Journal: bioRxiv

Article Title: Adenylyl cyclases combinatorially integrate opposing dopamine receptor signals

doi: 10.64898/2026.07.10.737756

Figure Lengend Snippet: (A, B). Single-cell dose-response curves for (A) Gαs-coupled DRD1 and (B) Gαi-coupled DRD2 in CHO-K1::cAMPinG1 cells transiently expressing HALO-tagged receptors. DRD2 was stimulated in the presence of 10 µM forskolin to elevate the baseline cAMP level. Cells are binned and colored by their measured surface receptor abundance (arbitrary flow-cytometry units; see color bars). Solid lines represent joint fits of the operational model (R 2 = 0.98 for DRD1; R 2 = 0.97 for DRD2) with binding affinity (Kd) locked to established IUPHAR values (fit values and 95% CIs are provided in ). Data represent per-bin medians, with error bars indicating ±1 SEM across independent replicate dates (n = 4 for DRD1, n = 5 for DRD2). The remaining subtypes (DRD3, DRD4, DRD5) are characterized in Fig. S3. (C, D) DRD1 dopamine dose-response under siRNA knockdown of Gαs at (C) 1 nM and (D) 33 nM siGNAS (ΔGs), overlaid on the wild-type Gαs response, binned by surface DRD1 abundance (colors as in A). Solid lines are the shared-parameter operational-model fit in which only the available transducer pool [Gs] varies per condition ; Gαs depletion right-shifts the curves along the dose axis without lowering the maximal response. (E). Schematic of the modified operational model. Ligand binding ([L]) yields a fractional receptor occupancy (ρ). This fraction is multiplied by the measured surface receptor abundance ([R]) to yield a composite signaling drive (D = [R] · [G] · ρ). The drive is processed through a saturable Hill-type transducer function (S) to compute the final integrated cAMP output (y). Full mathematical derivation is provided in the Supplementary Information. (F). Master-curve collapse across receptor abundances. Plotting the normalized transducer output (S) against the composite signaling drive (D) collapses the variable dose-response families onto a single, invariant master curve for each receptor subtype. This confirms that cellular cAMP responses are governed by the combined drive quantity rather than receptor density or ligand concentration independently.

Article Snippet: For experiments involving Gαi-coupled receptors (DRD2, DRD3, DRD4), adenylyl cyclase was activated with 10 μM forskolin (MedChemExpress, HY-15371) 5 minutes post-dopamine treatment.

Techniques: Single Cell, Expressing, Flow Cytometry, Binding Assay, Knockdown, Modification, Ligand Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: Adenylyl cyclases combinatorially integrate opposing dopamine receptor signals

doi: 10.64898/2026.07.10.737756

Figure Lengend Snippet: (A–C) Single-cell dose-response curves for (A) Gαo-coupled DRD3, (B) Gαi-coupled DRD4, and (C) Gαs-coupled DRD5 in CHO-K1::cAMPinG1 cells transiently expressing HALO-tagged receptors. The Gαi/o-coupled subtypes (DRD3, DRD4) were stimulated in the presence of 10 µM forskolin to elevate the baseline cAMP level; the Gαs-coupled DRD5 was measured from the unstimulated baseline. Cells are binned and colored by their measured surface receptor abundance (arbitrary flow-cytometry units; see color bar). Solid lines represent fits of the operational model with binding affinity (Kd) locked to established IUPHAR values (fit values and 95% CIs are provided in ). Data represent per-bin medians, with error bars indicating ±1 SEM across independent replicate dates (n = 2 for DRD3, n = 3 for DRD4, n = 4 for DRD5). (A) DRD3 produces no dopamine-evoked cAMP response, because its primary transducer Gαo is not endogenously expressed in CHO-K1 ; it therefore serves as a silent control. (B) DRD4 shows shallow, expression-dependent suppression of the forskolin-elevated baseline (R 2 = 0.91), consistent with DRD4’s canonical preference for Gαo over Gαi. (C) DRD5 shows expression-dependent cAMP stimulation (R 2 = 0.97). These subtypes are reported for completeness of the receptor panel and are not used in any quantitative downstream analysis; the identifiability of their fits is examined in .

