Glutamate sensor and calcium signals in dopamine neurons and dopamine release

We recorded glutamate input to dopamine neurons in the ventral tegmental area (VTA) with iGluSnFR(A184S), a glutamate sensor, somatodendritic dopamine neuron activity in the VTA with jGCaMP7f, a calcium indicator, and dopamine release at the ventral striatum (VS) with GrabDA2m, a dopamine sensor, using fiber fluorometry. Recordings were performed in two types of reward association tasks (classical conditioning and sequential conditioning), with presentation of aversive airpuff to the eye with or without buprenorphine treatment, and with optogenetic activation of glutamate input axons at the VTA.

Methods
Behavior

After 1 week of recovery from surgery, mice were water-restricted in their cages. All conditioning tasks were controlled by a NIDAQ board and LabVIEW. Mice were handled for 2 days, acclimated to the experimental setup for 1-2 days including consumption of water from the tube, and head-fixed with random interval water for 1-3 days until mice showed reliable water consumption. For odor-based classical conditioning, all mice were head-fixed, and the volume of water reward was constant for all reward trials (predicted or unpredicted) in all conditions (6 ml). Some sessions included mild air puff trials, directed at one of the eyes and the intensity of air puff was constant for all air puff trials (predicted or unpredicted; 2.5 psi). In classical conditioning, each association trial began with an odor cue (lasting 1 s) followed by a 2 s delay, and then an outcome (either water, nothing, or air puff) was delivered. In sequential conditioning, each association trial began with a distal odor cue (lasting 1 s) followed by a 2 s delay, and then a distal odor cue (lasting 1 s) followed by 1 s delay, and then an outcome (either water, or nothing) was delivered. Some trial types began with a proximal odor cue (lasting 1 s) followed by 1 s delay, and then an outcome (either water, or nothing) was delivered. Odors were delivered using a custom olfactometer. Each odor was dissolved in mineral oil at 1:10 dilution and 30 ml of diluted odor solution was applied to the syringe filter (2.7mm pore, 13mm; Whatman, 6823-1327). Odorized air was further diluted with filtered air by 1:8 to produce a 900 ml/min total flow rate. Different sets of odors (Ethyl butyrate, p-Cymene, Isoamyl acetate, Isobutyl propionate, 1-Butanol, 4-Methylanisole, Caproic acid, Eugenol, and 1-Hexanol) were selected for each animal. Some of the animals shared the same odor set (4 animals and 3 animals for DA sensor classical conditioning; 3 animals and 2 animals for sequential conditioning)

A variable inter-trial interval (ITI) of flat hazard function (minimum 10s, mean 13s, truncated at 20s) was placed between trials. Each session was composed of multiple blocks (12-24 trial/block) and all trial types were pseudorandomized in each block. Each day, the mice did about 70-350 trials over the course of 20-75 min, and with constant excitation from the laser and continuous recording in recording sessions.

Training for classical conditioning used 4 types of trials; odor cue predicting 100% water, odor cue predicting 40% water/60% no outcome (nothing), odor cue predicting nothing (29.4% of all trials for each odor), and water without cue (free water) (11.8%) for 7-10 days, and then odor cue predicting 80% water/20% nothing, odor cue predicting 40% water/60% nothing, odor cue predicting nothing (29.4% each), and free water (11.8%) for more than 2 days of training followed by recording sessions. The first 170 trials or trials before the animal stops licking for each session are used for analysis.  3 animals used for glutamate sensor recording (Figure 2) were trained with classical conditioning with air puff trial; odor cue predicting 100% water, odor cue predicting 100% air puff, odor cue predicting 40% water/60% no outcome (nothing), odor cue predicting nothing (20.8 % of all trials for each odor), and water without cue (free water), air puff without cue (free air puff)  (8.3 % of all trials for each stimulus) for 8-9 days, and then odor cue predicting 80% water/20% nothing, odor cue predicting 80% air puff/20% nothing, odor cue predicting 40% water/60% nothing, odor cue predicting nothing (20.8 % each), and water without cue (free water), air puff without cue (free air puff) (8.3% each) for more than 7 days of training followed by recording sessions. The first 192 trials or trials before the animal stops licking for each session are used for analysis. Of note these 3 animals are used only in the analysis for glutamate sensor activity pattern, and not included for comparison between different sensor recordings.

For sequential conditioning step1, mice were trained with proximal odor for 3-7 days, using 3 types of trials; proximal odor cue predicting 100% water, proximal odor cue predicting nothing (45.8 % of all trials for each odor), and water without cue (free water) (8.3 %). Then, for sequential conditioning step2, mice were further trained with distal odor and proximal odor using 6 types of trials; distal odor cue predicting proximal odor cue predicting 100% water (25 %), distal odor cue predicting 50% proximal odor cue predicting water/50% proximal odor cue predicting nothing (25 %), distal odor cue predicting proximal odor cue predicting nothing (25 %), proximal odor cue predicting 100% water (8.3 %), proximal odor cue predicting nothing (8.3 %), and water without cue (free water) (8.3 %). Sensor signals were recorded after 8-12 days of step2 training. The first 192 trials of each session are used for analysis.

For buprenorphine treatment, the mouse received unexpected air puff and water with a variable ITI of the flat hazard function (minimum 10s, mean 13s, truncated at 20s). Animals were first acclimated to head-fixation with water reward for 2 days. Then the animals were habituated to a test procedure for two days, which was composed of 40-70 trials of air puff and water presented in pseudorandomized order (the order of trials is pseudorandomized within each block of 10 trials composed of the same number of air puff trials and reward trials) with subcutaneous injection of saline (10 ml/g in body weights) at the end of sessions. After habituation sessions, buprenorphine sessions and control saline sessions were performed in pseudorandom order. Each session was separated into pre-injection sub-session and post-injection sub-session. The sub-session was composed of 48-75 trials with the air puff and water trials in the pseudorandom order. After pre-injection sub-sessions, either buprenorphine (Buprenorphine hydrochloride, Par Pharmaceutical: diluted in saline 0.03 mg/ml) or control saline was subcutaneously injected (10 ml/g; 0.3 mg/g buprenorphine or corresponding volume of saline). Post-injection sub-session started 5 min after injection. To prevent acute adverse effects, we used a smaller dose of buprenorphine (0.15 mg/g) in the first session and this session is not included in the analysis.

Optogenetic stimulation

Red light from 625 nm LED light (M625F2, Thorlab) was applied through an optical patch cord (400 mm, 0.39 NA, Thorlab). 12 mW single block pulse light of 20, 50, and 250ms duration was triggered pseudorandom order through custom software written in LabVIEW (National Instruments) via a NIDAQ board (PCI-e6321, National Instruments). A variable inter-trial interval (ITI) of flat hazard function (minimum 10s, mean 13s, truncated at 20s) was placed between trials.

Data analysis

Fiber-fluorometry

The noise from the power line in the voltage signal was cleaned by removing 58-62Hz signals through a band stop filter. The global change within a session was normalized using a moving median of 100 s. Then, the correlation between green and red signals during ITI was examined by linear regression when the red signal data was available. If the correlation is significant (p ≤ 0.05), fitted tdTomato signals were subtracted from green signals. Responses were calculated by subtracting the average baseline activity from the average activity of the target window. Z-scores of the signals were obtained using mean and standard deviation of signals in all ITI (from 5 s before odor onset to odor onset for classical conditioning, from 5 s before distal odor onset to odor onset for sequential conditioning) in each animal.

Licking

Licking from a water spout was detected by a photoelectric sensor that produces a change in voltage when the light path is broken. The timing of each lick was detected at the peak of the voltage signal above a threshold.