Subjects
In the research, both male and female C57BL/6J mice were utilized, with numbers kept approximately equal across experiments. Wild-type mice were bred with either DAT:IRES–Cre or ChAT:IRES–Cre mice. For the electrophysiology slices, ChAT:IRES–Cre mice were crossed with DAT–Flp mice. All transgenic mice underwent genotyping to confirm they were heterozygous for either Cre-recombinase or Flp-recombinase. Following weaning, mice were housed separately by sex before undergoing surgeries or behavioral tests. All experiments were conducted during the dark cycle, with mice aged between 10 to 24 weeks. They were kept at about 21°C in 30% to 70% humidity and were food restricted to around 85% of their normal body weight to encourage motivation during the experiments. The procedures adhered to NIH animal care guidelines and received Stanford University’s approval.
Behavioral training
Behavioral training and food restriction commenced at least two weeks post-virus injection and fiber implantation. Mice were acclimated for two days, involving handling and tethering to equipment, while allowing them to explore operant boxes for half an hour. Post-habituation, they took part in a Pavlovian task that rewarded them randomly every 25 to 35 seconds for 30 minutes. The operant chambers were designed with noise and nose-poke ports equipped with cue lights and sensors to track entries. Rewards were paired with two-second cues of sound and light, so the mice would learn to associate the cues with reward delivery. For rewards, a solution of 32% sucrose was typically used, although one experiment utilized 5% sucrose. After completing one day of Pavlovian training, mice moved on to the operant task using a fixed ratio (FR) schedule, beginning with FR1. The active nose-poke port alternated between left and right among mice, remaining the same throughout the experiments. The food port was initially baited to promote exploration. Mice who earned at least ten rewards at FR1 with over 80% accuracy progressed to FR5 for at least five sessions until their performance stabilized. Eventually, they were introduced to the sucrose effort task. The focus wasn’t necessarily on equalizing reward consumption, but on ensuring consistent performance among all subjects. For optogenetic self-stimulation, training was similar—focusing on using optogenetic stimulation in place of sucrose rewards.
Sucrose effort task
This task included five 10-minute blocks with varying FR schedules (FR46, FR21, FR10, FR5, and FR1). Mice had to poke in the active port the required number of times to receive sucrose rewards, which were delivered in the central magazine, again accompanied by the two-second light and sound cues. It’s important to note that the FR schedules were arranged in descending order to prevent early satiety among mice, with one control experiment featuring an ascending order. Additionally, block transitions were not indicated to the mice.
Modified sucrose effort task
In experiments using PBS or DHβE microinjections, a modified sucrose effort task was employed due to the possibility of the drug’s effects waning over time. Here, the poking requirement remained constant during the entire 30-minute session, set either at FR1 or FR21. The sequence of experimental days was balanced across the mice.
ChRmine effort task
The structure followed the sucrose effort task, but mice worked for optogenetic stimulation instead of sucrose. The stimulation was paired with the same cues as in the previous tasks.
Stereotaxic injections and viruses
Mice aged 8-12 weeks were anesthetized with isoflurane. They were secured in a stereotaxic frame, and small incisions were made in the skull to allow for virus injection or fiber implantation at specified coordinates. Virus solutions were injected at a controlled flow rate, with optic fibers implanted above the targeted brain areas, secured with screws and dental adhesive. Various adeno-associated viruses (AAVs) were employed for these injections, with different titers.
Fiber photometry
Mice with optical implants were linked to low-autofluorescence patch cords to record signals which were processed and analyzed using specialized software. Efforts were made to ensure precise recording conditions, with light sources adjusted for optimal performance.
Optogenetic manipulations
Optogenetic manipulations were executed alongside fiber photometry recordings, with varying equipment used based on the experimental needs. Control and experimental conditions were managed with stringent protocols to ensure reliability.
Drug administration
For systemic injections, various drugs were administered at specified doses, using solvents to ensure proper dosage delivery. Post-injection, mice were allowed to acclimate before undergoing the relevant tasks. In local injection experiments, specific drugs were infused into targeted regions, although some treatments led to side effects that hindered further behavioral tasks.
Validation of VTA DA cell body inhibition
To validate the effects of muscimol on VTA DA cell bodies, one cohort received injections and were prepared for real-time recordings during drug administration. Comparisons were made between conditions to assess neural activity comprehensively.
Slice electrophysiology
Acute brain slices were prepared from a specific cohort of mice, injected at various brain regions, and utilized for electrophysiological experiments after appropriate anesthetic and perfusion protocols. The resulting slices were analyzed for response to various stimulations.
Immunohistochemistry
Mice underwent perfusion for brain removal, followed by sectioning and specific antibody incubations to visualize various neural components. Visualization was conducted using advanced microscopy techniques.
Data analysis
Data was meticulously analyzed with specific criteria for exclusion to ensure accuracy. MATLAB was used for signal processing, and all statistical evaluations were carried out with care, following established protocols. Various statistical tests were chosen based on the data distribution and characteristics, ensuring thorough examination and representation of findings.
Reporting summary
More detailed information regarding the research design can be found in the supplementary materials linked to the article.
