Journal: Scientific reports

Article Title: Involvement of oxidative stress in orofacial mechanical pain hypersensitivity following neonatal maternal separation in rats.

doi: 10.1038/s41598-023-50116-1

Figure Lengend Snippet: Figure 3. Involvement of reactive oxygen species (ROS) in mechanical pain hypersensitivity following maternal separation (MS). (a, b) Mechanical head-withdrawal threshold in the whisker pad skin following intraperitoneal (a) or subcutaneous (b) administration of the ROS scavenger N-tert-butyl-α-phenylnitrone (PBN) in non-MS and MS rats at 7 weeks (n = 6–7 in each group). *P < 0.05, **P < 0.01, Mann–Whitney U test. (c) Expression of transient receptor potential ankyrin 1 (TRPA1) in the whisker pad skin in the non-MS and MS rats at 7 weeks. Red arrows denote TRPA1-immunoreactivity. Scale bars indicate 30 µm. (d) Immunoblot for TRPA1 in the whisker pad skin at 7 weeks in the non-MS and MS rats (n = 6–7). P = 0.77, unpaired t test. (e) Mechanical head-withdrawal threshold in the whisker pad skin 30 min after intraperitoneal administration of the TRPA1 channel antagonist HC-030031 (HC) to non-MS and MS rats at 7 weeks (n = 5–6 in each group). **P < 0.01, Mann–Whitney U test.

Article Snippet: Membranes were blocked with TBST (0.2% Tween-20 diluted in Tris-buffered saline) containing 5% Blocking-One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies for anti-TRPA1 (rabbit, Polyclonal, 1:500; NB110-40763, Novus Biologicals, LLC, Centennial, CO, USA), and anti-β-actin antibody (mouse monoclonal, 1:200; sc-69879; Santa Cruz, San Diego, CA, USA).

Techniques: Whisker Assay, MANN-WHITNEY, Expressing, Western Blot

Journal: Molecular Psychiatry

Article Title: The ion channel TRPA1 is a modulator of the cocaine reward circuit in the nucleus accumbens

doi: 10.1038/s41380-024-02623-4

Figure Lengend Snippet: A Representative confocal image of a coronal of the NAc obtained from Trpa1 -shRNA-injected mice. Scale bar: 500 μm. ac, anterior commissure. B Validation of TRPA1 KD in the NAc of Trpa1 -shRNA-injected mice by Western blotting (n = 10). C Representative heat maps of time spent in each CPP chamber during pre-test and post-test periods of the TRPA1 KD cocaine CPP tests in male mice. CPP score of each group in the TRPA1 KD cocaine CPP tests in male mice (n = 10). D CPP score of each group in the TRPA1 antagonist (A-967079) cocaine CPP tests in male mice [n = 10 (control), 10 (cocaine), 10 (A-967079 5 mg/kg, cocaine), 10 (A-967079 15 mg/kg, cocaine), 11 (A-967079 50 mg/kg, cocaine), and 10 (A-967079 50 mg/kg)]. E Representative heat maps of time spent in each CPP chamber during pre-test and post-test periods of the TRPA1 KD cocaine CPP tests in female mice. CPP score of each group in the TRPA1 KD cocaine CPP tests in female mice (n = 7). F CPP score of each group in the A-967079 cocaine CPP tests in female mice [n = 7 (control), 7 (cocaine), 8 (A-967079 5 mg/kg, cocaine), 8 (A-967079 15 mg/kg, cocaine), 8 (A-967079 50 mg/kg, cocaine), and 8 (A-967079 50 mg/kg)]. G CPP score of each group in the TRPA1 antagonist (HC-030031) cocaine CPP tests in male mice [n = 10 (control), 9 (cocaine), 8 (HC-030031 5 mg/kg, cocaine), 10 (HC-030031 15 mg/kg, cocaine), 9 (HC-030031 50 mg/kg, cocaine), and 6 (HC-030031 50 mg/kg)]. H CPP score of each group in the intra-NAc injection of A-967079 during cocaine CPP tests in male mice [n = 6 (control), 6 (cocaine), and 5 (A-967079, cocaine)]. I CPP score of each group in the intra-VTA injection of A-967079 during cocaine CPP tests in male mice [n = 4 (control), 5 (cocaine), and 5 (A-967079, cocaine)]. Data are presented as mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.001 versus control; # P < 0.05 versus cocaine; sal, saline; coc, cocaine; Scrambled or Scr, scrambled control; TRPA1 KD or KD, TRPA1 knockdown; HC, HC-030031; A-9, A-967079.

