TABLE 1.

Troubleshooting table.

Step	Problem	Possible reason	Solution
32	Some of the animals trained in the two-lever free-choice task may develop a strong bias toward one of the two levers during the training, making it difficult to obtain within-subject comparison of left-lever and right-lever pressing behaviors	Naturally occurring, though not often	Further train the mice in single-lever fixed-ratio 10 sessions during which either the left or right lever is presented. For each animal trained in this way, the optical measurements should be carried out in two consecutive sessions with either left or right lever presented. To minimize the effect of satiety levels between the two consecutive sessions, only ten rewards should be used in each session
54	Poor GCaMP spectrum (e.g., the spectrum shown in Fig. 6c) during the fiber implantation surgery	Low GCaMP expression	Use AAV serotypes that have high selectivity for the cells of interest. Use strong promoters such as the CAG promoter. Wait for a longer time (4 weeks or more after injection) or increase the injection dose
		Fiber tip is not in the targeted structure	Further lower the probe or retract the probe and change the x-y coordinates and lower the fiber again
		Poorly constructed probe	Make sure the fiber ends are precisely flat-cleaved, aligned parallel, without staggering ends
67, 80	Good GCaMP spectrum but no fluorescence transients are seen in freely moving animals, and isoflurane does not induce reversible decrease in GCaMP fluorescence	GCaMP expression is too high and causes toxicity	Choose a weaker promoter or AAV serotype. Reduce the AAV injection dose. Reduce the interval between injection and measurement. