs in Int and Les mice treated with VEH and LD. On the left, example traces of n = 3 neurons of Int (gray, top row) and n = 3 neurons of Les (red, bottom row) mice (scale bar: 200 sec). First column corresponds to traces at BL and after VEH and the second column shows traces at BL and after LD (note that BL and VEH/LD are separated by a dashed line). Bar graph represents the average of the event rates (events/s) per mouse and session when moving (move) ± SEM (n = 3-7 mice per group and 6 to 18 sessions per group). Ordinary 1-way ANOVA, F (3, 18) = 15.60, p < 0.001. Post hoc Bonferroni’s multiple comparisons test shows ***p < 0.001 Les-LD vs. Les-VEH; # p < 0.05 Les-LD vs. Int-LD; ## p < 0.005 Les-VEH vs. Int-VEH. The bar graph on the right shows the event rates when the mice are at rest. Note that the Les-LD group is not represented because Les-LD mice did not rest (see STAR Methods). Ordinary 1-way ANOVA, F (2, 12) = 1.889, p = 0.1936 (see Movie S3 for D1-SPN calcium imaging aligned to the video camera recording). (D) Event rate of D2-SPNs in Int and Les mice treated with VEH and LD. On the left, example traces of n = 3 neurons of Int and Les mice as in (C). Bar graph represents the average of event rates (events/s) per mouse and session ± SEM (n = 6 mice per group and 8 to 14 sessions per group). Ordinary 1-way ANOVA, F (3, 20) = 10.59, p = 0.0002. Post hoc Bonferroni’s multiple comparisons test shows ***p < 0.001 Les-LD vs. Les-VEH; # p < 0.05 Les-LD vs. Int LD. Bar plot on the right shows the event rates when mice are at rest. Ordinary 1-way ANOVA, F (2, 15) = 29.11, p < 0.001. Post hoc Bonferroni’s multiple comparisons test shows ### p < 0.001 Les-VEH vs. Int-VEH (see Movie S4 for D2-SPN calcium imaging aligned to the video camera recording). (E) Number of active (detected with CNMFe algorithm) D1-SPNs in Int and Les mice treated with VEH and LD when mice were moving. The plots show the number of active neurons ± SEM (n = 3-7 mice per group and 6 to 18 sessions per group). Ordinary 1-way ANOVA, F (3, 19) = 3.561, p = 0.0338. Post hoc Bonferroni’s multiple comparisons test shows *p < 0.05 Les-LD vs. Les-VEH. (F) Number of active D2-SPNs in Int and Les mice treated with VEH and LD. The plots show the number of active neurons ± SEM (n = 6 mice per group and 8 to 14 sessions per group). Ordinary 1-way ANOVA, F (3, 20) = 3.134, p = 0.0484. Post hoc Bonferroni’s multiple comparisons test shows *p < 0.05 Les-LD vs. Les-VEH. (G) Spatiotemporal cross-correlation between pairs of active SPNs of Les mice after VEH vs. LD. For D1-SPNs, two-way repeated measures ANOVA shows an effect of the distance , F (8,54) = 195.9, p < 0.001; no effect of the treatment , F (1,54) = 0.0088, p = 0.9257; and an effect of the interaction, F (8, 54) = 2.421, p = 0.0259. Post hoc Bonferroni’s multiple comparisons test shows **p < 0.01 at 31 µm distance between VEH and LD (squared inset from 30 to 73µm). For D2-SPNs, two-way repeated measures ANOVA shows an effect of the distance , F (8,45) = 108.5, p < 0.001; the treatment , F (1,45) = 8.651, p = 0.0051; and the interaction, F (8, 45) = 2.750, p = 0.0146. Post hoc Bonferroni’s multiple comparisons test shows ***p < 0.001 at 31 µm distance between VEH and LD (squared inset from 30 to 73µm). (H) Comparison of the spatiotemporal cross-correlation between pairs of active D1-SPNs vs. D2-SPNs of Les mice after LD. Two-way repeated measures ANOVA shows an effect of the distance , F (8,88) = 193.8, p < 0.001; a smaller effect of the treatment , F (1,11) = 4.984, p = 0.0473; and an effect of the interaction, F (8,88) = 4.741, p < 0.001. Post hoc Bonferroni’s multiple comparisons test shows ***p < 0.001 at 31 µm distance between D1-SPN and D2-SPN. (I) Comparison of the spatiotemporal cross-correlation between pairs of active D1-SPNs vs. D2-SPNs of Les mice after VEH. Two-way repeated measures ANOVA shows an effect of the distance , F (8,88) = 246.5, p < 0.0001; no effect of the treatment , F (1,11) = 0.4845, p = 0.5008; and an effect of the interaction, F (8,88) = 2.92, p < 0.01. Post hoc Bonferroni’s multiple comparisons test shows no significant difference at any distance between D1-SPN and D2-SPN.

