Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A)/(B) Coronal sections including the caudate putamen region from two Htr2a Cre BAC mouse lines (KM207 and KM208) with Cre-dependent GFP reporter distribution: Image S1A is from http://www.gensat.org/imagenavigator.jsp?imageID=69385 . Image S1B is from http://www.gensat.org/imagenavigator.jsp?imageID=83872 . Comparison of Htr2a mRNA , autoradiography and HTR2A-EGFP-CT protein : arrows shown in these 3 figures indicated the corresponding brain regions with these three different methods. (C) Expression of Htr2a mRNA in the adult C57 mouse brain: Images are from Allen Mouse Brain Atlas ( http://mouse.brainmap.org/experiment/siv?id=81671344 ). (D) [ 3 H] MDL100,907 (0.4 nM) autoradiography labeling of HTR2A binding sites in the Wistar rat brain section (V: layer V cortex, CPu: caudate putamen, Pn: pon, 7: facial nucleus and M05: motor trigeminal nucleus.) (E) Sagittal view of distribution of HTR2A-EGFP-CT fusion protein in the whole brain cleared tissue from Htr2a EGFP-CreERT2/+ mouse line. Comparison of HTR2A-EGFP-CT protein and Htr2a transcripts in the CPu: The red star with dashed line with arrows in and denoted the patch-like pattern (striosome) in the CPu. (F) The distribution of the HT2A-EGFP-CT fusion protein in coronal section at the level of the CPu from Htr2a EGFP-CreERT2/+ mouse line. (G) Distribution of Htr2a mRNA in the brain from C57 mice. Data resource from Allen Mouse Brain Atlas ( http://mouse.brainmap.org/experiment/siv?id=81671344 .

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Comparison, Autoradiography, Expressing, Labeling, Binding Assay

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A) Schema of the transgenic modifications at the murine Htr2a gene in exon 3. For Htr2a -EGFP-CT-IRES-CreERT2 ( Htr2a EGFP-CreERT2 ) mouse line, EGFP was inserted into the C terminus (CT) of the receptor, after residue 452. An IRES-CreERT2 cassette was inserted between the Htr2a stop codon and the 3’UTR. Blue boxes represent exons of murine Htr2a ; the green box is EGFP; the red box shows the STOP codon. The gray box is the IRES followed by a pink box for CreERT2. The schematic was created with https://www.biorender.com/ . (B) Validation of Egfp and Htr2a mRNA expressions in the mouse brain: RNAscope experiments were performed to probe Egfp and Htr2a mRNA in C57 ( Htr2a +/+ ) and Htr2a EGFP-CreERT2/EGFP-CreERT2 mice. The Htr2a (red) probe can be detected in C57 mice; Egfp (green) and Htr2a (red) probes showed co-localization (yellow) in Htr2a EGFP-CreERT2/EGFP-CreERT2 mice. Images were taken under an Olympus slide scanner with 10X objective and an Olympus confocal microscope with a 60X objective. (C) The expression pattern of HTR2A in cortical pyramidal neurons (soma, apical dendrites, and apical tufts). Images were taken with a 20X objective under an Olympus confocal microscope. (D) Paravalbumin is the GABAergic interneuron marker. The brain sections from Htr2a EGFP-CreERT2/+ mice were stained with anti-parvalbumin and anti-GFP antibodies. The yellow arrowhead denotes parvalbumin-positive and GFP-positive interneurons. Images were captured under a 60X objective with an Olympus confocal microscope. (E) Cortical L5a marker NECAB1 was used to locate the cortical layer 5a distribution. HTR2A-EGFP-CT fusion receptors with anti-GFP staining showed its distribution in the NECAB1-positive layer. (F) With CTIP2 as a cortical L5b/L6 marker, HTR2A-EGFP-CT did not show overlap with the Ctip2-positive layers. (G) An anti-HTR2A antibody showed the same pattern as the anti-GFP distribution associated with HTR2A-EGFP-CT. Note, all brain sections in - from Htr2a EGFP-CreERT2/+ mice were stained with an anti-GFP antibody for HTR2A-EGFP-CT and different cortical markers (NECAB1, CTIP2), as well as with an anti-HTR2A antibody. (H) The distribution pattern of HTR2A-EGFP-CT labeling in the dorsal striatum. The Mu opioid receptor (MOR) is rich in the striosome and subcallosal stria of the Caudate-Putamen (CPu). HTR2A-EGFP-CT signals showed the same expression pattern with MOR distribution in the patch-like (striosome) area and stria. Experiments in this figure were conducted with 3-4 mice with similar results. Images were taken using an Olympus VS120 slide scanner or an Olympus confocal microscope under 60X objective.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Transgenic Assay, Residue, Microscopy, Expressing, Marker, Staining, Labeling

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: Brain sections from Htr2a EGFP-CreERT2/+ mice were stained with a GFP antibody to amplify the HTR2A-EGFP-CT signal and then were acquisitioned with an Olympus slide scanner under 10X objective. The abbreviations for the brain areas are located in the Supplementary Figure. The raw images were uploaded to open-source website, A Mouse Imaging Server (AMIS, https://amis2.docking.org/ .

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Staining, Imaging

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A/B) Schematic representation of the cytoarchitectonic areas of the frontal lobe of the brain. (A) The sagittal view of medial and lateral brain region, as well as horizontal view of the ventral regions on the frontal lobe show mPFC (medial prefrontal cortex), OFC (orbitofrontal cortex, as ventral PFC), and insular cortex (dorsal and ventral agranular insular cortex, AId and AIv; posterior agranular insular cortex, AIp; dysgranular insular cortex, DI). The schema was adapted from (B) Coronal sections from rostral to caudal view for mPFC, OFC, and insular cortex. The schema was adapted from (C) AchE staining: sequential brain sections through the frontal lobe area were used to visualize the distribution of HTR2A-EGFP-CT in the mPFC. The cytoarchitecture features of AchE staining showed strongest staining in the dorsal prelimbic cortex that was absent in the ventral prelimbic cortex. The blue arrow indicates the boundary between the dorsal and ventral prelimbic cortex. HTR2A-EGFP-CT showed its distributions in the dorsal prelimic cortex but was not present in the ventral prelimbic cortex and infralimbic cortex. Images scale bar: 500 µm. Similar results were obtained with 3 animals. MOs: secondary motor cortex, MOp: primary motor cortex, AAC: anterior cingulate area, PL: prelimbic cortex, IL: infralimbic cortex, and fr: anterior forceps.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Staining

