Altered Dopamine Signaling in Extinction-Deficient Mice

Animals and husbandry

All experiments were carried out in compliance with national and international guidelines for animal welfare and appropriate guides for Care and Use of Laboratory Animals and approved by appropriate Animal Care and Use Committees. Male 129S1/SvImJ (S1) mice, C57BL/6J (BL6) mice, as well as DAT-Cre mice [B6.SJL-Slc6a3tm1.1(cre)Bkmn/J; JAX Stock No. 006660] on a C57BL/6J background (BL6-DAT-Cre) or backcrossed to a 129S1/SvImJ background (S1-DAT-Cre) for at least nine generations (11–16 weeks at the start of experiments or surgery) were bred in-house or purchased from JAX (The Jackson Laboratory) and habituated in the animal facility for at least 1 week. Animals were housed at constant temperature (22 ± 2°C) and humidity (40–60%) under a 12 h light/dark cycle (lights on/off at 07:00/19:00) with ad libitum access to food and water. All mice were held in groups (maximum of five per cage) and single-housed following surgery. Exact n numbers for each experiment are given in the corresponding figure legends.

Surgical procedures for fiber photometry

Surgical procedures were performed under isoflurane anesthesia (Forane, USP, Baxter) provided at a constant rate (2.0–5.0% isoflurane, oxygen flow rate 1 L/min; Anesthesia System, E-Z Systems), while the animal was head-fixed in a stereotaxic frame (Kopf Model 942, David Kopf Instruments). The animal was placed on a heating pad, and the breathing rate was constantly monitored. The eyes of the animal were covered with eye lubricant ointment (LubriFresh PM, Major Pharmaceuticals) to prevent desiccation. As analgesic, ketoprofen was subcutaneously injected (5 mg/kg).

Using a dental precision driller, one craniotomy was made at the following coordinates (from the bregma): AP −3.20 mm and ML ±0.40 mm. An AAV construct (Addgene) for the Cre-dependent expression of GCaMP6f (AAV5-pAAV-CAG-FLEX-GCaMP6f-WPRE-SV40, Addgene_100835; 1.3 × 1,013 vp/ml) was delivered unilaterally (0.2 µl) into the VTA at DV −4.6 mm. This was done via a 0.5 µl microliter syringe equipped with a 32 G needle (Neuros 7000.5, Hamilton) and at a rate of 20 nl/min [UMP3 with Micro4 Controller, World Precision Instruments (WPI)]. After the injection, the needle was left in place for another 5–10 min to ensure diffusion. Then, a single fiber (0.66 NA, ø 400 μm, 6 mm length; MFC_400/430-0.66_6mm_SM3_FLT, Doric Lenses) was implanted unilaterally directly above the VTA (coordinates relative to the bregma: AP −3.20, ML 0.40, DV −4.6, 0° angle). Two additional craniotomies were made in which jeweler's screws were mounted to build a fundament using dental cement (OrthoJet, Lang Dental Manufacturing) and ensure fixation of the implants to the skull. To reduce postoperative pain, ketoprofen was administered subcutaneously directly after surgery and for the following 3 d (5 mg/kg). After surgery, recovery of the animals and AAV expression were allowed for 4–5 weeks, before the start of behavioral experiments.

Surgical procedures for optogenetics

Surgical procedures were performed under isoflurane anesthesia (Vetflurane, Virbac) provided at a constant rate (1.9–2.5% isoflurane, air flow rate 190–220 ml/min; Univentor 410 Anesthesia Unit, AgnThos), while the animal was head-fixed in a stereotaxic frame (Kopf Model 962, David Kopf Instruments). The animal was placed on a heating pad, and the breathing rate was constantly monitored. The eyes of the animal were covered with eye cream (Bepanthen Augen- und Nasensalbe, Bayer) to prevent desiccation. As an analgesic, meloxicam was subcutaneously injected (0.5 mg/kg; Metacam, B. Braun Melsungen). A 1% lidocaine solution (Xylocaine 2% Ampullen, Aspen Pharmacare) was locally applied to the scalp before a rostrocaudal incision was made.