Article Snippet: The study was performed in bacterial artificial chromosome (BAC) transgenic mice expressing Cre recombinase under the control of the dopamine D1 receptor (D1-Cre, Tg(Drd1a-cre) FK150Gsat/Mmucd; MMRRC #029178-UCD) for targeting of direct-pathway SPNs, and adenosine A2a receptor (A2a-Cre, B6.FVB(Cg)-Tg(Adora2acre) KG139Gsat/Mmucd; MMRRC #036158-UCD) for targeting indirect-pathway SPNs.

Techniques: Transgenic Assay, Injection, Plasmid Preparation, Expressing, Transduction, Immunostaining, Fluorescence, Imaging, Comparison

Journal: bioRxiv

Article Title: Abnormal hyperactivity of specific striatal ensembles encodes distinct dyskinetic behaviors revealed by high-resolution clustering

doi: 10.1101/2024.09.06.611664

Figure Lengend Snippet: (A) Left: average event rate of all D1-SPNs and all D2-SPNs during each behavioral cluster group in Les-LD mice. The bar plots represent the mean ± SEM (n = 6-7 mice, 2-3 sessions per mouse) of D1-SPNs (green) and D2-SPNs (red) event rates (events/s) in axial+limb , axial alone , path rot , other N cluster groups. Two-way repeated measures ANOVA: SPN type , F (1, 11) = 49.31, p < 0.001; Cluster group , F (2.154, 23.69) = 3.004, p = 0.0654; interaction, F (3, 33) = 2.021, p = 0.13. Post hoc Bonferroni’s multiple comparisons test shows *p < 0.05 D1-SPN vs. D2-SPN in axial+limb cluster group; ***p < 0.001 D1-SPN vs. D2-SPN in axial alone and path rot cluster group, and **p < 0.01 D1-SPN vs. D2-SPN in other N . Right: ratio of the event rate in D1-SPN vs. D2-SPN per cluster group. Dashed line is the ratio between D1-SPN and D2-SPN average rate in Int-LD during move. (B) Left: percentage of behavior-related D1-SPNs and D2-SPNs in Les-LD mice (a neuron is defined as behavior-related if it showed a significant positive correlation between its activity and the behavior, see STAR Methods). The bar plots represent the mean ± SEM (n = 6-7 mice, 2-3 sessions per mouse) of the percentage of neurons significantly modulated in axial+limb , axial alone , path rot , and other N cluster groups. Dashed line is the ratio between D1-SPN and D2-SPN’s % of behavior-related neurons in Int-LD during other N cluster groups. Two-way repeated measures ANOVA: SPN type , F (1, 11) = 61.44, p < 0.001; Cluster group , F (1.650, 18.15 ) = 23.20, p < 0.001; interaction, F (3, 33) = 2.253, p = 0.1005. Post hoc Bonferroni’s multiple comparisons test shows: ***p < 0.001 D1-SPN vs. D2-SPN in axial alone and path rot cluster group; *p < 0.05 D1-SPN vs. D2-SPN in other N cluster group; in D2-SPN # p < 0.05 axial+limb vs. other N ; ### p < 0.001 axial alone vs. other N ; ## p < 0.01 path rot vs. other N . Right: ratio of the behavior-related D1-SPN over D2-SPN per cluster group. Dashed line represents no changes in the ratio. (C) Percentage of shuffle-corrected overlap between behavior-related neuronal groups for D1-SPNs and D2-SPNs. The overlap was calculated as the number of neurons shared over the total number of neurons in the two respective behavioral cluster groups. This number varies between 0 (no shared neuron) and 100% (complete overlap). Shuffle corrected overlap is the overlap minus the shuffle overlap (see STAR Methods for details). Box and whiskers diagrams show the values for the overlap between all the different comparisons. Individual unpaired t-tests showed a significant difference (compared to 0), **p < 0.01: in axial+limb vs path rot and