Journal: ACS Omega

Article Title: Receptor-Selective Modulation of Cannabinoid Signaling by Cardamonin: Integrating Molecular Dynamics, Free Energy Calculations, and Behavioral Validation

doi: 10.1021/acsomega.5c10026

Figure Lengend Snippet: Schematic overview of the study design combining computational and behavioral approaches. Cardamonin, a chalcone derivative, was evaluated for its interaction with cannabinoid receptors CB1 and CB2 using molecular dynamics simulations and MM/PBSA analyses. Parallel in vivo behavioral assaysVon Frey and Hargreaves testswere conducted in mice to assess mechanical and thermal antinociception, respectively.

Article Snippet: Cardamonin (MedChemExpress LLC, USA) and selective cannabinoid receptor 1 (CB1) antagonists SR141716 (MedChemExpress LLC, USA) and selective cannabinoid receptor 2 (CB2) antagonist SR144528 (MedChemExpress LLC, USA) were dissolved in dimethyl sulfoxide [DMSO], Tween 20 and diluted with saline at a ratio of 5:5:90 for intraperitoneal (i.p.) administration.

Techniques: In Vivo

Journal: ACS Omega

Article Title: Receptor-Selective Modulation of Cannabinoid Signaling by Cardamonin: Integrating Molecular Dynamics, Free Energy Calculations, and Behavioral Validation

doi: 10.1021/acsomega.5c10026

Figure Lengend Snippet: Docking poses of cardamonin with CB1 (A) and CB2 (B) receptors. (A) Cardamonin binds to CB1 with −8.7 kcal/mol, forming hydrogen bonds (SER 505 , ILE 267 ), hydrophobic contacts (PHE 170 , VAL 196 , LEU 193 , PHE 200 ), and a π-cation interaction (HIS 178 ). (B) In CB2 (−8.0 kcal/mol), cardamonin engages in hydrophobic interactions and a π-stacking (PHE117). Interaction types: hydrogen bonds (blue), hydrophobic (gray dashed), π-cation (orange dashed), π-stacking (green dashed).

Article Snippet: Cardamonin (MedChemExpress LLC, USA) and selective cannabinoid receptor 1 (CB1) antagonists SR141716 (MedChemExpress LLC, USA) and selective cannabinoid receptor 2 (CB2) antagonist SR144528 (MedChemExpress LLC, USA) were dissolved in dimethyl sulfoxide [DMSO], Tween 20 and diluted with saline at a ratio of 5:5:90 for intraperitoneal (i.p.) administration.

Techniques:

Journal: ACS Omega

Article Title: Receptor-Selective Modulation of Cannabinoid Signaling by Cardamonin: Integrating Molecular Dynamics, Free Energy Calculations, and Behavioral Validation

doi: 10.1021/acsomega.5c10026

Figure Lengend Snippet: Hydrogen bond interaction snapshots between cardamonin and cannabinoid receptors CB1 (A) and CB2 (B) at selected time points from molecular dynamics simulations. Each frame corresponds to a representative structure at 100, 250, 500, and 750 ns. Hydrogen bonds are depicted as red dashed lines with annotated distances (Å). In CB1, dynamic repositioning of the ligand allows alternating interactions with residues such as HSD 178 , ILE 267 , PHE 189 , ASP 184 , SER 173 , and LEU 193 . In contrast, the CB2 complex demonstrates a more consistent hydrogen bonding profile, particularly with SER 285 and GLY 284 . These interactions reflect the temporal stability and flexibility of ligand–receptor engagement during the simulation trajectory.

Article Snippet: Cardamonin (MedChemExpress LLC, USA) and selective cannabinoid receptor 1 (CB1) antagonists SR141716 (MedChemExpress LLC, USA) and selective cannabinoid receptor 2 (CB2) antagonist SR144528 (MedChemExpress LLC, USA) were dissolved in dimethyl sulfoxide [DMSO], Tween 20 and diluted with saline at a ratio of 5:5:90 for intraperitoneal (i.p.) administration.