Using a dental precision driller, two (VTA fiber implantations) or four [infralimbic cortex (IL) fiber implantations] craniotomies were made at the following coordinates (from the bregma): AP −3.20 mm, ML ±0.5 mm (VTA) and AP +1.40 mm, ML ±1.3 mm (IL). AAV constructs (UNC Vector Core) for the Cre-dependent expression of channelrhodopsin 2 (ChR2) and mCherry (AAV5-EF1a-DIO-ChR2(H134R)-mCherry, Addgene_20297; 3.4 × 1,012 vp/ml) and mCherry only (AAV5-EF1a-DIO-mCherry, Addgene_114471; 5.4 × 1,012 vp/ml) were delivered bilaterally (0.35 µl/hemisphere) into the VTA at DV −4.6 mm. This was done via a 0.5 µl microliter syringe equipped with a 32 G needle (Neuros 7000.5, Hamilton) and at a rate of 35 nl/min (UMP3 with Micro4 Controller, WPI).

After the injection, the needle was left in place for another 5–10 min to ensure diffusion. Then, single (IL, 0.39 NA, ø 200 μm, cleaved to 4 mm length; CFMLC12U-20, Thorlabs) or dual fibers (VTA, 0.22 NA, ø 200 μm, 8.5 mm length; DFC_200/240-0.22_8.5mm_GS1.0_FLT, Doric Lenses) were implanted bilaterally above IL (DV −1.90 mm, 20° angle) or VTA (DV −4.10 mm, 0° angle). Two additional craniotomies were made in which jeweler's screws were mounted to build a fundament using dental cement (Paladur, Heraeus-Kulzer) and ensure fixation of the implants to the skull. To reduce postoperative pain, a single dose of buprenorphine was administered subcutaneously directly after surgery (0.5 mg/ml; Bupaq, Richter Pharma) and meloxicam was provided via drinking water (1 mg/ml; Metacam, B. Braun Melsungen) for the following 3 d. After surgery, recovery of the animals and AAV expression were allowed for 4–5 weeks, before beginning behavioral experiments.

Behavioral testing for S1-DAT-Cre phenotyping

Before the start of behavioral experiments, all animals were habituated to the experimenter and the handling procedure for at least 3 d. Fear conditioning (FC) and extinction procedures were conducted based on previously employed methods (Gunduz-Cinar et al., 2019) and here controlled using ANY-maze behavioral tracking software (Stoelting Europe) with a compatible FC system (Ugo Basile). All sessions were digitally video recorded for later analysis.

FC (Context A): The conditioning chamber was a 17 × 17 × 25 cm cubicle with transparent walls, a Lucite lid, and a metal rod floor that was cleaned with water and illuminated with white light to 600 lux. The room in which FC was performed was brightly illuminated with white light. After a 120 s acclimation period, three pairings of a CS (10 kHz sine tone, 75 dB, 30 s) with a coterminating US (scrambled footshock, 0.6 mA, 2 s) were delivered with a variable (30–120 s) intertone interval. After the final pairing, a 120 s no-stimulus consolidation period followed, before the mice were returned to their home cage.

Fear extinction training (EXT; Context B): The extinction context was a 17 × 17 × 25 cm cubicle with black and white checked walls, an open top and a solid gray floor that was cleaned with 1% acetic acid and illuminated with red light to 5 lux. The room in which fear extinction training was performed was dimly illuminated with white light. After a 120 s acclimation period, 20 CS presentations with a variable (5–30 s) intertone interval were delivered. After the final presentation, a 120 s no-stimulus consolidation period followed, before the mice were returned to their home cage.

Extinction retrieval (RET; Context B): For extinction retrieval, the mice were again placed in the extinction context (Context B) 24 h following extinction training. After a 120 s acclimation period, 5 CS presentations with a variable (5–30 s) intertone interval were delivered. After the final presentation, a 120 s no-stimulus consolidation period followed, before the mice were returned to their home cage.

For quantification of freezing behavior, the time spent freezing was manually scored and quantified using a custom-written MATLAB script (MathWorks). All freezing values are presented as the percentage of the analyzed time period or trial blocks (e.g., pre-CS, CS, five CS trial blocks).

Behavioral testing for optogenetics and fiber photometry

Before the start of behavioral experiments, all animals were habituated to the experimenter and the handling procedure for at least 3 d. For optogenetic manipulation and fiber photometry measurements, the animals were connected to the fiber cables via their implants and habituated for 5 (optogenetics) or 10–20 min (fiber photometry) in their home cage before being placed in the corresponding context for behavioral testing.