Article Snippet: For experiments involving Gαi-coupled receptors (DRD2, DRD3, DRD4), adenylyl cyclase was activated with 10 μM forskolin (MedChemExpress, HY-15371) 5 minutes post-dopamine treatment.

Techniques: Single Cell, Expressing, Flow Cytometry, Binding Assay, Control

Journal: bioRxiv

Article Title: Adenylyl cyclases combinatorially integrate opposing dopamine receptor signals

doi: 10.64898/2026.07.10.737756

Figure Lengend Snippet: (A) Per-receptor scatter of dopamine binding measurements before and after the stringent curation that produced . Each colored point (orange = Gαs-coupled, blue = Gαi-coupled) is one of the 24 measurements retained in the main figure; each light-grey point is a measurement excluded by one or more curation criteria (see Methods and panel B of this figure). All rows that map to one of the five dopamine receptors are included, with descriptions like “D2L”, “D2A”, and “D2long” mapped to DRD2 (long isoform) by an extended description-matching rule applied in this SI figure but not in the main-text curation, in order to expose the full per-receptor scatter that the database contains. n (kept/total): DRD1 = 6/39, DRD5 = 1/2, DRD2 = 5/116, DRD3 = 10/50, DRD4 = 2/21. Solid colored bars, stringent median (matches ); dashed black ticks, unfiltered median across all rows shown. The two medians lie within 0.1 log unit (≈25%) of each other for every receptor that has more than one entry, indicating that the stringent filter narrows the database but does not systematically bias the central tendency. (B) Counts of GtoPdb rows excluded by each curation criterion. Each row is assigned to the first applicable category in the order: functional assay (Assay Type = “F” in GtoPdb; signaling readouts such as cAMP, β-arrestin recruitment, or [ S]GTPγS binding rather than direct ligand binding), species not specified (assay description does not explicitly state the species), non-human species (description names rat, mouse, bovine, dog, or porcine receptor), other / multiple (combinations of the above or unparseable values, value reported as a range, or “unknown origin” phrase in description), unrecognized assay type (Assay Type column does not contain the standard B/F values).

Article Snippet: For experiments involving Gαi-coupled receptors (DRD2, DRD3, DRD4), adenylyl cyclase was activated with 10 μM forskolin (MedChemExpress, HY-15371) 5 minutes post-dopamine treatment.

Techniques: Binding Assay, Produced, Functional Assay, Ligand Binding Assay

Journal: Cell Reports Medicine

Article Title: Role of dopamine in the development of impaired counterregulation and impaired awareness of hypoglycemia