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A-I) Motor activities in the open field Baseline activities (0-30 min; pre-administration) and post-injection activities following administration (31-120 min) of the vehicle (Veh), LSD (0.3 mg/kg), DOI (1 mg/kg) or psilocin (Psil, 1 mg/kg); n=9-10 mice/genotype/treatment. Note, the C57BL/6J and Htr2a EGFP-CreERT2/EGFP-CreERT2 mice are termed C57 and Htr2a EGFP-CreERT2 mice, respectively, below and in the figure panels. All data represented as means ±SEMs; N=9-10 mice/genotype/treatment. (A-C) RMANOVA for baseline: time [F(23,1587)=23.922, p <0.001], time by genotype [F(23,1587)=8.900, p <0.001], time by treatment [F(69,1587)=14.677, p <0.001], time by genotype by treatment [F(69,1587)=7.859, p <0.001], genotype [1,69)=30.277, p <0.001], treatment [F(3,69)=24.114, p <0.001], and genotype by treatment interaction [F(3,69)=11.598, p <0.001]. RMANCOVA for post-injection: time [F(17,1071)=2.600, p <0.001], time by genotype [F(17,1071)=12.309, p <0.001], time by treatment [F(51,1071)=10.977, p <0.001], time by genotype by treatment [F(51,1071)=8.914, p <0.001], genotype [1,63)=16.278, p <0.001], treatment [F(3,63)=26.809, p <0.001], and genotype by treatment interaction [F(3,63)=8.972, p <0.001]. (D-F) Rearing activities in the same mice. RMANOVA for baseline: time [F(23,1587)=23.548, p <0.001], time by genotype [F(23,1587)=9.221, p <0.001], time by treatment [F(69,1587)=7.544, p <0.001], time by genotype by treatment interaction [F(69,1587)=1.840, p =0.020], genotype [1,69)=5.228, p =0.025], treatment [F(3,69)=12.397, p <0.001]. RMANCOVA for post-injection: time by genotype [F(17,1071)=2.670, p <0.001], time by treatment [F(51,1071)=6.033, p <0.001], time by genotype by treatment [F(51,1071)=1.699, p =0.002], genotype [1,63)=15.125, p <0.001], treatment [F(3,63)=11.016, p <0.001], and genotype by treatment interaction [F(3,63)=4.016, p =0.011]. (G-I) Stereotypical activities in these same mice. RMANOVA for baseline: time [F(23,1587)=139.510, p <0.001], time by genotype [F(23,1587)=7.485, p <0.001], time by treatment [F(69,1587)=5.973, p <0.001], time by genotype by treatment interaction [F(69,1587)=2.485, p <0.001], genotype [1,69)=7.491, p =0.008], and treatment [F(3,69)=7.632, p <0.001]. RMANCOVA for post-injection: time by genotype [F(17,1071)=3.353, p <0.001], time by treatment [F(51,1071)=7.105, p <0.001], time by genotype by treatment interaction [F(51,1071)=3.910, p <0.001], and treatment [F(3,63)=7.226, p <0.001].