FC procedures were controlled using ANY-maze behavioral tracking software (Stoelting Europe) with a compatible FC system (Ugo Basile; optogenetics) or Med Associates software and FC system (Med Associates; fiber photometry). Extinction procedures were controlled using ANY-maze behavioral tracking software (Stoelting Europe) and corresponding AMi-2 audio and optogenetics interfaces (optogenetics) or Ethovision software (Noldus) with compatible hardware (fiber photometry). All sessions were digitally video recorded for later analysis.

FC (Context A): The conditioning chamber was a 26 × 26 × 35 cm (optogenetics) or a 30 × 25 × 25 cm (fiber photometry) cubicle with transparent walls, an open top and a metal rod floor that was cleaned with water and illuminated with white light to 600 lux. The room in which FC was performed was brightly illuminated with white light. After a 180 s acclimation period, five parings of a CS (10 kHz sine tone, 75 dB, 28 s) followed by an US (scrambled footshock, 0.6 mA, 2 s) were delivered with a 90 s intertone interval. After the final pairing, a 120 s no-stimulus consolidation period followed, before the mice were returned to their home cage.

Fear extinction training (EXT; Context B): The extinction context was a 24 × 32 × 30 cm cubicle with white walls, an open top, and a solid yellow floor (optogenetics) or a transparent 27 × 27 × 14 cm container with some bedding material covering the floor (fiber photometry) that was cleaned with 1% acetic acid and illuminated with red light to 5 lux. The room in which fear extinction training was performed was dimly illuminated with red light. After a 180 s acclimation period, 25 CS presentations with a 7 s (optogenetics) or 20 s (fiber photometry) intertone interval were delivered. After the final presentation, a 120 s no-stimulus consolidation period followed, before the mice were returned to their home cage.

Extinction retrieval (RET; Context B): For extinction retrieval, the mice were again placed in the extinction context (context B) 24 h following extinction training. After a 180 s acclimation period, five CS presentations with a 7 s (optogenetics) or 20 s (fiber photometry) intertone interval were delivered. After the final presentation, a 120 s no-stimulus consolidation period followed, before the mice were returned to their home cage.

For quantification of freezing behavior, the time spent freezing was either manually scored and quantified using a custom-written MATLAB script (MathWorks) or measured automatically using ANY-maze behavioral tracking software (Stoelting Europe) according to predefined thresholds that were validated by manual scoring. All freezing values are presented as the percentage of the analyzed time period or trial blocks (e.g., pre-CS, CS, five CS trial blocks).

Fiber photometry

For recording of fluorescent signals during all behavioral tests, the implanted optical fibers were connected to mono fiber-optic patch chords with low autofluorescence (0.57 NA, ø 400 µm, 0.60 m length; Doric Lenses). The patch cords were attached to a fiber-optic rotary joint (FRJ_1 × 1_PT_FCM-FCM, Doric Lenses) that was connected to the photometry system. The photometry system consisted of a fluorescence mini cube [FMC6_E1(400–410)_F1(420–450)_E2(460–490)_F2(500–540)_O(570–650)_S, Doric Lenses] that was connected to photoreceivers (NPM_2151_FOA_FC, Doric Lenses) and 465 and 405 nm LED lights (M470F3 and M405F1, Thorlabs). A real-time signal processor was used to sinusoidally modulate LED outputs and collect fluorescent signals at a sampling rate of 1,017 Hz (RZ5P with Synapse Software, Tucker-Davis Technologies).

All data analysis was performed using MATLAB scripts (MathWorks) with minor modifications as previously described (Gunduz-Cinar et al., 2023). Recordings obtained from the 405 nm channel were used to control for autofluorescence, bleaching, and motion artifacts. For this, the 405 nm signal was first aligned to the 465 nm signal by scaling and linear fitting. The change in fluorescence (dF) for each time point (t) was calculated by subtracting the fitted control from the raw 465 nm channel signal (ΔF = 465 nm signal − fitted 405 nm signal). Z-Scores of each time point [Z(t)] were calculated by normalizing to a predefined baseline period (which was taken 5 s before the first CS onset, otherwise subtracting the average stated in figure legends) by dF at all time points during baseline period from the dF at each time point and dividing to the standard deviation (SD) of all baseline dF:Zscoreattimepointt=Z(t)=dF(t)−mean(dF(baselineperiod))SD(dF(baselineperiod)). Time-normalized area under the curve (AUC) values were calculated using MATLAB's built-in “trapz” function, which uses trapezoidal numerical integration to calculate the AUC (Z-score) between inputted x-values (time) on a graph of z-score versus time. Time-normalized AUC values for the US (2 s post-US) during conditioning, for the CS onset and CS offset (5 s post CS onset or offset) during conditioning extinction and retrieval were compared with each event's respective (5 s) pre-event baseline. Data analyses were performed using MATLAB R2023b (MathWorks), and graphs were prepared by Graph Pad Prism10.