doi: 10.1016/j.xcrm.2026.102710

Figure Lengend Snippet: Sympathoadrenal response and dopaminergic action with agonist bromocriptine (A–C) Recurrent BROMO administration in i.c.v. reduced food intake and BG response. (A) Schematic diagram of recurrent aCSF ( n = 7) or recurrent BROMO ( n = 7, 2 μg) for 3 days into i.c.v. (B) BG on day 4 following singular injection of INS (15 U/kg NPH) Groups were compared via two-way ANOVA. (C) Cumulative food intake after INS injection (∗ p < 0.005 RBROMO i.c.v. vs. RCSF i.c.v. + INS). Groups were compared via unpaired t test. Each bar represents the mean + SEM. (D–H) Recurrent BROMO administration in i.c.v. and effects on CRR. (D) Schematic diagram of recurrent aCSF ( n = 7) or recurrent BROMO ( n = 7, 2 μg) into i.c.v.. (E) BG during hyperinsulinemic-hypoglycemic clamp where BG is held at 40–45 mg/dL for 90 min. Groups were compared via two-way ANOVA. (F) Ginf. Rate during clamp (∗∗∗∗ p < 0.0001 vs. RBROMO). Groups were compared via unpaired t test. (G) Peak epinephrine concentrations at basal and hypoglycemia (∗ p < 0.005 RCSF vs. RBROMO). Groups were compared via two-way ANOVA. (H) Peak glucagon concentrations at basal and hypoglycemia (∗ p < 0.005 RCSF vs. RBROMO). Groups were compared via two-way ANOVA. Each bar represents the mean + SEM. (I) Dopaminergic gene expression in VTA. Relative gene product expression of COMT, DDC, DRD2, SLC6A3 in the ventral tegmental area following 3-day treatment with RS ( n = 6), RH ( n = 7), or RH + MET ( n = 6) (∗ p < 0.04 RS vs. RH + MET; # p < 0.04 RH vs. RH + MET). Groups were compared via one-way ANOVA. Each bar represents the mean + SEM. (J–O) Recurrent BROMO infusion in VTA effects on CRR. (J) Schematic diagram of recurrent aCSF ( n = 7) or recurrent BROMO (n = 7–9, 5 μg/day; infusion rate 0.1 μL/min). (K) BG concentration during hyperinsulinemic-hypoglycemic clamp, which held BG at 40–50 mg/dL for 90 min. Groups were compared via two-way ANOVA. (L) Ginf. rate during clamp (∗∗∗∗ p < 0.001 RCSF vs. RBROMO). Groups were compared via two-way ANOVA. (M) Peak epinephrine concentrations at basal and hypoglycemia (∗ p < 0.05 BROMO vs. SAL). Groups were analyzed via unpaired t test. (N) Relative gene expression of DRD2 in the ventral tegmental area following a 3-day treatment with either aCSF or BROMO directly into the VTA (∗ p < 0.05 aCSF vs. BROMO). Groups were compared via unpaired t test. (O) Relative gene expression of SLC6A3 in the ventral tegmental area following a 3-day treatment with either aCSF or BROMO directly into the VTA ( p = 0.21). Groups were compared via unpaired t test. Each bar represents the mean + SEM.

Article Snippet: In brief, total RNA was extracted and purified from brain regions using miRNeasy Mini Kit (Qiagen, CA) which was reverse transcribed and then amplified by real-time PCR using Taqman gene assays (Thermo Fisher) for dopamine receptor D2 ( Drd2 ; assay ID: Rn00561126_m1), dopamine transporter or solute carrier family 6, member 3 ( Dat or Slc6a3 ; assay ID: Rn00562224_m1), dopa decarboxylase ( Ddc : assay ID: Rn01401189_m1), catechol- O -methyltransferase ( Comt ; assay ID: Rn00561037_m1) and the housekeeping gene beta-actin ( Actb ; assay ID: Rn00667869_m1).

Techniques: Injection, Gene Expression, Expressing, Concentration Assay

Journal: iScience

Article Title: The role of dopamine-sensitive motor cortical circuits in the development and execution of skilled forelimb movements