Techniques:

Journal: ACS Omega

Article Title: Receptor-Selective Modulation of Cannabinoid Signaling by Cardamonin: Integrating Molecular Dynamics, Free Energy Calculations, and Behavioral Validation

doi: 10.1021/acsomega.5c10026

Figure Lengend Snippet: Principal Component Analysis (PCA) of CB1 and CB2 receptor complexes. (A) PCA projection of CB1 control (black) and cardamonin-bound complex (red) onto the first two principal components. Cardamonin binding induces broader conformational sampling. (B) PCA projection of CB2 control (black) and cardamonin-bound complex (red), showing overlapping conformational space and reduced ligand impact.

Article Snippet: Cardamonin (MedChemExpress LLC, USA) and selective cannabinoid receptor 1 (CB1) antagonists SR141716 (MedChemExpress LLC, USA) and selective cannabinoid receptor 2 (CB2) antagonist SR144528 (MedChemExpress LLC, USA) were dissolved in dimethyl sulfoxide [DMSO], Tween 20 and diluted with saline at a ratio of 5:5:90 for intraperitoneal (i.p.) administration.

Techniques: Control, Binding Assay, Sampling

Journal: ACS Omega

Article Title: Receptor-Selective Modulation of Cannabinoid Signaling by Cardamonin: Integrating Molecular Dynamics, Free Energy Calculations, and Behavioral Validation

doi: 10.1021/acsomega.5c10026

Figure Lengend Snippet: (A) Mechanical paw withdrawal threshold (mean ± SEM) in the Von Frey test. SHM and CDM groups displayed high thresholds (normal sensitivity), while VHC, CB1 – , and CB2 – groups exhibited mechanical allodynia. Co-treatment with cardamonin (CDM+CB1 – and CDM+CB2 – ) partially restored pain thresholds. (B) Thermal paw withdrawal latency (mean ± SEM) in the Hargreaves test. SHM group showed normal latency. CB1 – , CB2 – , and VHC groups exhibited thermal hyperalgesia. Cardamonin (CDM) increased latency significantly, and cotreatment with CB1 – or CB2 – partially restored thermal sensitivity.

Article Snippet: Cardamonin (MedChemExpress LLC, USA) and selective cannabinoid receptor 1 (CB1) antagonists SR141716 (MedChemExpress LLC, USA) and selective cannabinoid receptor 2 (CB2) antagonist SR144528 (MedChemExpress LLC, USA) were dissolved in dimethyl sulfoxide [DMSO], Tween 20 and diluted with saline at a ratio of 5:5:90 for intraperitoneal (i.p.) administration.

Techniques:

Journal: bioRxiv

Article Title: Layer-5 Pyramidal Cell tLTD Requires Astrocytic Ca 2+ and CB1 Receptor Signaling

doi: 10.64898/2026.04.21.719669

Figure Lengend Snippet: (A) Neonatal AAV injections were performed at P0–P2 in heterozygous or homozygous CB1 f mice, or CB1 f mice were bred with Aldh1l1-Cre mice. Experiments were conducted at P19–P23. (B) Representative quadruple-patch experiment used to assess monosynaptic PC → PC connections (blue, Alexa 594) in slices with astrocyte-specific Cnr1 deletion (red, EGFP). Presynaptic PC1 was connected to postsynaptic PC2. (C) Example recording from tLTD induction experiments in Cnr1 fl/+ slice. Gray bar indicates the tLTD induction period. Inset scale bars: 25 ms and 0.2 mV. (D) Example recording from the same PC1 → PC2 connection as in (B), showing failed tLTD in a Cnr1 fl/fl slice. Gray bar indicates the tLTD induction period. Inset scale bars: 25 ms and 0.2 mV. (E) tLTD was robust in Cnr1 fl/+ (blue) but not in Cnr1 fl/fl slices (red). (F) Homozygous astrocyte-specific CB1 receptor deletion abolished tLTD (Cnr1 fl/fl : 114 ± 7% vs 100%, one-sample t-test p = 0.1), whereas heterozygous deletion did not (Cnr1 fl/+ : 68 ± 9% vs 100%, one-sample t-test p < 0.05; genotype comparison t-test p < 0.01). (G) ΔPPR for Cnr1 fl/+ group was consistent with presynaptically expressed tLTD (ΔPPR Cnr1 fl/+ 0.14 ± 0.08, n = 6 vs Cnr1 fl/fl -0.14 ± 0.07, n = 4, t-test p < 0.05). (H) CV analysis in the Cnr1 fl/+ group showed most data points below the identity line, consistent with a presynaptic expression (φ = 13 ± 6°, Wilcoxon signed rank p = 0.09). In addition, 1⁄𝐶𝑉 2 norm was reduced (45 ± 9%, t-test p < 0.01, n = 6), suggesting reduced release probability .