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Injection

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A-C) Cumulative motor activities Cumulative baseline activities (0-30 min; pre-administration) and cumulative post-injection activities following administration (31-120 min) of the vehicle (Veh), LSD (0.3 mg/kg), DOI (1 mg/kg) or psilocin (Psil, 1 mg/kg); n=9-10 mice/genotype/treatment. Note, the C57BL/6J and Htr2a EGFP-CreERT2/EGFP-CreERT2 mice are termed C57 and Htr2a EGFP-CreERT2 mice, respectively, below and in the figure panels. The symbol “*” is for genotype difference (black); “+” is for within C57 effect (blue); “^” for within Htr2a EGFP-CreET2 effect (green), and “#” for overall treatment effects (purple). All data are represented as means ± SEMs. (A) Locomotor activities in C57 and Htr2a EGFP-CreERT2 mice. Two-way ANOVA for baseline: genotype [F(1,69)=8.968, p =0.004]; for C57 vs . Htr2a EGFP-CreERT2 mice (black): ** p <0.01, overall genotype effects. Two-way ANCOVA for post-injection: genotype [F(1,68)=19.433, p <0.001], treatment [F(3,68)=29.894, p <0.001], and genotype by treatment interaction [F(3,68)=11.143, p <0.001]; Bonferroni post-hoc tests; for C57 vs . Htr2a EGFP-CreERT2 mice (black): *** p <0.001, DOI; for C57 mice (blue): + p <0.05, Veh vs. DOI; for Htr2a EGFP-CreERT2 mice (green): ^^^ p<0.001, Veh, LSD, Psil vs. DOI. (B) Rearing activities in the same mice. Two-way ANOVA for baseline: genotype [F(1,69)=9.307, p =0.003]; for C57 vs . Htr2a EGFP-CreERT2 mice (black): ** p <0.01, overall genotype effects. Two-way ANCOVA for post-injection: genotype [F(1,68)=17.900, p <0.001] and treatment [F(3,68)=13.765, p <0.001]; Bonferroni post-hoc tests; for C57 vs . Htr2a EGFP-CreERT2 mice (black): ** p <0.01, overall genotype effects; overall treatment effects (purple): ## p <0.01, Psil vs. LSD and DOI; ### p <0.001, Veh vs. LSD and DOI. (C) Stereotypical activities in these same mice. Two-way ANOVA for baseline: genotype [F(1,69)=44.086, p <0.001]; for C57 vs . Htr2a EGFP-CreERT2 mice (black): *** p <0.001, overall genotype effects. Two-way ANCOVA for post-injection: treatment [F(3,68)=8.338, p <0.001]; Bonferroni post-hoc tests; overall treatment effects (purple): # p<0.05, Veh vs. DOI; ## p<0.01, Veh vs. LSD or Psil vs. DOI; ### p<0.001, Psil vs. LSD. (D-F) Head twitch responses, grooming, and retrograde walking These responses were scored beginning immediately after vehicle or psychedelic administration and followed over the next 30 min; n=9-10 mice/genotype/treatment. (D) Head twitch responses in C57 and Htr2a EGFP-CreERT2 animals following administration (31-60 min) of the Veh, 0.3 mg/kg LSD, 1 mg/kg DOI, or 1 mg/kg Psil. Two-way ANOVA: genotype [F(1,69)=9.236, p =0.003], treatment [F(3,69)=51.113, p <0.001], and genotype by treatment interaction [F(3,69)=2.878, p =0.042]; Bonferroni post-hoc tests: for C57 vs. Htr2a EGFP-CreERT2 mice (black): *** p <0.001, DOI; for C57 mice (blue): + p <0.05, LSD vs. DOI; +++ p <0.001, Veh vs. all psychedelics, or Psil vs. DOI; for Htr2a EGFP-CreERT2 mice (green): ^ p <0.05, Psil vs. LSD; ^^ p <0.01, Veh vs. Psil; ^^^ p <0.001, Veh vs. LSD and DOI. (E) Groom time in the same mice. Two-way ANOVA: genotype [F(1,69)=11.465, p <0.001], treatment [F(3,69)=15.125, p <0.001], and genotype by treatment interaction [F(3,69)=6.855, p <0.001]; Bonferroni post-hoc tests: for C57 vs Htr2a EGFP-CreERT2 mice (black): *** p <0.001, LSD and DOI; for C57 mice (blue): +++ p <0.001; Veh vs. LSD and DOI or or Psil vs. LSD and DOI; for Htr2a EGFP-CreERT2 mice (green): ^^ p <0.01, Psil vs. Veh and LSD. (F) Retrograde walking in these same mice. Two-way ANOVA for treatment [F(3,69)=21.713, p <0.001]; Bonferroni post-hoc tests: overall treatment effects (purple): ### p <0.001, Veh vs. LSD and DOI, or Psil vs. LSD and DOI. (G) Prepulse inhibition C57 and Htr2a EGFP-CreERT2 mice were injected with the Veh, 0.3 mg/kg LSD, 1 mg/kg DOI, or 1 mg/kg Psil and tested 10 min later; n=10-15 mice/genotype/treatment. RMANOVA: PPI [F(2,210)=329.097, p <0.001], PPI by genotype interaction [F(2,210)=23.856, p <0.001], PPI by treatment interaction [F(6,210)=3.342, p =0.004], genotype [F(1,105)=45.808, p <0.001], and treatment [F(3,105)=19.562, p <0.001]; Bonferroni post-hoc tests: for C57 vs Htr2a EGFP-CreERT2 mice (black): *** p <0.001, overall genotype effects; for treatment effects (purple): # p <0.05, Veh vs. Psil; ## p <0.01, Veh vs. LSD; ### p <0.001, Veh vs. DOI. Some schematics were created with https://www.biorender.com/ .

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Injection, Inhibition

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A-B) Null and startle activities in prepulse inhibition Mice were injected with the Veh, 0.3 mg/kg LSD, 1 mg/kg DOI, or 1 mg/kg Psil and tested 10 min later; n=10-15 mice/genotype/treatment. Note, the C57BL/6J and Htr2a EGFP-CreERT2/EGFP-CreERT2 mice are termed C57 and Htr2a EGFP-CreERT2 mice, respectively, below and in the figure panels. The results are presented as means ± SEMs; n=10-15 mice/genotype/treatment. (A) Null activities in C57 and Htr2a EGFP-CreERT2 mice. Two-way ANOVA: genotype [F(1,105)=33.886, p <0.001] and treatment [F(3,105)=32.101, p <0.001]; Bonferroni post-hoc tests: for C57 vs . Htr2a EGFP-CreERT2 mice (black): *** p <0.001, overall genotype effects; overall treatment effects (purple): ### p <0.001, Veh vs. LSD and DOI or LSD and Psil vs. DOI. (B) Startle activities in C57 and Htr2a EGFP-CreERT2 mice. Two-way ANOVA: genotype [F(1,105)=51.786, p <0.001], treatment [F(3,105)=12.810, p <0.001], and genotype by treatment interaction [F(3,105)=3.282, p =0.024]; Bonferroni post-hoc tests: for C57 vs . Htr2a EGFP-CreERT2 mice (black): *** p <0.001, Veh, LSD, and Psil; for C57 (blue): + p <0.05, Veh vs. Psil; +++ p <0.001, LSD vs. Psil; for Htr2a EGFP-CreERT2 mice (green): ^ p <0.05, Veh vs. DOI; ^^^ p <0.001, LSD vs. DOI.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Inhibition, Injection

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A) [ 3 H] ketanserin saturation binding assay was conducted to examine the affinity (Kd, nM) and receptor levels (Bmax, pmole/mg) of HTR2A in the whole cortex of C57 and Htr2a EGFP-CreERT2 mice. Non-specific binding was determined with 10 µM clozapine. Data are means ± SEMs (n=4 samples/genotype) and analyzed by unpaired t-test. Kd values: C57(1.09 ± 0.324 nM) vs. Htr2a EGFP-CreERT2 (0.75 ± 0.13nM), [t(6)=1.005, p =0.354]; Bmax values: C57 (0.25 ±0.018 pmole/mg) vs. Htr2a EGFP-CreERT2 (0.46 ± 0.025 pmole/mg), [t(6)=6.745, p =0.0005] (*** p <0.001, C57 vs. Htr2a EGFP-CreERT2 ). (B) Real-time qPCR was used to determine Htr2a mRNA levels from the whole cortex of C57 mice ( Htr2a +/+) , Htr2a EGFP-CreERT2/+ , and Htr2a EGFP-CreERT2/EGFP-CreERT2 mice. Data are means ± SEMs (n=4 samples/genotype). One-way ANOVA [(F(2,9)=129.4, p <0.0001] followed by Tukey’s post-hoc test showed *** p <0.001, C57 vs. Htr2a EGFP-CreERT2/+ ; ****p <0.0001, C57 vs. Htr2a EGFP-CreERT2/EGFP-CreERT2 . (C) LSD-mediated HTR2A downregulation: Mice (C57 and Htr2a EGFP-CreERT2 ) were injected with vehicle or LSD (0.5 mg/kg, i.p.) for 5 consecutive days and then were euthanized 24 hr later. The whole cortices were dissected followed by receptor purification using Wheat-Germ beads pull-down. Western blot against anti-HTR2A antibody detected the receptor expression levels. Data represent the means ± SEMs (n=5-7). Two-way ANOVA: treatment [F(1,21)=27.11, p <0.0001], genotype [F(1,21)=24.09, p <0.0001], and treatment by genotype interaction [F(1,21)=0.1832, p =0.673] (overall genotype effects (black): ****p<0.0001, C57 vs. Htr2a EGFP-CreERT2/EGFP-CreERT2 ; overall treatment effect (purple): #### p <0.0001, vehicle vs. LSD). The schematic was created with https://www.biorender.com/ .

