Figures and data
In vivo fiber photometry in the mesolimbic dopamine system captures phasic dopamine responses to primary taste stimuli.
(A) Representative images of GRABDA_2h and Cre-dependent GCamp6f expression in the NAc and VTA, respectively. Top Row: Dopamine release recordings from the lateral subregion of the NAc shell are confirmed via viral expression of dopamine-sensor, GRABDA2h (green), probed against DAPI (blue) to visualize sensor location targeted to the lateral shell of the NAc (NAcLS), which borders the NAc core (NAcC). Bottom row: Dopamine cellular activity was recorded in TH Cre+ rats in the VTA (paranigral nucleus (PN), the parabrachial pigmented area; (PBP)). TH+ (red) colocalized with intracellular calcium-sensor, GCaMP6f (green) to demonstrate isolation of the VTADA population. (B) Real-time dopamine release in the NAc across processing steps from a representative rat receiving 5s 0.3M sucrose intraoral infusions (light green bars). Fluorescence excited by the 465nm (Ca2+- and GRABDA2h-dependent, green), and 405nm LED (Ca2+-independent, purple) was captured. 465 and 405nm traces were scaled and subtracted to remove motion artifacts and photobleaching (blue). Fluorescence was then normalized to the whole recording session and represented as a z-score (black). (C) Top: heat maps for NAc dopamine release for 30 trials/session in which 200µL of 0.3M sucrose (green) or 0.001M quinine (orange) was delivered over 5s. Bottom: Average dopamine release aligned to the onset of intraoral delivery. Dotted line represents onset of infusion and gray shading reflects infusion duration and time window for statistical analysis. Bottom inset: Average z-score during infusion period. (D) Relative frequency histogram of dopamine responses (mean z-score) to sucrose and quinine from every trial reported. (E) ROC of D determined a discriminable difference between dopamine responses. Dark lines and shading in C are represented as means; *p<0.05 in paired t-test.
Aversive taste stimuli are linked to increased movement of nose and forepaws.
(A) Representative image of nose, forepaws, tail base, and chamber legs tracking of a rat in a cylindrical chamber from a below chamber perspective. Positional coordinates of selected features were obtained using model created via DeepLabCut, an open-source deep-learning pose estimation program. Custom MATLAB scripts were used to analyze movement from the positional data. (B) Representative movement of nose and forepaws tracked by a DeepLabCut model during 5s pre-infusion, sucrose or quinine infusion, and post-infusion periods. (C&D) Behavioral reactivity was measured as the average change in nose movement or forepaw movement from baseline to infusion period. Intraoral infusion of quinine produces a greater behavioral reactivity. (E&F) Relationship between average change in behavioral reactivity and mean z-score of NAc dopamine during infusion averaged by session. Data in C & D are represented as means; *p<0.05 in paired t-tests. Line in E & F denote the linear relationship between parameters with dotted lines as 95% confidence intervals. P-value of linear regressions indicate slope’s deviation from zero.
Pairing of LiCl-induced malaise to sucrose suppresses phasic dopamine responses to intraoral sucrose delivery.
(A) Schematic of Single-pairing CTA timeline. Subjects were first habituated to 30 brief intraoral infusions (200µL/trial) of water at varying intertrial intervals (35-55s) daily for two days. On Conditioning Day (CD), rats received intraoral infusions of 0.3M sucrose and were then injected i.p. with saline (Unpaired) or 0.15M LiCl (Paired). Rats received the counterbalanced injection in the home cage the next day and were untreated the following day. On Test Day (TD), rats received intraoral infusions (parameters identical to CD). (B) NAc dopamine across trials and sessions before and after intraoral sucrose delivery onset. Top: NAc dopamine release (in z-score) across 30 trials/session during both CD and TD. Bottom: Average traces of dopamine release aligned to the onset of intraoral delivery of sucrose on CD and TD. Insets: Mean z-score quantified during 5s intraoral delivery of sucrose. (C-D) Relative frequency histogram of dopamine release responses to sucrose on CD and TD for every trial reported as mean z-score for both Unpaired and Paired subjects. (E) ROC of relative frequency distributions of mean z-score acquired from each trial of on CD and TD between treatment groups. (F) Plotted area under the curve (AUCROC) values of E. (G-K) Recordings of VTADA activity from Unpaired and Paired rats reported with same conventions as B-F. Data in B & G are represented as means; *p<0.05, ** p<0.01, paired t-test.