Article Snippet: Cryorecovered heterozygous CB1 f mice (JAX 036107) were used to conditionally delete the cannabinoid receptor 1 Cnr1 gene ( ).

Techniques: Comparison, Expressing

Journal: bioRxiv

Article Title: Tetrahydrocannabinol exposure to postejaculatory sperm compromises sperm structure, function, the epigenome, and early embryo development

doi: 10.64898/2026.03.23.713385

Figure Lengend Snippet: A) Immunocytochemistry images of bovine sperm, human placenta, bovine placenta, and mouse brain depicting nuclear staining with DAPI (blue), CB1 (red), and merged images. FITC-PSA fluorescence can be observed in the merged image of sperm (green), for identification of the sperm acrosome. B) Western blot image of bovine sperm and equine pituitary identifying a single and specific band at the expected 75 kD value. Scale bar = 50 μM.

Article Snippet: Protein detection was performed with Anti-CB1(1:1500 proteintech Cat No: 17978-1-AP) in TBST buffer overnight at 4°C followed by detection with anti-rabbit secondary antibody HRP-linked (1:2000; Cat. #7074P2, Cell Signaling Technology ® ), in TBST buffer.

Techniques: Immunocytochemistry, Staining, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: Tetrahydrocannabinol exposure to postejaculatory sperm compromises sperm structure, function, the epigenome, and early embryo development

doi: 10.64898/2026.03.23.713385

Figure Lengend Snippet: Localization of CB1 in non-capacitated sperm (upper panel), to the post-acrosomal sheath as compared to loss of abundance when sperm are held in capacitating conditions (middle panel) as indicted by tyrosine phosphorylation staining (YPO 3-, yellow). Sperm exposed to THC under non-capacitating conditions exhibit decreased YPO 3 , CB1 and acrosome detection (green) (lower panel). Scale bar = 50 μm.

Article Snippet: Protein detection was performed with Anti-CB1(1:1500 proteintech Cat No: 17978-1-AP) in TBST buffer overnight at 4°C followed by detection with anti-rabbit secondary antibody HRP-linked (1:2000; Cat. #7074P2, Cell Signaling Technology ® ), in TBST buffer.

Techniques: Phospho-proteomics, Staining

Journal: bioRxiv

Article Title: Tetrahydrocannabinol exposure to postejaculatory sperm compromises sperm structure, function, the epigenome, and early embryo development

doi: 10.64898/2026.03.23.713385

Figure Lengend Snippet: A) Immunocytochemistry images of bovine sperm, human placenta, bovine placenta, and mouse brain depicting nuclear staining with DAPI (blue), CB1 (red), and merged images. FITC-PSA fluorescence can be observed in the merged image of sperm (green), for identification of the sperm acrosome. B) Western blot image of bovine sperm and equine pituitary identifying a single and specific band at the expected 75 kD value. Scale bar = 50 μM.