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Saturation Assay, Binding Assay, Injection, Purification, Western Blot, Expressing

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A) Htr2a EGFP-CreERT2 mice crossed with the Cre-dependent reporter line (Ai9 mice here) were used to visualize HTR2A expressing cells with tdTomato expression. The bicistronic design of Htr2a-EGFP-IRES-CreERT2 produced a HTR2A-EGFP-CT fusion protein and CreERT2 recombinase. Tamoxifen activates CreERT2 recombinase, leading to Cre-loxP recombination and then turning on tdTomato expression. The schema was created with https://www.biorender.com/ . (B) The expression pattern of tdTomato (red) and HTR2A-EGFP-CT fusion proteins (green) in the mPFC subregion were identified using immunohistochemistry staining for HTR2A expression patterns and AchE staining for the cytoarchitecture. MOs: secondary motor cortex; AAC: anterior cingulate area; PL: prelimbic cortex; IL: infralimbic cortex; and fr: anterior forceps (C) L5a pyramidal neurons were used for recording neuronal firing activity during focal 5-HT (10 µM) application. Addition of 5-HT resulted in an initial decrease in firing followed by increased firing shown 3 min after application. Data are presented as means ± SEMs (n=7) and were analyzed using paired t-test to compare basal activity and firing after 5-HT application, [t(6)=3.177, p=0.0191] (*p<0.05) and one sample t-test was used for normalization of basal activity for the 5-HT response, p=0.0469 (*p<0.05). (D) Representative recording of HTR2A + /L5a pyramidal neuronal firing after M100907 (200 nM) and 5-HT (10 µM). M100907 effects on firing were analyzed using paired t-test, [t(10)=2.667, p=0.0236] (*p<0.05 vs. baseline) and normalized M100907 effects on firing were analyzed with one-sample t-test, [t(11)=4.421, p =0.001] (***p=0.001). Data are represented as means ± SEMs (n=11). The effects of M100907 + 5-HT on firing were analyzed with paired t-test, [t(11)=2.036, p=0.067] and normalized effects were analyzed using one-sample t-test. Data are presented as means ± SEMs (n=12). (E) Representative recording of HTR2A + /L5a neuronal firing after administration of WAY100635 (100 nM) and 5-HT (10 μM). WAY100635 effects on firing were analyzed with paired t-test, [t(6)=3.115, p=0.021] (*p<0.05, vs baseline) and normalized effects on firing were analyzed with one-sample t-test, [t(6)=7.228, p=0.0004] (***p<0.001). Data are presented as means ± SEMs (n=7). WAY100635 + 5-HT effects on firing were analyzed with paired t-test, [t(8)=2.159, p=0.0629] and normalized WAY100635 + 5-HT effects on firing were analyzed with one sample t-test p =0.0039 (**p=0.01). Data are represented as means ± SEMs (n=9). (F) A representative biocytin-labeled L5 pyramidal neuron showed its location in the HTR2A-EGFP-CT positive cortical band layer in the dorsal prelimbic cortex. After recording, the patched neuron was injected with biocytin. The brain sections were stained with an anti-GFP antibody to locate the L5a cortical band. Images were taken under 20X objective with an Olympus confocal microscope.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Expressing, Produced, Immunohistochemistry, Staining, Activity Assay, Labeling, Injection, Microscopy

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: Htr2a EGFP-CreERT2/+ mice were crossed with Ai9 mice. Mice were treated with tamoxifen (100 mg/kg, i.p.) at p39-p42 for 4 days and then brains were perfused at 14 days post-injection (14 dpi) at p56. The brain sections were stained with GFP antibody. Images were captured in two channels for tdTomato (red) and GFP (green) signals under an Olympus slide scanner with 10X objective. The GFP signal as a HTR2A-EGFP-CT fusion protein and the tdTomato signal as HTR2A-positive cells were shown here. Experiments were conducted on 4 brains with similar results. The raw images have been uploaded to open-source website, A Mouse Imaging Server (AMIS, https://amis2.docking.org/ ).

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Injection, Staining, Imaging

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: Htr2a EGFP-CreERT2/+ x Ai9 or C57 x Ai9 ( Htr2a +/+ x Ai9) mice were treated with vehicle (corn oil) or tamoxifen (100 mg/kg) at p39 to p42 with 4 injections and then brains were collected at p56. A whole brain mapping was conducted to determine whether CreERT2 recombinase turned on tdTomato protein expression after TAM treatment. The brain sections were stained with DAPI and then images were rendered by an Olympus VS120 slide scanner. Experiments were conducted with 4 animals with similar results.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: Htr2a EGFP-CreERT2/+ x Ai9 mice were used for this experiment. Individual recording neurons (HTR2A-tdTomato-positive) from different brain sections of each treated group (purple: 5-HT; blue: M100907+5-HT; and Green: WAY100365+5-HT) are depicted in the ACC or dPL. (A) HTR2A signals with anti-HTR2A staining from Htr2a Cre/+ mouse (B) HTR2A - A242S-EGFP-CT signals with anti-GFP staining from Htr2a A242S-EGFP-Cre/+ (C-D) Comparison of different Cre-dependent reporters (Ai9 mice vs PHP.eB.flex.tdT AAV virus) among Htr2a mouse lines