Article Snippet: The membranes were then immunoblotted with primary antibodies against TRPA1 (rabbit, Novus, NB110-40763, 1:1000), DAT (rabbit, Millipore, AB2231, 1:3000), p-DAT (rabbit, Abcam, ab183486, 1:1000), TH (rabbit, Abcam, ab112, 1:5000), GluA1 (rabbit, Abcam, ab31232, 1:1000), GluA2 (rabbit, Abcam, ab20673, 1:1000) and ß-actin (mouse, Santa Cruz, Dallas, TX, USA, SC-47778, 1:2000) overnight at 4 °C.

Techniques: shRNA, Injection, Western Blot, Control, Saline, Knockdown

Journal: Molecular Psychiatry

Article Title: The ion channel TRPA1 is a modulator of the cocaine reward circuit in the nucleus accumbens

doi: 10.1038/s41380-024-02623-4

Figure Lengend Snippet: A Representative raw traces of spontaneous excitatory post-synaptic current (sEPSC) recordings from the NAc core of male mice. B Left, sEPSC amplitude in NAc core MSNs of saline- and cocaine-injected scrambled control and TRPA1 KD groups [n = 16 (scrambled, saline), 13 (scrambled, cocaine), 9 (KD, saline), and 15 (KD, cocaine)]. Right, sEPSC frequency in NAc core MSNs of saline- and cocaine-injected scrambled control and TRPA1 KD groups [n = 16 (scrambled, saline), 13 (scrambled, cocaine), 9 (KD, saline), and 15 (KD, cocaine)]. C Representative raw traces of spontaneous excitatory post-synaptic current (sEPSC) recordings from the NAc shell of male mice. D Left, sEPSC amplitude in NAc shell MSNs of saline- and cocaine-injected scrambled control and TRPA1 KD groups [n = 8 (scrambled, saline), 10 (scrambled, cocaine), 11 (KD, saline), and 11 (KD, cocaine)]. Right, sEPSC frequency in NAc shell MSNs of saline- and cocaine-injected scrambled control and TRPA1 KD groups [n = 8 (scrambled, saline), 10 (scrambled, cocaine), 11 (KD, saline), and 11 (KD, cocaine)]. E Representative raw traces of spontaneous excitatory post-synaptic current (sEPSC) recordings from the NAc core of female mice. F Left, sEPSC amplitude in NAc core MSNs of saline- and cocaine-injected scrambled control and TRPA1 KD groups [n = 10 (scrambled, saline), 9 (scrambled, cocaine), 9 (KD, saline), and 9 (KD, cocaine)]. Right, sEPSC frequency in NAc core MSNs of saline- and cocaine-injected scrambled control and TRPA1 KD groups [n = 10 (scrambled, saline), 9 (scrambled, cocaine), 9 (KD, saline), and 9 (KD, cocaine)]. G Representative raw traces of spontaneous excitatory post-synaptic current (sEPSC) recordings from the NAc shell of female mice. H Left, sEPSC amplitude in NAc shell MSNs of saline- and cocaine-injected scrambled control and TRPA1 KD groups [n = 8 (scrambled, saline), 10 (scrambled, cocaine), 10 (KD, saline), and 8 (KD, cocaine)]. Right, sEPSC frequency in NAc shell MSNs of saline- and cocaine-injected scrambled control and TRPA1 KD groups [n = 8 (scrambled, saline), 10 (scrambled, cocaine), 10 (KD, saline), and 8 (KD, cocaine)]. I Representative images of Western blot from each protein assayed. J Relative GluA1 protein expression in the NAc of the scrambled control and TRPA1 KD groups following cocaine CPP tests in male mice (n = 8). K Relative GluA2 protein expression in the NAc of the scrambled control and TRPA1 KD groups following cocaine CPP tests in male mice (n = 8). L Relative TRPA1 protein expression in the NAc of the scrambled control and TRPA1 KD groups following cocaine CPP tests in male mice [n = 8 (scrambled) and 9 (KD)]. Data are presented as mean ± standard error of the mean. * P < 0.05, ** P < 0.01 and *** P < 0.001; S or sal Saline, C or coc cocaine, Scr Scrambled control, KD TRPA1 knockdown.

Article Snippet: The membranes were then immunoblotted with primary antibodies against TRPA1 (rabbit, Novus, NB110-40763, 1:1000), DAT (rabbit, Millipore, AB2231, 1:3000), p-DAT (rabbit, Abcam, ab183486, 1:1000), TH (rabbit, Abcam, ab112, 1:5000), GluA1 (rabbit, Abcam, ab31232, 1:1000), GluA2 (rabbit, Abcam, ab20673, 1:1000) and ß-actin (mouse, Santa Cruz, Dallas, TX, USA, SC-47778, 1:2000) overnight at 4 °C.