doi: 10.1016/j.isci.2026.114983

Figure Lengend Snippet: Head-fixed mice learn to perform skilled forelimb movements to obtain a reward (A) Representation of injecting AAV9-hSyn-GRAB DA2h and AAV1-Syn-Flex-NES-jRGECO1a into M1. Coronal sections obtained from D1Cre and D2Cre mice showing expression of GRAB DA2h (green) and jRGECO1a (red) in the M1 forelimb area (brain slices are 0.5 mm anterior to the bregma). Scale bars, 500 μm. Magnification: 20×. See also for the histological map of GRAB DA2h and jRGECO1a expression and fiber tip placements. (B) Representation of the task in which head-fixed mice perform skilled forelimb movements with a joystick to obtain a delayed reward, and a fiber photometry setup for simultaneous recording of DA dynamics and population level Ca 2+ activity in the M1 forelimb area. See also for representative traces of neuronal Ca 2+ and DA signals acquired during training. (C) Increase in the number of rewarded joystick movements in D1Cre mice during training. (D) Movement kinematic parameters obtained from the early and late sessions in D1Cre mice showing that the average movement amplitude and velocity increased between training phases. (Left to right) Distribution of movement amplitudes, movement amplitude, and joystick velocity at the time of threshold crossing. (E) Individual movement trajectories of an example D1Cre mouse aligned to the initial joystick position (black dot), and preferred movement direction in the group of D1Cre animals (based on all extracted trajectories). (F) Change in movement similarity in D1Cre mice (based on the average distance between pairwise trajectories). (Left to right) Similarity between the early and late sessions; similarity within the late session. (G–J) Same as (C)–(F) for D2Cre mice. In (C)–(J), data were obtained from 14 D1Cre (10 Cre+ and 4 Cre-) and 13 D2Cre (10 Cre+ and 3 Cre-) animals. Data are represented as mean ± SEM. Paired t test; ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: D2Cre: B6.FVB(Cg)-Tg(Drd2-cre)ER44Gsat/Mmucd , MMRRC: UCD , RRID: MMRRC_032108-UCD.

Techniques: Expressing, Activity Assay

Journal: iScience

Article Title: The role of dopamine-sensitive motor cortical circuits in the development and execution of skilled forelimb movements

doi: 10.1016/j.isci.2026.114983

Figure Lengend Snippet: DA dynamics in the M1 encode execution of skilled forelimb movement and reward consumption (A) Peri-event plots of the average normalized DA signal in D1Cre mice aligned to threshold cross (top) and binned data analyzed at 1-s intervals (bottom). (Left to right) Data are presented as within-session (early or late) comparisons with respect to the baseline (BL) and between-session comparisons of movement- and reward-related periods (early vs. late). (B) Peri-event plots of the average normalized Ca 2+ signal in D1Cre mice aligned to threshold cross (top) and binned data analyzed at 1-s intervals (bottom). (Left to right) Data are presented as within-session (early or late) comparisons with respect to the BL and between-session comparisons of movement- and reward-related periods (early vs. late). (C) Peri-event plots of the average lick rate (licks/s) in D1Cre mice aligned to threshold cross (top) and binned data analyzed at 1-s intervals (bottom). (Left to right) Data are presented as within-session (early or late) comparisons with respect to the BL and between-session comparisons of movement- and reward-related periods (early vs. late). (D–F) Same as (A)–(C) for D2Cre mice. In (A)–(F), data were obtained from 14 D1Cre (10 Cre+ and 4 Cre-) and 13 D2Cre (10 Cre+ and 3 Cre-) animals. Data are represented as mean ± SEM. Repeated-measure one-way ANOVA; multiple comparisons: BL mean vs. every other mean, movement (0–1 s) mean vs. reward (1–2 s) mean; Bonferroni’s post hoc test; ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; or paired t test; ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. Dashed lines indicate joystick movement (threshold crossing) and reward delivery. See also for comparisons between D1Cre and D2Cre mice and cross-correlations between DA and Ca 2+ signals.

Article Snippet: D2Cre: B6.FVB(Cg)-Tg(Drd2-cre)ER44Gsat/Mmucd , MMRRC: UCD , RRID: MMRRC_032108-UCD.

Techniques:

Journal: iScience

Article Title: The role of dopamine-sensitive motor cortical circuits in the development and execution of skilled forelimb movements