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Staining, Comparison, Virus

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A) The schema depicts the transgenic designs of each mouse line. As described previously, the Htr2a -EGFP-CT-IRES-CreERT2 ( Htr2a EGFP-CreERT2 ) was inserted with EGFP in the C-terminus of the Htr2a followed by adding the IRES-CreERT2 sequence after exon 3 as an inducible Htr2a mouse line. Htr2a -IRES-Cre mouse line ( Htr2a Cre ) was engineered IRES-Cre sequence at the end of exon 3. A humanized mouse line, Htr2a -A242S-EGFP-CT-IRES-Cre ( Htr2a A242S-EGFP-Cre ), was created with one point mutation at the alanine 242 residue of the murine Htr2a to the serine residue of the human HTR2A and this was followed at the murine 452 residue by an EGFP insertion; the IRES-Cre was inserted after the exon 3 coding sequence. In the diagrams, the blue boxes represent exons of the murine Htr2a gene; the green box is Egfp ; the red box is the stop codon. The gray box is the IRES (internal ribosome entry site) followed by a pink box for Cre or CreERT2 recombinase. The white box is the A242S point mutation. The schematic was created with https://www.biorender.com/ . (B) HTR2A distribution in the brain among four mouse lines ( Htr2a +/+ , Htr2a Cre/+ , Htr2a EGFP-CreERT2/+ , and Htr2a A242S-EGFP-Cre/+ ). For this study, mice were perfused, and the brain sections were immunostained with an anti-HTR2A antibody to visualize the HTR2A distribution. (C) The HTR2A receptor from four mouse lines ( Htr2a +/+ , Htr2a Cre/Cre , Htr2a EGFP- CreERT2/EGFP-CreERT2 , and Htr2a A242S- EGFP-Cre/A242S-EGFP-Cre ). Here, mice were euthanized and then cortices were dissected followed by receptor purification using Wheat-Germ beads pull-down. Western blot against anti-HTR2A antibody to detect receptor expression level. In Htr2a +/+ and Htr2a Cre/Cre mice, the HTR2A was detected between 50-75 KD; for EGFP insertion lines, the HTR2A-EGFP-CT fusion protein was detected around 100 KD. (D) Representative image of Htr2a Cre/+ mice expressing DIO-eYFP in the mPFC was stained with anti-GFP for eYFP signal amplification at 20x (left) and 63x magnification (left inset). Example electrophysiological trace of a HRT2A + neuron expressing DIO-eYFP during bath application of DCZ. Representative image of Htr2a Cre/+ mice expressing DIO-HA-hM3Dq-IRES-mCitrine in the mPFC was stained with anti-GFP for mCitrine at 20x (right) and 63x magnification (right inset). Example electrophysiological trace of a HRT2A + neuron expressing DIO-HA-hM3Dq-IRES-mCitrine during bath application of DCZ.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Transgenic Assay, Sequencing, Mutagenesis, Residue, Purification, Western Blot, Expressing, Staining, Amplification

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A) The HTR2A proteins in the whole brain mapping from Htr2a Cre/+ mice were shown using an anti-HTR2A antibody. (B) The distribution of HTR2A-A242S-EGFP-CT fusion proteins from Htr2a A242S-EGFP-Cre/+ mice were mapped in the whole brain using an anti-GFP antibody. The raw images have been uploaded to open-source website, A Mouse Imaging Server (AMIS, https://amis2.docking.org/ ). (C) Cre reporter mice (Ai9) crossed with three Htr2a mouse lines. The distribution patterns of HTR2A and HTR2A + tdTomato from an inducible Htr2a EGFP-CreERT2/+ x Ai9 mouse and from constitutive Cre lines ( Htr2a Cre/+ xAi9 and Htr2a A242S-EGFP-Cre/+ x Ai9) are shown. All mice were perfused at p56. The brain sections were stained with anti-HTR2A or anti-GFP antibodies. Images were captured in two channels for tdTomato (red) and anti-HTR2A or anti-GFP (green) signals under and Olympus slide-scanner with 10X objective. Experiments were conducted with 3-4 brains with similar results. (D) Cre reporter virus (PHP.eB.flex.tdT) with inducible CreERT2 and constitutive Cre-mouse Htr2a lines. The Htr2a mouse lines were retro-orbitally injected with PHP.eB.flex.tdTomato . The tdTomato patterns of expression among the 3 mouse lines from virally-treated groups are compared with Htr2a EGFP-CreERT2/+ x Ai9 mice in the area of the somatosensory cortex . Mice were perfused and then stained with anti-HTR2A for Htr2a Cre/+ mice and with anti-GFP for Htr2a EGFP-CreERT2/+ and Htr2a A242S-EGFP-Cre/+ mice. Images were captured under an Olympus slide-scanner with 10X objective. (CPu-s: striosome, TUO: olfactory tubercle, IG: induseum griseum, SH: septohippocampal nucleus).

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Imaging, Staining, Virus, Injection, Expressing