Techniques: Saline, Injection, Control, Western Blot, Expressing, Knockdown

Journal: Molecular Psychiatry

Article Title: The ion channel TRPA1 is a modulator of the cocaine reward circuit in the nucleus accumbens

doi: 10.1038/s41380-024-02623-4

Figure Lengend Snippet: A Timeline of TRPA1 antagonist cocaine self-administration acquisition test on a fixed-ratio 2 (FR2) schedule. B Mean number of infusions from 3 days before test sessions and 2 days of FR2 test sessions following injection of a TRPA1 antagonist (A-967079) or vehicle in a cocaine self-administration test (n = 6). Active lever presses from 2 days of FR2 tests (n = 6). Inactive lever presses from 2 days of FR2 tests (n = 6). Cocaine intake from 1 day before test sessions and 2 days of tests (n = 6). C Rate of response to sucrose pellets from 1 day before the test sessions and 2 days of test sessions following injection of A-967079 or vehicle in a sucrose reinforcement test (n = 5). D Timeline of TRPA1 antagonist cocaine self-administration test on a progressive ratio (PR) and reinstatement (RI) schedule. E Mean number of infusions from 3 days before PR test sessions (n = 5) and breakpoint during the PR test following injection of A-967079 or vehicle in a cocaine self-administration test (n = 5). F Mean number of infusions from 12 days of extinction sessions (n = 8). G Numbers of infusions (n = 4), active lever presses (n = 4) and inactive lever presses (n = 4) during cocaine-induced reinstatement following injection of A-967079 or vehicle. Data are presented as mean ± standard error of the mean. * P < 0.05 and ** P < 0.01 versus control; Veh vehicle, A-9 A-967079.

Article Snippet: The membranes were then immunoblotted with primary antibodies against TRPA1 (rabbit, Novus, NB110-40763, 1:1000), DAT (rabbit, Millipore, AB2231, 1:3000), p-DAT (rabbit, Abcam, ab183486, 1:1000), TH (rabbit, Abcam, ab112, 1:5000), GluA1 (rabbit, Abcam, ab31232, 1:1000), GluA2 (rabbit, Abcam, ab20673, 1:1000) and ß-actin (mouse, Santa Cruz, Dallas, TX, USA, SC-47778, 1:2000) overnight at 4 °C.

Techniques: Injection, Control

Journal: Molecular Psychiatry

Article Title: The ion channel TRPA1 is a modulator of the cocaine reward circuit in the nucleus accumbens

doi: 10.1038/s41380-024-02623-4

Figure Lengend Snippet: A Timeline of cocaine and TRPA1 antagonist (HC-030031) application and current recording in FSCV. Representative dopamine voltammetry traces in the NAc core during FSCV. B Relative dopamine area under the curve (AUC) in the NAc core of cocaine responses following treatment with HC-030031 or vehicle [n = 7 (control), 5 (cocaine), 8 (HC-030031, cocaine), and 8 (HC-030031)]. C Dopamine peak in the NAc core of mice with cocaine responses following treatment with HC-030031 or vehicle [n = 7 (control), 5 (cocaine), 8 (HC-030031, cocaine), and 8 (HC-030031)]. D Relative ratio of human dopamine transporter (hDAT) uptake efficacy in hDAT and mouse TRPA1 (mTRPA1) stably transfected HEK293 cells following treatment with cocaine and TRPA1 agonist (AITC) (n = 3). E Relative ratio of hDAT uptake efficacy in hDAT and mTRPA1 stably transfected HEK293 cells following treatment with cocaine and TRPA1 antagonist (A-967079) (n = 3). F Timeline of cocaine and A-967079 injections, with the NAc dissected 1 day after the last dose. Representative images of Western blot from each protein assayed. G Relative DAT protein expression in the NAc of cocaine- and A-967079-injected groups (n = 9). H Relative ratio of p-DAT to total DAT protein expression in the NAc of cocaine- and A-967079-injected groups (n = 9). I Relative TH protein expression in the NAc of cocaine- and A-967079-injected groups (n = 9). Data are presented as mean ± standard error of the mean. * P < 0.05 and *** P < 0.001 versus control; # P < 0.05, ## P < 0.01, and ### P < 0.001 versus cocaine; sal saline, coc cocaine, Veh vehicle, HC HC-030031.

Article Snippet: The membranes were then immunoblotted with primary antibodies against TRPA1 (rabbit, Novus, NB110-40763, 1:1000), DAT (rabbit, Millipore, AB2231, 1:3000), p-DAT (rabbit, Abcam, ab183486, 1:1000), TH (rabbit, Abcam, ab112, 1:5000), GluA1 (rabbit, Abcam, ab31232, 1:1000), GluA2 (rabbit, Abcam, ab20673, 1:1000) and ß-actin (mouse, Santa Cruz, Dallas, TX, USA, SC-47778, 1:2000) overnight at 4 °C.