axial+limb vs other N ; *p < 0.05: in path rot vs other N . (D) Percentage of overlap between the different behavioral clusters in the original data (i.e., without shuffle correction). The bar plots represent the mean ± SEM (n = 6-7 mice, 2-3 sessions per mouse). (E) Example heatmaps of individual D1-SPN (left) and D2-SPNs (right) aligned to the beginning of the behavioral events (at time point 0 s) for axial+limb , axial alone and path rot behavioral clusters. Note the increase in activity during the second after the start of the behavioral events, especially notable in D1-SPNs. (F) Behavior-related D1-SPN and D2-SPN event rate during the different behavioral cluster groups. Each bar represents the average event rate ± SEM (n = 6-7 mice, 2-3 sessions per mouse) of the behavior-related neurons for each behavioral cluster group. Black dashed line is the average rate in Les-LD during move, and the gray dashed line is the average rate in Int-LD during move. Note that the behavior-related D1-SPNs and D2-SPNs show the highest activity during their respective behavior. Kruskal-Wallis nonparametric test was performed for each of the 4 cluster groups individually. Left: for D1-SPN, Kruskal-Wallis test showed significance for axial+limb (**p < 0.01), axial alone (**p < 0.01) and path rot (*p < 0.05) cluster groups, but not for other N . Post hoc Dunn’s multiple comparison shows: in axial+limb cluster group, **p < 0.01 ‘axial+limb neurons’ vs. ‘path rot neurons’ and *p < 0.05 vs. ‘other N neurons’; in axial alone cluster group, **p < 0.01 ‘axial alone neurons’ vs. ‘other N neurons’; and in path rot cluster group, **p < 0.01 ‘path rot neurons’ vs. ‘axial+limb neurons’. Right: for D2-SPN, Kruskal-Wallis test showed significance for axial alone cluster group (**p < 0.01), but not for axial+limb (p = 0.05, n.s.), path rot or other N . Post hoc Dunn’s multiple comparison shows: in axial+limb cluster group, *p < 0.05 ‘axial+limb neurons’ vs. ‘axial alone neurons’; in axial alone cluster group, **p < 0.01 ‘axial alone neurons’ vs. ‘axial+limb neurons’ and *p < 0.05 vs. other N cluster group. (G) Ratio of the event rate in D1-SPN vs. D2-SPN per cluster group. Dashed line is the ratio between D1-SPN and D2-SPN average rate in Int-LD during move. (H) Percentage of overlap between axial+limb , axial alone , path rot SPNs under LD and other N SPNs during BL, using the original data, i.e.,without shuffle correction (shown are the mean ± SEM; n = 6-7 mice, 2-3 sessions per mouse). (I) Percentage of overlap between the neuron groups in (H) with shuffle correction, calculated as in (C). Box and whiskers diagrams show the values for the overlap between all the different comparisons. Individual unpaired t-tests showed no significant difference (compared to 0) in any of the compared groups. (J) Behavior-related D1-SPNs and D2-SPNs event rate during other N during BL average event rate ± SEM; (n = 6-7 mice, 2-3 sessions per mouse). Dashed line is the average rate in Int-BL for D1-SPN and D2-SPN.

Article Snippet: The study was performed in bacterial artificial chromosome (BAC) transgenic mice expressing Cre recombinase under the control of the dopamine D1 receptor (D1-Cre, Tg(Drd1a-cre) FK150Gsat/Mmucd; MMRRC #029178-UCD) for targeting of direct-pathway SPNs, and adenosine A2a receptor (A2a-Cre, B6.FVB(Cg)-Tg(Adora2acre) KG139Gsat/Mmucd; MMRRC #036158-UCD) for targeting indirect-pathway SPNs.