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A-D) Head twitch responses and prepulse inhibition (PPI) Head twitch responses were scored over 30 min beginning after injection of the vehicle (Veh), LSD (0.3 mg/kg), DOI (1 mg/kg), or Psil (1 mg/kg). For PPI, mice received the same regimen of treatment and were tested in PPI 10 min later as described in the Methods section. Note, the C57BL/6J and Htr2a A242S-EGFP-Cre/A242S-EGFP-Cre mice are termed C57 and Htr2a A242S-EGFP-Cre mice, respectively, below and in the figure panels. All data are represented as means ± SEMs. (A) Head twitch responses in C57 and Htr2a A242S-EGFP-Cre animals immediately following administration of the Veh, LSD, DOI, or Psil over the 30 min following administration; n=9-10 mice/genotype/treatment. Two-way ANOVA: treatment [F(3,70)=43.033, p <0.001], and genotype by treatment interaction [F(3,70)=6.784, p <0.001]; Bonferroni post-hoc tests: for C57 vs. Htr2a A242S-EGFP-Cre mice (black): * p <0.05, Psil; *** p <0.001, DOI; for C57 mice (blue): +++ p <0.05, Veh vs. all psychedelics or Psil vs. DOI; for Htr2a A242S-EGFP-Cre mice (green): ^^^ p <0.05, Veh vs . all psychedelics. (B) Prepulse inhibition: C57 and Htr2a A242S-EGFP-Cre mice were injected with the Veh, LSD, DOI, or Psil and tested 30 min later; n=9-10 mice/genotype/treatment. RMANOVA: PPI [F(2,140)=113.340, p <0.001], genotype [F(1,70)=4.989, p =0.029], and treatment [F(3,70)=23.203, p <0.001]; Bonferroni post-hoc tests: for C57 vs Htr2a A242S-EGFP-Cre mice (black): ** p <0.01, Veh; for C57 mice (blue): + p <0.05, Psil vs. Veh; ++ p ≤0.001, LSD vs. Veh; +++ p ≤0.001, DOI vs. Veh; for Htr2a A242S-EGFP-Cre mice (green): ^^^ p <0.001, all psychedelics vs. Veh. (C) Null activities in C57 and Htr2a A242S-EGFP-Cre mice. In the two-way ANOVA Levene’s test of homogeneity was violated and it was still violated when null activity was standardized to genotype or vehicle. Neither the Mann-Whitney U test nor the Wilcoxin W test for genotype (two-tailed) were significant for any treatment. Kruskal-Wallis one-way ANOVA for treatment (two-tailed): [h(3)=27.582, p <0.001]; Bonferroni post-hoc tests for C57 mice (blue); + p <0.05; Veh vs. Psil; ++ p <0.01, Psil vs. DOI; +++ p <0.001, Veh vs. LSD and DOI; for Htr2a A242S-EGFP-Cre mice (green): ^ p <0.05; Veh vs. Psil or Psil vs. DOI; ^^ p <0.01, Veh vs. LSD; ^^^ p <0.001, Veh vs. DOI. (D) Startle activities in C57 and Htr2a A242S-EGFP-Cre mice. Two-way ANOVA: genotype [F(1,70)=4.295, p =0.042], treatment [F(3,70)=7.547, p <0.001], and genotype by treatment interaction [F(3,70)=3.740, p =0.015]; ANOVA followed by Bonferroni post-hoc tests: for C57 vs . Htr2a A242S-EGFP-Cre mice (black): * p <0.05, Psil; ** p <0.01, Veh; for C57 (blue): ++ p <0.05, LSD vs. Psil; for Htr2a A242S-EGFP mice (green): ^^^ p <0.001, Veh vs. DOI.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Inhibition, Injection, Activity Assay, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: Htr2a EGFP-CreERT2/+ x Ai9 x Thy1 GFP/+ mice were used and were treated with tamoxifen (100 mg/kg for 4 days) to induce tdTomato expression. Since HTR2A-EGFP-CT is very dim without antibody amplification, it was difficult to directly detect. Thus, in this experiment we used tdTomato to label HTR2A-containing neurons and Thy1-GFP to observe their distribution in the cortex. Images were captured under an Olympus slide-scanner with 10X objective. Experiments were conducted on 3 brains with similar results.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Expressing, Amplification

Journal: bioRxiv

Article Title: A suite of engineered mice for interrogating psychedelic drug actions

doi: 10.1101/2023.09.25.559347

Figure Lengend Snippet: (A) RNAscope was used to detect Htr2a and Egfp transcripts in the Htr2a EGFP-CreERT2/EGFP-CreERT2 mice. Brain sections were also stained with anti-GFP antibody to detect protein signals of the HTR2A-EGFP-CT fusion protein. Experiments were conducted with 4 animals containing similar results. Images were taken under a 10X objective with an Olympus VS120 slide-scanner (scale bar 1mm). (B) In the motor cortex area, Egfp transcripts were co-localized with Lamp5 in Layer2/3 and Layer5b, and with parvalbumin positive interneurons. Cplx3 as a layer6b marker showed its co-localization with Egfp transcripts. Images were taken under 10X objective with an Olympus VS120 slide-scanner and under a 20X objective with an Olympus FV3000RS confocal microscope. Experiments were conducted with 3 animals achieving similar results.

Article Snippet: The anti-rabbit HTR2A antibody (1:250, Neuromics, #RA24288) and anti-rat CTIP2 (1:1000, Abcam, #ab18465) primary antibodies were incubated at room temperature (RT) overnight.

Techniques: Staining, Marker, Microscopy

Journal: Respiratory Research

Article Title: Apoptosis of viral-infected airway epithelial cells limit viral production and is altered by corticosteroid exposure

doi: 10.1186/1465-9921-7-78

Figure Lengend Snippet: Representative images of airway epithelial immunostaining of Cam Hartley Guinea pigs Semi-quantitative scoring was utilized to determine the expression for DR4(A), DR5 (C) and p85-PARP (E) in immunohistochemically stained lung sections. Panels B, D and F were the isotype controls for the respective antibodies. Arrows indicate the stained epithelial cells. Scale bar represent 10 μm in panels A through F.

Article Snippet: Polyclonal rabbit anti-DR4 and -DR5 antibodies (Cell Sciences Inc, MA) and rabbit anti- PARP p85 fragment antibody (Promega, MA) were used along with normal Rabbit IgG as negative control to measure receptor expression and apoptosis respectively. p85-PARP antibody is specific for the p85 fragment of PARP generated by caspase cleavage and provides a reliable measure of in situ apoptosis [ ].