Optogenetic manipulations

For optogenetic manipulations during fear extinction, the implanted optical fibers were connected to mono fiber-optic patch chords (0.22 NA, ø 200 µm, 0.75 m length; Doric Lenses). The patch cords were attached to a light-splitting fiber–optic rotary joint (FRJ_1 × 2i_FC-2FC, Doric Lenses) that was connected to a 473 nm laser (MBL-F-473 (150 mW), CNI Optoelectronics Technology). Laser power was adjusted before each animal to 8–10 mW at the fiber tip (PM100D power meter with S120C sensor, Thorlabs). To phasically activate DA neurons on VTA-IL terminals at the time of US omission, 5 ms light pulses were delivered at 20 Hz (5% duty cycle) for 2 s after the offset of the CS. For VTA and VTA-IL activation experiments, the laser was controlled using the ANY-maze behavioral tracking software (Stoelting Europe) with a corresponding AMi-2 optogenetic interface.

Immunohistochemistry

Tissue extraction and sectioning: After the behavioral experiments involving fiber photometry or optogenetic manipulations, all animals were transcardially perfused with ice-cold 0.9% saline (5 min) and PFA solution (4% in 0.1 M PB; 8 min), pH 7.4, at a flow rate of 5 ml/min (Peri-Star Pro Peristaltic Pump, WPI). The brains were extracted, placed in PFA solution for 24 h for postfixation and then transferred into PB (0.1 M), pH 7.4, until sectioning. All brains were sectioned on a vibrating microtome (VT1000S, Leica Biosystems) at a thickness of 40 µm, and the free-floating sections were stored in PB until further processing.

For mCherry immunostaining, all sections were first washed in a single washing step with phosphate-buffered saline (PBS; 0.05 M), pH 7.4, for 10 min. Then, the sections were incubated with blocking buffer (10% normal goat serum and 1% BSA in 0.05 M PBS with 0.3% Triton X-100), pH 7.4, for 2 h. Next, the primary antibody targeting mCherry was added (polyclonal rabbit, 632496, Takara Bio; 1:500 in blocking buffer diluted 1:10 in 0.05 M PBS, with 0.3% Triton X-100), pH 7.4, overnight at 4°C. On the next day, the primary antibody solution was removed, and the sections were washed with PBS (three times for 10 min) and incubated with the secondary antibody (Alexa Fluor 555, goat anti-rabbit, A-21428, Invitrogen/Thermo Fisher Scientific; 1:500 in blocking buffer diluted 1:10 in 0.05 M PBS, with 0.3% Triton X-100), pH 7.4, for 2 h at room temperature. After removal of the secondary antibody solution, again a washing step with PBS followed (two times for 10 min). Thereafter, the sections were washed once with PB and then mounted onto glass slides. After the slides were dry, they were immediately coverslipped with a DAPI-containing mounting medium (ProLong Gold Antifade Mountant with DAPI, P-36931, Invitrogen/Thermo Fisher Scientific), dried at room temperature overnight and then stored at 4°C.

GCaMP6f expression was sufficiently visible without immunostaining; therefore sections were mounted onto glass slides in PB and counterstained with Hoechst 33342 (5 µg/ml; H1399, Invitrogen/Thermo Fisher Scientific). After the slides were dry, they were immediately coverslipped with a glycerol-based aqueous mounting medium, sealed with nail polish and then stored at 4°C.