Article Snippet: Primary antibody incubation for detection of CB1 include the addition of 50 μL of Anti-CB1(proteintech ® Cat No: 17978-1-AP) at a dilution of 1:50 in BB for 15 h at 4 ° C. Slides were then washed and 100μL of secondary antibody (Donkey-AntiRabbit Abcam Cat No: ab6799) was added at a dilution of 1:100 (in BB) for 1 h in the dark followed by washing.

Techniques: Immunocytochemistry, Staining, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: Tetrahydrocannabinol exposure to postejaculatory sperm compromises sperm structure, function, the epigenome, and early embryo development

doi: 10.64898/2026.03.23.713385

Figure Lengend Snippet: Localization of CB1 in non-capacitated sperm (upper panel), to the post-acrosomal sheath as compared to loss of abundance when sperm are held in capacitating conditions (middle panel) as indicted by tyrosine phosphorylation staining (YPO 3-, yellow). Sperm exposed to THC under non-capacitating conditions exhibit decreased YPO 3 , CB1 and acrosome detection (green) (lower panel). Scale bar = 50 μm.

Article Snippet: Primary antibody incubation for detection of CB1 include the addition of 50 μL of Anti-CB1(proteintech ® Cat No: 17978-1-AP) at a dilution of 1:50 in BB for 15 h at 4 ° C. Slides were then washed and 100μL of secondary antibody (Donkey-AntiRabbit Abcam Cat No: ab6799) was added at a dilution of 1:100 (in BB) for 1 h in the dark followed by washing.

Techniques: Phospho-proteomics, Staining

Journal: bioRxiv

Article Title: Loss of dystrophin reduces CB1 receptor expression and endocannabinoid-dependent synaptic plasticity in the cerebellar cortex

doi: 10.64898/2026.03.20.713279

Figure Lengend Snippet: A. Representative Pinceaux staining showing a PC Soma (green, calbindin) along with the characteristic pinceaux formation on the axon side (red, CB1R) in both genotypes. B. Quantification of average CB1R pinceaux intensity across both genotypes and slide sets (VGAT, VGLUT1, VGLUT2). C. Same as B, but area of pinceaux. D-F. Proportion of CB1R puncta that colocalize with the respective vesicular marker before and after a 100 pixel shift of the CB1R channel along the X axis.

Article Snippet: Slides were incubated for 48 hours at 4°C under agitation with the following primary antibodies in blocking buffer: calbindin (1:3000, Sigma-Aldrich, Cat# C9848), CB1R (1:1000, Cayman Chemical Company, Cat# 10006590), and one of the following vesicular transporters - VGAT (1:250, Synaptic Systems, Cat# 131004), VGLUT2 (1:500, Synaptic Systems, Cat# 135418), or VGLUT1 (1:5000, Synaptic Systems, Cat# 135304).

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: Loss of dystrophin reduces CB1 receptor expression and endocannabinoid-dependent synaptic plasticity in the cerebellar cortex

doi: 10.64898/2026.03.20.713279

Figure Lengend Snippet: A. Representative confocal images from WT, DMD mdx and CB1R KO cerebella demonstrating calbindin (green), raw CB1R (red), and filtered CB1R puncta (see methods: image analysis). The right column shows an expanded and merged view of the area indicated in the filtered CB1R column. Quantification of mean CB1R puncta intensity ( B ), density ( C ), and area ( D ) in WT (black) and DMDmdx (blue) cerebella.

Article Snippet: Slides were incubated for 48 hours at 4°C under agitation with the following primary antibodies in blocking buffer: calbindin (1:3000, Sigma-Aldrich, Cat# C9848), CB1R (1:1000, Cayman Chemical Company, Cat# 10006590), and one of the following vesicular transporters - VGAT (1:250, Synaptic Systems, Cat# 131004), VGLUT2 (1:500, Synaptic Systems, Cat# 135418), or VGLUT1 (1:5000, Synaptic Systems, Cat# 135304).