Techniques: Immunostaining, Expressing, Staining

Journal: Respiratory Research

Article Title: Apoptosis of viral-infected airway epithelial cells limit viral production and is altered by corticosteroid exposure

doi: 10.1186/1465-9921-7-78

Figure Lengend Snippet: Acutely infected GPTEC demonstrate apoptosis coordinate with DR4 and DR5 expression Semi-quantitative scoring was utilized to determine the expression of p85-PARP, DR4 and DR5 in the Guinea pig lung sections by immunohistochemistry. p85-PARP was significantly higher in 1 -4 dPi lung sections compared to Sham controls, peaked at 4 dPi and decreasing significantly by 7 dPi (Figure 4A). This trend in apoptosis in the Acute model was coordinate with the changes in DR4 and DR5 expression (Figure 4B). * p < 0.05 compared to Sham and †p < 0.05 compared to 4 dPi.

Article Snippet: Polyclonal rabbit anti-DR4 and -DR5 antibodies (Cell Sciences Inc, MA) and rabbit anti- PARP p85 fragment antibody (Promega, MA) were used along with normal Rabbit IgG as negative control to measure receptor expression and apoptosis respectively. p85-PARP antibody is specific for the p85 fragment of PARP generated by caspase cleavage and provides a reliable measure of in situ apoptosis [ ].

Techniques: Infection, Expressing, Immunohistochemistry

Journal: Respiratory Research

Article Title: Apoptosis of viral-infected airway epithelial cells limit viral production and is altered by corticosteroid exposure

doi: 10.1186/1465-9921-7-78

Figure Lengend Snippet: Guinea pig AEC apoptosis and DR expression as detected by Immunohistochemistry of the Chronic model of Guinea pig viral infection and airway inflammation Semi-quantitative scoring was utilized to determine the expression of p85-PARP, DR4 and DR5 in the Guinea pig lung sections by immunohistochemistry. Significant reduction in the detection of p85-PARP for Ad5+OVA+Bud group (** p < 0.005) was observed when compared to Ad5 alone group (Figure 7A). However, DR4/DR5 expression for Ad5+OVA+Bud group was higher compared to Ad5 alone group * p < 0.05 (Figure 7B).

Article Snippet: Polyclonal rabbit anti-DR4 and -DR5 antibodies (Cell Sciences Inc, MA) and rabbit anti- PARP p85 fragment antibody (Promega, MA) were used along with normal Rabbit IgG as negative control to measure receptor expression and apoptosis respectively. p85-PARP antibody is specific for the p85 fragment of PARP generated by caspase cleavage and provides a reliable measure of in situ apoptosis [ ].

Techniques: Expressing, Immunohistochemistry, Infection

Journal: Translational Psychiatry

Article Title: Intranasal delivery of shRNA to knockdown the 5HT-2A receptor enhances memory and alleviates anxiety

doi: 10.1038/s41398-024-02879-y

Figure Lengend Snippet: A – D Representative immunofluorescence images in human neurons following a 10-day treatment with scrambled AAV9 shRNA-AAV9 viral particles ( A , B ) or hu-HTR2A shRNA-AAV9 viral particles at MOI of 3 × 10 5 ( C , D ). Green fluorescence represents green fluorescence protein expression, while red fluorescence is indicative of 5HT-2A receptor protein following immunocytochemistry using an anti-mouse 5HT-2A receptor antibody at 1:50. Low level GFP expression, which served as a proxy for scrambled shRNA expression was observed in all cases (Fig. 1A). Panel B indicates robust expression of the 5HT-2A receptor protein in both cell bodies and neurites following treatment with the scrambled control. Panels C and D are representative images following treatment with hu-HTR2A shRNA-AAV9 and revealed stronger GFP expression ( C ). D Following hu-HTR2A shRNA-AAV9 treatment, a decrease in 5HT-2A fluorescent intensity was apparent. E ImageJ quantification of 5HT-2A immunofluorescence indicated a significant 27% decrease in hu-HTR2A shRNA-AAV9-treated neurons as compared to scrambled-treated (* p -value = 0.0019, N = 3 different samples for each condition). F Data show the results of qPCR real-time assays to analyze mRNA levels of Htr2a following extraction of RNA iPSC differentiated neurons in either vehicle-controls (blue bar) or scrambled treated (green bar), and hu-HTR2A shRNA (red bar). Neurons were treated on day 1 at a cell density of 200 K/well and cells pelleted and frozen on day 16. Results display the relative change in expression using GAPDH as an internal control. Real-time PCR results represent a total of 3 separate treatments for each condition in which cells were pooled and frozen at −80 °C. PCR experiments were performed in triplicate. The results indicated a significant 34% decrease in htr2a mRNA expression as compared to vehicle controls, where *denotes significant difference between the two groups, p = 0.012. Relative mRNA levels were not significantly different between vehicle control and scrambled-treated cells ( p -value = 0.502) or between scrambled- and shRNA-treated neurons ( p -value = 0.094).

Article Snippet: Briefly, all tissue sections were labeled with anti-GFP antibody (rabbit mAB #2956) 1:1000 (Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-5HT-2A receptor antibody (rabbit polyclonal, #24288) at 1:500 dilution (Immunostar, Hudson, WI).

Techniques: Immunofluorescence, shRNA, Fluorescence, Expressing, Immunocytochemistry, Control, Extraction, Real-time Polymerase Chain Reaction

Journal: Translational Psychiatry

Article Title: Intranasal delivery of shRNA to knockdown the 5HT-2A receptor enhances memory and alleviates anxiety