Suppressed dopamine responses correlate with enhanced behavioral reactivity to intraoral sucrose delivery after CTA formation.
(A&B) Behavioral reactivity was quantified as the change in movement from baseline to infusion period. In Unpaired subjects, the average behavioral reactivity of nose (left) or forepaw (right) movement did not change from CD to TD. In Paired subjects, behavioral reactivity increased from CD to TD. (C) Relationship between behavioral reactivity of nose movement and mean z-score of NAc dopamine during infusion averaged by session. (D) Relationship between average change in behavioral reactivity of forepaw movement and mean z-score of VTADA activity responses during infusion averaged by session. Data in A, B, E & G are represented as means; *p<0.05, **p<0.01; ***p<0.005, Two-Way RM ANOVA with Uncorrected Fisher’s LSD post-hoc. Line in C, D, F & H denote the linear relationship between parameters with dotted lines as 95% confidence intervals. P-value of linear regressions indicate slope’s deviation from zero.
Sucrose exposure under extinction conditions ameliorates CTA’s suppression of phasic dopamine responses to intraoral sucrose.
(A) Schematic of the CTA paradigm consisting of a single-pairing of LiCl or saline pairing to sucrose followed by five consecutive sessions intraoral sucrose not subject to additional US (malaise) exposure. CTA training as in Figure 3 was conducted followed by five Extinction sessions (E1-E5), in which rats received intraoral infusions (parameters identical to TD) without any additional injections. (B) VTADA across trials and sessions before and after intraoral sucrose delivery onset. Top: VTADA activity (in z-score) across 30 trials of the CD and E1-E5 sessions. Bottom: Average traces of VTADA activity aligned to the onset of intraoral delivery of sucrose. (C) Mean z-score during 5s intraoral delivery of sucrose. (D) Relative frequency histogram of VTADA responses to sucrose on all test days for every trial reported as mean z-score for both Unpaired and Paired subjects. (E) ROC of relative frequency distributions of mean z-score acquired from each trial of on all test sessions between treatment groups. (F) Plotted area under the curve (AUCROC) values of E. Data in C are represented as means; ** p<0.01, One-Way RM ANOVA with Dunnett’s multiple comparisons test post hoc.
Suppressed dopamine responses to sucrose predict conditioned taste avoidance.
(A) Each Extinction session was followed by a two-hour Two-Bottle Preference test with access to both sucrose and water. Sucrose preference scores were calculated as the percent of sucrose solution consumed from the sum of sucrose and water consumed. Paired (red, closed circles) rats showed decreased sucrose preference on E1 and E2 relative to Unpaired rats (blue, open circles). (B) Average VTADA responses during intraoral sucrose delivery were positively correlated to sucrose preference scores calculated from the Two-Bottle Preference test. Data in A are represented as means; ****p<0.001, Two-Way RM ANOVA with Šidák multiple comparisons test post hoc. Line in B denotes the linear relationship between parameters with dotted lines as 95% confidence intervals. P-value of linear regression indicates slope’s deviation from zero.
Delayed testing after sucrose pairing to LiCl-induced malaise suppresses dopamine response to sucrose.
(A) Schematic of the Delayed-test CTA paradigm. CTA training as in Figure 3 was conducted. To match the timeline of Single-pairing CTA with Extinction, rats had five additional untreated days followed by Test Day (TD), which corresponded to the delay from CD to E5. On TD, rats received intraoral infusions (parameters identical to E1). (B) VTADA across trials and sessions before and after intraoral sucrose delivery onset. Top: VTADA activity (in z-score) across 30 trials during CD and TD sessions. Bottom: Average traces of VTADA activity aligned to the onset of intraoral delivery of sucrose. Inset: Average z-score quantified during 5s intraoral delivery of sucrose. (C-D) Relative frequency histogram of dopamine release responses to sucrose on CD and TD for every trial reported as mean z-score for both Unpaired and Paired subjects. (E) ROC of relative frequency distributions of mean z-score acquired from each trial of on CD and TD between treatment groups. (F) Plotted area under the curve (AUCROC) values of E. Data in B are represented as means; ***p<0.005, paired t-test.
Phasic dopamine responses to intraoral sucrose scale to the strength of the CTA.