Techniques: Activity Assay, Comparison

Journal: bioRxiv

Article Title: YTHDF1 mediates translational control by m6A mRNA methylation in adaptation to environmental challenges

doi: 10.1101/2024.08.07.607063

Figure Lengend Snippet: (A) A schematic showing the timeline to examine cocaine’s sensitization effect in the open field box. (B) A schematic showing the in vivo fiber photometry recording setup. (C, D) Cocaine-induced locomotor sensitization. (C) D1-Cre;Mettl14 f/+ (Ctrl, blue) and D1-Cre;Mettl14 f/f (KO, green) mice, n=7/genotype. (D) A2A-Cre;Mettl14 f/+ (Ctrl, blue) and A2A-Cre;Mettl14 f/f (KO, red) mice, n=7/genotype. Locomotor activity was recorded for 60 min after saline/cocaine injection. Total distance traveled was recorded. (E) Fiber photometry recordings from D1 striatal neurons. Left: representative Ca 2+ traces from D1-Cre;Mettl14 f/+ mice (Ctrl, blue) after saline and cocaine injection using fiber photometry. Right: representative Ca 2+ traces from D1-Cre;Mettl14 f/f mice (KO, green) after saline and cocaine injection using fiber photometry. (F) Left bar graph: Mean Ca 2+ activity of D1-Cre;Mettl14 f/+ mice (Ctrl, blue) and D1-Cre;Mettl14 f/f (KO, green) mice from 15 min fiber photometry recording after saline (S) and cocaine (C) injection. *: P=0.0163, paired T-test. ns: P=0.0702, paired T-test. **: P=0.0010, 2-way ANOVA. Right bar graph: Peak Ca 2+ transients level comparison. **: P=0.0029, paired T-test. Ns: P=0.1250, paired T-test. ****: P<0.0001, 2-way ANOVA, n=5. (G) Fiber photometry recordings from D2 striatal neurons. Left: representative Ca 2+ traces from A2A-Cre;Mettl14 f/+ mice (Ctrl, blue) after saline and cocaine injection using fiber photometry. Right: representative Ca 2+ traces from A2A-Cre;Mettl14 f/f mice (KO, red) after saline and cocaine injection using fiber photometry. (H) Left bar graph: Mean Ca 2+ activity of A2A-Cre;Mettl14 f/+ mice (Ctrl, blue) and A2A-Cre;Mettl14 f/f mice (KO, red) from 15 min fiber photometry recording after saline (S) and cocaine (C) injection. **: P=0.0020, paired T-test. Ns: P=0.0690, paired T-test. ***: P=0.0007, 2-way ANOVA. Right bar graph: Peak Ca 2+ transients level comparison. **: P=0.0011, paired T-test. *: P=0.0150, paired T-test. ***: P=0.0007, 2-way ANOVA, n=5. All data expressed as mean ± SEM. Overall, Mettl14 deficiency blunted the cellular responses in both D1 and D2 SPNs, but resulted in opposite behavioral outcomes observed in mice after cocaine treatment.

Article Snippet: To selectively delete Ythdf1 in D1 or D2 SPNs, we crossed Ythdf1 f/f mice to a D1 receptor promoter-driven Cre recombinase (D1-Cre) transgenic line (B6.FVB(Cg)-Tg(Drd1-cre)EY262Gsat/Mmucd, RRID: MMRRC-030989-UCD) or an adenosine 2A receptor promoter-driven Cre recombinase (A2A-Cre) transgenics line (B6.FVB(Cg)-Tg(Adora2a-cre)KG139Gsat/Mmucd, RRID: MMRRC_036158-UCD).

