Additional file 1 of Chronic morphine regulates TRPM8 channels via MOR-PKCβ signaling
Bibliographic record
Abstract
Additional file 1: Figure S1. Morphine treatment increases the evoked neuronal activity. (a) Representative AP discharges evoked by 100, 200 and 300 pA current injections (1 s) in DRG neurons from control (black) and morphine-treated (red) animals (2.75 ± 0.6 Hz in control (black) vs 16.00 ± 3.5 Hz (red) evoked by the injection of 300 pA of current, n = 10 and 9, respectively. (b) Mean values of the data presented in (a). (c) Expression of TRPM8, MOR, TREK-1 and TRAAK mRNA in total DRGs harvested from control (black) and morphine treated mice (red). (d) Mean ratio (R1/baseline) of the amplitude response to menthol or cold stimulation in control (3.51 ± 0.23, light green; 3.29 ± 0.15 light blue), after overnight morphine treatment in DRG culture (3.47 ± 0.26, dark green; 3.6 ± 0.16, dark blue), or chronic morphine treatment in mice (3.18 ± 0.27, dark green; 3.28 ± 0.32, dark blue); n = 32, 30 and 30 for menthol and 30, 31 and 30 for cold. Figure S2. Morphine potentiates TRPM8 sensitivity to cold and menthol in transfected HEK cells. (a) Temperature-response curve measured by calcium imaging during cooling ramp on HEK cells transfected with TRPM8 and MOR from untreated (open symbols) versus morphine-treated cells (closed symbols). Sensitization of the temperature-response relationship is indicated by the shift towards warmer temperature; median temperature value 21.64 ± 0.65 °C, control vs 25.41 ± 0.58 °C, MS; n = 52 and 91, respectively). (b) Dose-response curve evoked by menthol, measured by calcium imaging on HEK cells transfected with TRPM8 and MOR from untreated (open symbols) versus morphine-treated cells (closed symbols). The shift towards lower concentration indicates that morphine potentiates the sensitivity of TRPM8 to menthol; EC50 = 11.44 ± 3.4 μM, control vs EC50 = 0.59 ± 0.02 μM, MS; n = 91 and 76, respectively. (c) R2/R1 amplitude ratio of two repeated applications of menthol in HEK cells transfected with TRPM8 and MOR and exposed to DAMGO (0.05 μM) overnight (63.84 ± 1.81%, n = 46 vs. 60.18 ± 1.47% control, n = 58). (d) R2/R1 amplitude ratio of two repeated applications of menthol in HEK cells transfected with TRPM8 and G protein-coupled receptors (PAR2, α1AR or α2AR, CB1) and exposed to their specific agonists (61.25 ± 7.13%, n = 25 for 1 μM 2fLI, 58.46 ± 5.45%, n = 32 for 1 μM clonidine, 58.25 ± 4.51%, n = 29 for 1 μM phenylephrine, and 67.42 ± 2.96%, n = 34 for 0.5 μM CBD) overnight. (e) Current density -Voltage curves in control or menthol stimulation in HEK cells transfected with TRPM8-WT (black) and SS1040-1041AA mutant (orange). Data are mean values, error bars indicate ±SEM. Statistical analysis was performed using unpaired t-test, One and Two-Way ANOVA followed by Bonferroni post hoc test (**p < 0.01). Figure S3. Acute treatment with morphine did not prevent TRPM8 desensitization in response to cold and menthol. (a) Representative whole cell current-clamp recording of the menthol-evoked current at + 100 mV and − 100 mV from a holding potential of 0 mV, in response to an acute (5 min) treatment with morphine (1 μM). (b) R2/R1 amplitude ratio of two repeated applications of menthol (green) or cold (blue) after an acute morphine treatment, in HEK cells transfected with TRPM8 and MOR.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.016 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.002 | 0.001 |
| Bibliometrics | 0.002 | 0.004 |
| Science and technology studies | 0.002 | 0.001 |
| Scholarly communication | 0.002 | 0.002 |
| Open science | 0.003 | 0.001 |
| Research integrity | 0.002 | 0.002 |
| Insufficient payload (model declined to judge) | 0.910 | 0.208 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".