(A) Schematic of the Repeated-pairing CTA paradigm. The three-day CTA conditioning process (as in Figure 3) was repeated three times (C1-C3). Following conditioning, subjects received eight daily Extinction sessions (E1-E8) with intraoral infusions of sucrose followed by no US (malaise) exposure. After the behavioral sessions, rats had access to sucrose and water consumption for two-hours (Two-Bottle Preference test). (B) VTADA across trials and sessions before and after intraoral sucrose delivery onset. Top: Heat maps for VTADA activity (in z-score) for 30 trials in each session during the three conditioning sessions (C1-C3). Bottom: Associated average traces of VTADA activity aligned to the onset of intraoral delivery of sucrose. (C) Conventions as in B but for all eight non-reinforced sucrose sessions (E1-E8). (D) Average z-score quantified during 5s intraoral delivery of sucrose for all conditioning and subsequent test sessions. (E) Relative frequency histogram of VTADA responses to sucrose on all test days for every trial reported as mean z-score for both Unpaired and Paired subjects. (F) ROC of relative frequency distributions of mean z-score acquired from each trial of on all conditioning (left) and extinction (right) days between treatment groups. (G) Plotted area under the curve (AUCROC) values of F. Data in D are represented as means; * p<0.05, One-Way RM ANOVA with Dunnett’s multiple comparisons test post hoc.
Intraoral sucrose driven dopamine responses are negatively correlated to behavioral reactivity and predict sucrose preference.
(A) Head movement to intraoral sucrose averaged across trials. Top: Unpaired rats showed comparable average change in head movement behavioral reactivity across testing sessions. Bottom: Paired subjects showed an increase in average head movement responses to intraoral sucrose from C1 to C2-E3. (B) Relationship between average head movement behavioral reactivity and VTADA responses (in mean z-score) during infusion averaged by session. (C) After each E1-E8 session, rats were administered the Two-Bottle Preference test. Paired (red, closed circles) rats showed decreased sucrose preference on E1-E5 relative to Unpaired rats (blue, open circles). (D) Average VTADA responses during intraoral sucrose delivery were positively correlated to sucrose preference scores calculated from the Two-Bottle Preference test. Data in A are represented as means; *p<0.05, **p<0.01, One-Way RM ANOVA with Dunnett’s multiple comparisons test post hoc. Data in C are represented as means; *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, Two-Way RM ANOVA with Šidák multiple comparisons test post hoc. Line in B & D denotes the linear relationship between parameters with dotted lines as 95% confidence intervals. P-value of linear regression indicates slope’s deviation from zero.
NAc lateral shell recording placements from Primary Taste experiment.
The fiber optic implant locations in the NAc lateral shell were verified via post-experimental histological reconstruction. Measurement values denote anterior-posterior coronal plane in relation to Bregma. Reconstructed images do not represent the specific hemisphere where fiber optics were implanted.
NAc lateral shell and VTA recording placements from Single Paring CTA experiment.
The fiber optic implant locations in the NAc lateral shell and VTA were verified via post-experimental histological reconstruction. Measurement values denote anterior-posterior coronal plane in relation to Bregma. Treatment group is indicated by outline color: Unpaired (blue outline) and Paired (red outline). Reconstructed images do not represent the specific hemisphere where fiber optics were implanted.
VTA recording placements from Delayed Test CTA experiment.
The fiber optic implant locations in the VTA were verified via post-experimental histological reconstruction. Measurement values denote anterior-posterior coronal plane in relation to Bregma. Treatment group is indicated by outline color: Unpaired (blue outline) and Paired (red outline). Reconstructed images do not represent the specific hemisphere where fiber optics were implanted.
VTA recording placements from Single Paring CTA with Extinction experiment.
The fiber optic implant locations in the VTA were verified via post-experimental histological reconstruction. Measurement values denote anterior-posterior coronal plane in relation to Bregma. Treatment group is indicated by outline color: Unpaired (blue outline) and Paired (red outline). Reconstructed images do not represent the specific hemisphere where fiber optics were implanted.
VTA recording placements from Repeated Paring CTA with Extinction experiment.
The fiber optic implant locations in the VTA were verified via post-experimental histological reconstruction. Measurement values denote anterior-posterior coronal plane in relation to Bregma. Treatment group is indicated by outline color: Unpaired (blue outline) and Paired (red outline). Reconstructed images do not represent the specific hemisphere where fiber optics were implanted.