For microscopy and histological verification of fiber placements, all sections of interest were 2D-scanned (Stereo Investigator, MBF Bioscience) at 4× magnification in DAPI and CY3 channels using a fluorescent microscope (BX-40, Olympus) with a motorized stage (MAC6000 System, Ludl Electronic Products) or imaged at 4× magnification in DAPI and CY2 channels on a fluorescent microscope with manual stage and cellSens software (BX-41, Olympus). AAV expression and fiber placements were verified according to distinct anatomical landmarks corresponding to the Paxinos stereotaxic mouse brain atlas (Paxinos and Franklin, 2019). Only mice with sufficient AAV expression and correct fiber placements were included in the analysis.

Immunohistochemistry and stereological analysis of VTA-DA neurons

Tissue extraction and sectioning was performed as described above for the verification of fiber placements.

For immunostaining of tyrosine hydroxylase positive (TH+) neurons, all sections were placed in Tris-buffered saline (TBS; 0.05 M), pH 7.4, and incubated with H₂O₂ at a concentration of 0.03% for 30 min. After washing with TBS (three times for 10 min), the sections were incubated with blocking buffer (20% normal goat serum in 0.05 M TBS, with 0.3% Triton X-100), pH 7.4, for 1.5 h. Next, the primary antibody targeting TH was added (polyclonal rabbit, AB152, Merck Millipore; 1:4,000 in 0.05 M TBS, with 2% normal goat serum and 0.3% Triton X-100), pH 7.4, at 4°C for ∼72 h. Again, after three washing steps in TBS (three times for 10 min), the secondary antibody (biotinylated goat anti-rabbit, BA-1000, Vector Laboratories; 1:500 in 0.05 M TBS, with 2% normal goat serum and 0.3% Triton X-100), pH 7.4, at 4°C overnight. After washing in TBS (three times for 10 min), sections were incubated with an avidin–biotin–peroxidase complex solution (Vectastain ABC Kit, Vector Laboratories; in 0.05 M TBS, with 2% normal goat serum), pH 7.4, for 1 h at room temperature. Following three washing steps in Tris-borate buffer (TB; 0.05 M), pH 7.4, the sections were preincubated in 3,3′diaminobenzidine tetrahydrochloride solution (0.5 mg/ml in 0.05 M TB), pH 7.4, for 10 min at RT. The immunoreaction was started by the addition of 3% H₂O₂ to a final concentration of 0.003%. The reaction was stopped after 2 min by washing with TBS (three times for 10 min). Finally, the sections were mounted onto gelatine-coated glass slides, air-dried, and dehydrated in ethanol (5 min in each 50, 70, 95, 100, 100%) and xylol (two times for 10 min) before they were immediately coverslipped with Eukitt (Millipore Sigma).

TH+ cell bodies in the VTA were quantified in one per three coronal brain sections of S1 and BL6 mice between bregma −2.92 and −3.80 mm (according to Paxinos and Franklin, 2019). This was done via unbiased stereological analysis (optical fractionator method) on a microscope (BX-40, Olympus) with a motorized stage (MAC6000 System, Ludl Electronic Products) and a computer-assisted image analysis system (Stereo Investigator Software, MBF Biosciences). The following counting parameters and settings were used: counting frame 50 × 50 µm; grid size 150 × 80 µm; section evaluation interval 3; and magnification 2× and 100× (oil). TH+ cell bodies are reported per hemisphere. The Gundersen coefficient errors (m = 1) were ≤0.4.

Statistical analysis

The GraphPad Prism 9 and 10 software (GraphPad Software) or MATLAB (MathWorks) were used for generating graphs and performing statistical analysis. Results from all statistical analysis are summarized in a table as Extended Data Figure 1-1. All datasets were tested for normality using a Shapiro–Wilk test or a D’Agostino and Pearson’s test. Two-tailed, unpaired Student's t test (two-group comparisons) and one-way ANOVA with a Šídák post hoc test (multigroup comparisons) was used for normally distributed data without or with repeated measures. The Kruskal–Wallis test (multigroup comparisons) was used for not normally distributed data with or without repeated measures. The Kruskal–Wallis test (multigroup comparisons) were used for data that were not normally distributed, with or without repeated measures. Experiments with two independent variables were analyzed by using two-way ANOVA or two-way RM-ANOVA if one of the variables was time. Šídák post hoc tests were applied where multiple comparisons were of interest. The significance level was set to p < 0.05 (significance levels denoted as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). All data are presented as means ± SEM unless stated otherwise.