doi: 10.1038/s41398-024-02879-y

Figure Lengend Snippet: Following behavioral analysis, a subset of brains from these mice were fixed, and 4 µm paraffin-embedded sagittal tissue sections were stained with a specific antibody against GFP (green fluorescence, 1:1000), anti-5HT2A receptor (red fluorescence, 1:500), or Hoechst nuclear labeling (blue fluorescence). Whole slide imaging was performed using a Pannoramic Midi II scanner (see “Materials and methods” for details). A Representative, low-field immunofluorescence sagittal image following treatment with MeCP2-GFP-mouse HTR2A-shRNA identified neuronal labeling in most brain regions including the olfactory bulb (OB), cortex, cerebellum, and numerous sub-cortical areas including the interpeduncular nucleus (IPN), a major connectome for stress-mediated pathways. Three different brain regions were examined at higher magnification including the cerebellum ( B – G ), IPN ( H – M ), and olfactory bulb ( N – S ). Data are representative images from either vehicle controls in each brain region ( B – D , H – J , or N – P ) and AAV-treated mice ( E – G , K – M , or Q – S ). Treatment with MeCP2-GFP-mouse HTR2A-shRNA led to a general pattern of less robust staining profile of the 5HT2A receptor in cell body regions and apical dendrites (merged image Panel G as an example). All scale bars represent 50 µm. T – V Quantitative analysis using ImageJ software indicated a significant decrease in 5HT-2A receptor fluorescence intensity of AAV-treated mice (red bar) versus vehicle-controls (green bar) in both total brain sections ( T ) and olfactory bulb ( U ). Although there was a trend for a decrease in 5HT-2A receptor fluorescence intensity in the cerebellum, data did not reach significance. Data represent the mean gray value ± SD in three different cases. *Denotes significant difference, p -value < 0.05. W , X Data show the results of qPCR real-time assays to analyze mRNA levels of Htr2a following extraction of total brain RNA from frozen brain ( W ) or olfactory tissue ( X ) in either vehicle-controls (black bars) or shRNA treated (red bars). Results display relative mRNA levels after 8-weeks of treatment with AAV9-MeCP2-GFP-mouse HTR2A as compared to vehicle-controls. Real-time PCR results represent a total of N = 5 animals for each group performed in triplicate ±SEM. *Denotes significant difference between the two groups, p = 0.006 in olfactory mRNA.

Article Snippet: Briefly, all tissue sections were labeled with anti-GFP antibody (rabbit mAB #2956) 1:1000 (Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-5HT-2A receptor antibody (rabbit polyclonal, #24288) at 1:500 dilution (Immunostar, Hudson, WI).

Techniques: Staining, Fluorescence, Labeling, Imaging, Immunofluorescence, shRNA, Software, Extraction, Real-time Polymerase Chain Reaction

Journal: Translational Psychiatry

Article Title: Intranasal delivery of shRNA to knockdown the 5HT-2A receptor enhances memory and alleviates anxiety

doi: 10.1038/s41398-024-02879-y

Figure Lengend Snippet: A Aged male C57Bl6 mice were treated intranasally with vehicle or with 2.0 × 10 11 viral particles on day 1, and 5-weeks later tested behaviorally using a spontaneously alteration memory test. Mice treated with AAV9-CRISPR/Cas9 showed a significant increase in the percent spontaneous alterations ( p -value = 0.0007, N = 15 mice per group, asterisk, blue bar). B Representative, merged immunofluorescence image of vehicle-control animals depicting the presence of 5HT-2A receptor protein labeling in apical dendrites in the CA2/CA3 region of the hippocampus. The blue staining reflects nuclear staining with DAPI. As expected, there was no expression of GFP in vehicle controls. C Identical to Panel B with the exception that the merged image is from a AAV9-CRISPR/Cas9-treated mouse brain. In this case, strong GFP labeling was observed in cell bodies while there was an observed decrease in 5HT-2A receptor fluorescence. Images are representative of 3 separate mice for each group.

Article Snippet: Briefly, all tissue sections were labeled with anti-GFP antibody (rabbit mAB #2956) 1:1000 (Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-5HT-2A receptor antibody (rabbit polyclonal, #24288) at 1:500 dilution (Immunostar, Hudson, WI).

Techniques: CRISPR, Immunofluorescence, Control, Labeling, Staining, Expressing, Fluorescence

Journal: Translational Psychiatry

Article Title: Intranasal delivery of shRNA to knockdown the 5HT-2A receptor enhances memory and alleviates anxiety

doi: 10.1038/s41398-024-02879-y

Figure Lengend Snippet: The target sequence used to synthesize the shRNA is 100% conserved between mice and rats. To test whether shRNA-knockdown of the rat 5HT-2A receptor improves memory, Wistar rats (12 animals per group) were randomly assigned to two different groups consisting of vehicle- or AAV9-MeCP2-GFP-mHTR2A-shRNA. Following treatment on day 1, animals were assessed behaviorally 3- ( A – D ) and 5-weeks later ( E – H ). Details of the novel object recognition test can be found in the methods. At 3 weeks, there was no significance difference in the time spent or the number of physical contacts with the novel object during the acquisition (training) period ( p > 0.05) ( A , B ). Rats were tested 24-h later, and AAV9-treated rats (blue bars) showed a significant increase in both the contact-recognition index ( p < 0.000003) ( C ) and the time recognition-index ( p < 0.0003) ( D ). The same groups of rats were retested at 5-weeks and again no significant difference was noted in the time spent or the number of physical contacts with the novel object during the acquisition (training) period ( p > 0.05) ( E , F ). Twenty-four hours later there was a significant increase in the contact-recognition index ( p < 0.01) ( G ), however, there was no significant difference in the contact-recognition index ( p = 0.114) ( H ). I Data show the results of qPCR real-time assays to analyze mRNA levels of Htr2a following extraction of rat brain RNA. Results display relative mRNA levels after 5-weeks post-treatment with AAV9-HTR2A-shRNA. Real-time PCR results represent a total of N = 5 animals for each group performed in triplicate ±SEM. *Denotes significant difference between the two groups, p = 0.038. J Representative, merged immunofluorescence image of vehicle-control animals depicting the presence of 5HT-2A receptor protein labeling (red fluorescence) within the olfactory bulb. K Representative, merged immunofluorescence image of AAV9-HTR2A-shRNA-treated animals. The blue staining reflects nuclear staining with DAPI. As expected, there was no expression of GFP in vehicle controls ( J , top panel) while strong GFP labeling was observed in cell bodies of neurons of shRNA-treated rats ( K , bottom panel). Images are representative of 3 separate mice for each group.

Article Snippet: Briefly, all tissue sections were labeled with anti-GFP antibody (rabbit mAB #2956) 1:1000 (Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-5HT-2A receptor antibody (rabbit polyclonal, #24288) at 1:500 dilution (Immunostar, Hudson, WI).

Techniques: Sequencing, shRNA, Knockdown, Extraction, Real-time Polymerase Chain Reaction, Immunofluorescence, Control, Labeling, Fluorescence, Staining, Expressing