doi: 10.1016/j.isci.2026.114983

Figure Lengend Snippet: The neural response in the M1 reflects the movement vigor (A and B) Movement kinematic parameters obtained from the low- and high-amplitude trials during regular threshold session in D1Cre and D2Cre mice showing the difference in response vigor. (Left to right) Distribution of movement amplitudes, movement amplitude, and joystick velocity at the time of threshold crossing. (C) Peri-event plots of the average normalized DA signal in D1Cre mice aligned to threshold cross (top) and comparisons of movement- and reward-related periods (analyzed at 1-s intervals) between low- and high-amplitude trials (bottom). (D) Peri-event plots of the average normalized Ca 2+ signal in D1Cre mice aligned to threshold cross (top) and comparisons of movement- and reward-related periods (analyzed at 1-s intervals) between low- and high-amplitude trials (bottom). (E) Peri-event plots of the average lick rate (licks/s) in D1Cre mice aligned to threshold cross (top) and comparisons of movement- and reward-related periods (analyzed at 1-s intervals) between low and high amplitude trials (bottom). (F–H) Same as (C)–(E) for D2Cre mice. In (A)–(H), data were obtained from 14 D1Cre (10 Cre+ and 4 Cre-) and 13 D2Cre (10 Cre+ and 3 Cre-) animals. Data are represented as mean ± SEM. Paired t test; ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. Dashed lines indicate joystick movement (threshold crossing) and reward delivery. See also for comparisons between D1Cre and D2Cre mice.

Article Snippet: D2Cre: B6.FVB(Cg)-Tg(Drd2-cre)ER44Gsat/Mmucd , MMRRC: UCD , RRID: MMRRC_032108-UCD.

Techniques:

Journal: iScience

Article Title: The role of dopamine-sensitive motor cortical circuits in the development and execution of skilled forelimb movements

doi: 10.1016/j.isci.2026.114983

Figure Lengend Snippet: The neural response in the M1 reflects the movement vigor during the increased threshold session (A and B) Movement kinematic parameters obtained from the low- and high-amplitude trials during the increased threshold session in D1Cre and D2Cre mice. (Left to right) Distribution of movement amplitudes, movement amplitude, and joystick velocity at the time of threshold crossing. (C) Peri-event plots of the average normalized DA signal in D1Cre mice aligned to threshold cross (top) and comparisons of movement- and reward-related periods (analyzed at 1-s intervals) between low- and high-amplitude trials (bottom). (D) Peri-event plots of the average normalized Ca 2+ signaling D1Cre mice aligned to threshold cross (top) and comparisons of movement- and reward-related periods (analyzed at 1-s intervals) between low- and high-amplitude trials (bottom). (E) Peri-event plots of the average lick rate (licks/s) in D1Cre mice aligned to threshold cross (top) and comparisons of movement- and reward-related periods (analyzed at 1-s intervals) between low- and high-amplitude trials (bottom). (F–H) Same as (C)–(E) for D2Cre mice. In (A)–(H) Data were obtained from 14 D1Cre (10 Cre+ and 4 Cre-), and 12 D2Cre (10 Cre+ and 2 Cre-) animals. Data are represented as mean ± SEM. Paired t test; ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. Dashed lines indicate joystick movement (threshold crossing) and reward delivery. See also for comparisons between D1Cre and D2Cre mice.

Article Snippet: D2Cre: B6.FVB(Cg)-Tg(Drd2-cre)ER44Gsat/Mmucd , MMRRC: UCD , RRID: MMRRC_032108-UCD.

Techniques:

Journal: iScience

Article Title: The role of dopamine-sensitive motor cortical circuits in the development and execution of skilled forelimb movements

doi: 10.1016/j.isci.2026.114983

Figure Lengend Snippet: DA in the M1 signals actual reward availability during skilled performance (A) Peri-event plots of the average normalized DA signal in D1Cre mice aligned to threshold cross (top) and binned data analyzed at 1-s intervals (bottom). (Left to right) Data are presented as within-session (delay or omission) comparisons with respect to the baseline (BL). (B) Peri-event plots of the average normalized Ca 2+ signal in D1Cre mice aligned to threshold cross (top) and binned data analyzed at 1-s intervals (bottom). (Left to right) Data are presented as within-session (delay or omission) comparisons with respect to the BL. (C) Peri-event plots of the average lick rate (licks/s) in D1Cre mice aligned to threshold cross (top) and binned data analyzed at 1-s intervals (bottom). (Left to right) Data are presented as within-session (delay or omission) comparisons with respect to the BL. (D–F) Same as (A)–(C) for D2Cre mice. In (A)–(F), data for “delayed reward” analysis were obtained from 14 D1Cre (10 Cre+ and 4 Cre-) and 13 D2Cre (10 Cre+ and 3 Cre-) animals and for “omission” analysis from 9 D1Cre (7 Cre+ and 2 Cre-) and 8 D2Cre (8 Cre+) animals. Data are represented as mean ± SEM. Repeated-measure one-way ANOVA; multiple comparisons: BL mean vs. every other mean, movement (0–1 s) mean vs. expected reward (1–2 s) mean, expected reward (1–2 s) mean vs. actual reward (3–4 s) mean; Bonferroni’s post hoc test; ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05 See also for comparisons between D1Cre and D2Cre mice.