Techniques:

Journal: bioRxiv

Article Title: Loss of dystrophin reduces CB1 receptor expression and endocannabinoid-dependent synaptic plasticity in the cerebellar cortex

doi: 10.64898/2026.03.20.713279

Figure Lengend Snippet: A. Representative images demonstrating calbindin (green), filtered CB1R (red), and filtered VGAT (cyan) staining across WT and DMD mdx cerebella. The right column shows an expanded and merged view of the area indicated in the VGAT image. Quantification of mean intensity ( B ), density ( C ), and area ( D ) of VGAT puncta in the molecular layer. E. Percentage of VGAT puncta colocalized with CB1R puncta. F. Intensity of CB1R puncta colocalized with VGAT puncta. G. Intensity of CB1R puncta not colocalized with VGAT puncta.

Article Snippet: Slides were incubated for 48 hours at 4°C under agitation with the following primary antibodies in blocking buffer: calbindin (1:3000, Sigma-Aldrich, Cat# C9848), CB1R (1:1000, Cayman Chemical Company, Cat# 10006590), and one of the following vesicular transporters - VGAT (1:250, Synaptic Systems, Cat# 131004), VGLUT2 (1:500, Synaptic Systems, Cat# 135418), or VGLUT1 (1:5000, Synaptic Systems, Cat# 135304).

Techniques: Staining

Journal: bioRxiv

Article Title: Loss of dystrophin reduces CB1 receptor expression and endocannabinoid-dependent synaptic plasticity in the cerebellar cortex

doi: 10.64898/2026.03.20.713279

Figure Lengend Snippet: A. Representative images demonstrating calbindin (green), filtered CB1R (red), and filtered VGLUT1 (cyan) staining across WT and DMD mdx cerebella. The right column shows an expanded and merged view of the area indicated in the VGLUT1 image. Quantification of mean intensity ( B ), density ( C ), and area ( D ) of VGLUT1 puncta in the molecular layer. E. Percentage of VGLUT1 puncta associated with CB1R puncta. F. Intensity of CB1R puncta colocalized with VGLUT1 puncta. G. Intensity of CB1R puncta not colocalized with VGLUT1 puncta

Article Snippet: Slides were incubated for 48 hours at 4°C under agitation with the following primary antibodies in blocking buffer: calbindin (1:3000, Sigma-Aldrich, Cat# C9848), CB1R (1:1000, Cayman Chemical Company, Cat# 10006590), and one of the following vesicular transporters - VGAT (1:250, Synaptic Systems, Cat# 131004), VGLUT2 (1:500, Synaptic Systems, Cat# 135418), or VGLUT1 (1:5000, Synaptic Systems, Cat# 135304).

Techniques: Staining

Journal: bioRxiv

Article Title: Loss of dystrophin reduces CB1 receptor expression and endocannabinoid-dependent synaptic plasticity in the cerebellar cortex

doi: 10.64898/2026.03.20.713279

Figure Lengend Snippet: A. Representative images demonstrating calbindin (green), filtered CB1R (red), and filtered VGLUT2 (cyan) staining across WT and DMD mdx cerebella. The right column shows an expanded and merged view of the area indicated in the VGLUT2 image. Quantification of mean intensity ( B ), density ( C ), and area ( D ) of VGLUT2 puncta in the molecular layer. E. Percentage of VGLUT2 puncta associated with CB1R puncta. F. Average intensity of CB1R puncta colocalized with VGLUT2 puncta. G. Intensity of CB1R puncta not colocalized with VGLUT2 puncta.

Article Snippet: Slides were incubated for 48 hours at 4°C under agitation with the following primary antibodies in blocking buffer: calbindin (1:3000, Sigma-Aldrich, Cat# C9848), CB1R (1:1000, Cayman Chemical Company, Cat# 10006590), and one of the following vesicular transporters - VGAT (1:250, Synaptic Systems, Cat# 131004), VGLUT2 (1:500, Synaptic Systems, Cat# 135418), or VGLUT1 (1:5000, Synaptic Systems, Cat# 135304).

Techniques: Staining