Techniques: In Vivo, Activity Assay, Saline, Injection, Comparison

Journal: bioRxiv

Article Title: YTHDF1 mediates translational control by m6A mRNA methylation in adaptation to environmental challenges

doi: 10.1101/2024.08.07.607063

Figure Lengend Snippet: (A) Cocaine-induced locomotor sensitization in D1-Cre;Ythdf1 f/+ mice (Ctrl, blue) and D1-Cre;Ythdf1 f/f mice (KO, cyan). Total distance traveled (cm) was recorded for 60 min after saline/cocaine injection. n=8. (B) Cocaine-induced locomotor sensitization in A2A-Cre;Ythdf1 f/+ mice (Ctrl, blue) and A2A- Cre;Ythdf1 f/f mice (KO, magenta). n=8. (C) The rotarod motor learning in D1-Cre;Ythdf1 f/+ mice (Ctrl, blue) and D1-Cre;Ythdf1 f/f mice (KO, cyan). Performance was recorded as latency to fall (s), n=5. (D) The rotarod motor learning in A2A-Cre;Ythdf1 f/+ mice (Ctrl, blue) and A2A-Cre;Ythdf1 f/f mice (KO, magenta). N=5. (E) The sensitization of haloperidol-induced catalepsy response in A2A-Cre;Ythdf114 f/+ mice (Ctrl, blue) and A2A-Cre;Ythdf1 f/f mice (KO, magenta). Catalepsy duration was recorded (s). ***: P=0.0003, 2-way ANOVA, n=7. All data expressed as mean ± SEM. Ythdf1 deletion resembles impairment caused by Mettl14 deletion in a cell type specific manner. YTHDF1 is potentially the main downstream reader protein that regulating translation in response to stimulation and during learning in the striatum.

Article Snippet: To selectively delete Ythdf1 in D1 or D2 SPNs, we crossed Ythdf1 f/f mice to a D1 receptor promoter-driven Cre recombinase (D1-Cre) transgenic line (B6.FVB(Cg)-Tg(Drd1-cre)EY262Gsat/Mmucd, RRID: MMRRC-030989-UCD) or an adenosine 2A receptor promoter-driven Cre recombinase (A2A-Cre) transgenics line (B6.FVB(Cg)-Tg(Adora2a-cre)KG139Gsat/Mmucd, RRID: MMRRC_036158-UCD).

Techniques: Saline, Injection

Journal: bioRxiv

Article Title: YTHDF1 mediates translational control by m6A mRNA methylation in adaptation to environmental challenges

doi: 10.1101/2024.08.07.607063

Figure Lengend Snippet: (A) Representative images of de novo protein synthesis measured by HPG incorporation in the striatal neurons from wild type and Ythdf1 KO P1 mice. Three experimental conditions were compared: HPG+CHX group as negative control, HPG group as baseline condition and HPG+SKF (dopamine D1 receptor agonist) group to test the response after cAMP elevation. Blue: DAPI, red: MAP2, green: HPG tagged newly synthesized protein. Scale bar, 20um (B) Quantification of the HPG expression intensity in CHX, HPG and SKF group in wild type (Ctrl) and Ythdf1 KO striatal neurons. Genotype main effect, p<0.0001, genotype x time interaction, p<0.0001, 2-way ANOVA. HPG vs. SKF treatment: ****: P<0.0001(ctrl), ns: P=0.8390 (KO), paired t-test. Each group contained 3 replicates. (C) Gene ontology (GO) analysis of the upregulated YTHDF1 transcripts after cocaine treatment. (D) Venn diagram depicting the number of YTHDF1 targets after saline and cocaine treatment. (E) UHPLC-MS/MS analysis of m 6 A level in the striatum after saline and cocaine treatment. Striatal neurons with Ythdf1 deficiency have a higher baseline de novo protein synthesis rate but are incapable of responding to stimulations. At the molecular level, boosting dopamine release by cocaine drastically increased YTHDF1 binding to many mRNA targets in the striatum.

Article Snippet: To selectively delete Ythdf1 in D1 or D2 SPNs, we crossed Ythdf1 f/f mice to a D1 receptor promoter-driven Cre recombinase (D1-Cre) transgenic line (B6.FVB(Cg)-Tg(Drd1-cre)EY262Gsat/Mmucd, RRID: MMRRC-030989-UCD) or an adenosine 2A receptor promoter-driven Cre recombinase (A2A-Cre) transgenics line (B6.FVB(Cg)-Tg(Adora2a-cre)KG139Gsat/Mmucd, RRID: MMRRC_036158-UCD).

Techniques: Negative Control, Synthesized, Expressing, Saline, Tandem Mass Spectroscopy, Binding Assay