Article Snippet: D2Cre: B6.FVB(Cg)-Tg(Drd2-cre)ER44Gsat/Mmucd , MMRRC: UCD , RRID: MMRRC_032108-UCD.

Techniques:

Journal: iScience

Article Title: The role of dopamine-sensitive motor cortical circuits in the development and execution of skilled forelimb movements

doi: 10.1016/j.isci.2026.114983

Figure Lengend Snippet: A subset of D1+ and D2+ neurons of the M1 contacts long-range targets (A) Representation of the four major areas of rAAV2-Retro-EYFP and Fluoro-Green injections: primary motor cortex (M1), dorsolateral striatum (DLS), thalamus (Thal), and pontine nucleus (PN). See also for representative histology of the injection sites. (B and C) Coronal sections obtained from D1-tdTomato and D2Cre::Ai14 (tdTomato) mice showing laminar distribution of tdTomato (red) expressing D1+ or D2+ neurons and EYFP or FGr (green) expressing retrogradely labeled projection neurons in the M1 forelimb area (brain slices are 0.5 mm anterior to the bregma). Scale bars, 50 μm. See also for analysis of laminar distributions. (D) Cell density of D1+ neurons plotted as a function of distance from pia (where pia = 0 and white matter = 1; therefore, <0.5 indicates cells clustered in the upper layers, while >0.5 indicates cells clustered in the deep layers). (E) Colocalization of D1+ neurons with different populations of projection neurons. Percentage of D1+ neurons indicates colabeled cells across all layers that are also EYFP+ (% [D1+ EYFP+] / D1+) or FGr+ (% [D1+ FGr+] / D1+). Percentage of EYFP+ neurons indicates cells that are also D1+ (% [D1+ EYFP+] / EYFP+), and percentage of FGr+ neurons indicates cells that are D1+ (% [D1+ FGr+] / FGr+). (F) Comparison of labeling specificity of rAAV2-retro and FGr tracers measured by the number of cells labeled by each tracer. (G–I) Same as (D)–(F) for D2Cre mice. In (D)–(I), data were obtained from 21 D1-tdTomato and 19 D2Cre::Ai14 animals (2–3 mice per injection site, 4 slices per animal, slices were collected from −0.10 mm posterior to 1.00 mm anterior to the bregma). Data are represented as mean ± SEM. Paired t test; ∗∗∗ p < 0.001. Two-way ANOVA; multiple comparisons: EYFP mean vs. FGr mean; Bonferroni’s post hoc test; ∗∗∗ p < 0.001, ∗∗ p < 0.01. (B–I) cM1 (primary motor cortex injection, M1 forelimb area contralateral to the injection site); cDLS (DLS injection, M1 forelimb area contralateral to the injection site); iDLS (dorsolateral striatum injection, M1 forelimb area ipsilateral to the injection site); iThal (thalmus injection, M1 forelimb area ipsilateral to the injection site); iPN (pontine nucleus injection, M1 forelimb area ipsilateral to the injection site).

Article Snippet: D2Cre: B6.FVB(Cg)-Tg(Drd2-cre)ER44Gsat/Mmucd , MMRRC: UCD , RRID: MMRRC_032108-UCD.

Techniques: Injection, Expressing, Labeling, Comparison