Techniques: Control, Stable Transfection, Transfection, Western Blot, Expressing, Injection, Saline

Journal: Molecular Psychiatry

Article Title: The ion channel TRPA1 is a modulator of the cocaine reward circuit in the nucleus accumbens

doi: 10.1038/s41380-024-02623-4

Figure Lengend Snippet: A Representative dot plots and histograms from flow cytometry present the surface expression of TRPA1 of D1R- and D2R- cells in the striatum (n = 3). Bar graphs presented as median defined as the normalized mean fluorescence intensity (MFI) of TRPA1 [n = 3 (WT mice for D1R-MSNs) and 3 (WT mice for D2R-MSNs)]. B Representative images of RNAscope showing the distributions of Trpa1 (red), Drd1 (yellow), Drd2 (green), and DAPI (blue) in the NAc. Scale bar: 20 μm. C Quantification of the fluorescence of TRPA1 mRNA puncta signals in the NAc subregions of saline- or cocaine-injected mice (n = 4). D Quantification of the percentage of D1R- or D2R-MSNs co-localized with TRPA1 signal (n = 4). E Quantification of the Drd1 - or Drd2 -positive cells (n = 4). Data are presented as mean ± standard error of the mean. sal Saline, coc cocaine.

Article Snippet: The membranes were then immunoblotted with primary antibodies against TRPA1 (rabbit, Novus, NB110-40763, 1:1000), DAT (rabbit, Millipore, AB2231, 1:3000), p-DAT (rabbit, Abcam, ab183486, 1:1000), TH (rabbit, Abcam, ab112, 1:5000), GluA1 (rabbit, Abcam, ab31232, 1:1000), GluA2 (rabbit, Abcam, ab20673, 1:1000) and ß-actin (mouse, Santa Cruz, Dallas, TX, USA, SC-47778, 1:2000) overnight at 4 °C.

Techniques: Flow Cytometry, Expressing, Fluorescence, RNAscope, Saline, Injection

Journal: Molecular Psychiatry

Article Title: The ion channel TRPA1 is a modulator of the cocaine reward circuit in the nucleus accumbens

doi: 10.1038/s41380-024-02623-4

Figure Lengend Snippet: A Representative confocal image of a coronal slice of the NAc obtained from AAV-DIO-hChR2-EYFP-injected D1-Cre mice. Scale bar: 500 μm. ac, anterior commissure. B Timeline of TRPA1 KD CPP tests with optogenetic activation of D1R MSNs. Schematic representation of the cocaine/blue laser-paired conditioning paradigm. C Representative heat map images of time spent in each CPP chamber during pre-test and post-test periods of the TRPA1 KD optogenetic cocaine (5 mg/kg, sub-threshold dose) CPP tests. D CPP score of each group in the TRPA1 KD optogenetic cocaine (5 mg/kg, sub-threshold dose) CPP tests (n = 7). E CPP score of each group in the TRPA1 KD optogenetic saline CPP tests [n = 5 (control), 6 (scrambled, stimulation), and 6 (TRPA1 KD, stimulation)]. F Representative raw traces of spontaneous excitatory post-synaptic current (sEPSC) recordings from D1R-MSNs in the NAc core. G Amplitude and frequency of sEPSCs in NAc D1R-MSNs of scrambled control and TRPA1 KD animals by saline and cocaine [n = 8 (D1R-MSNs, scrambled, saline), 6 (D1R-MSNs, scrambled, cocaine), 8 (D1R-MSNs, TRPA1 KD, saline), and 11 (D1R-MSNs, TRPA1 KD, cocaine)]. H Representative raw traces of spontaneous excitatory post-synaptic current (sEPSC) recordings from nonD1R-MSNs in the NAc core. I Amplitude and frequency of nonD1R-MSNs of scrambled control and TRPA1 KD animals by saline and cocaine [n = 12 (nonD1R-MSNs, scrambled, saline), 8 (nonD1R-MSNs, scrambled, cocaine), 8 (nonD1R-MSNs, TRPA1 KD, saline), and 9 (nonD1R-MSNs, TRPA1 KD, cocaine)]. Data are presented as mean ± standard error of the mean. * P < 0.05 and ** P < 0.01 versus control; ## P < 0.01 versus Scr light, sal saline, coc cocaine, Scr Scrambled control, TRPA1 KD or KD TRPA1 knockdown, w week.

Article Snippet: The membranes were then immunoblotted with primary antibodies against TRPA1 (rabbit, Novus, NB110-40763, 1:1000), DAT (rabbit, Millipore, AB2231, 1:3000), p-DAT (rabbit, Abcam, ab183486, 1:1000), TH (rabbit, Abcam, ab112, 1:5000), GluA1 (rabbit, Abcam, ab31232, 1:1000), GluA2 (rabbit, Abcam, ab20673, 1:1000) and ß-actin (mouse, Santa Cruz, Dallas, TX, USA, SC-47778, 1:2000) overnight at 4 °C.

Techniques: Injection, Activation Assay, Saline, Control, Knockdown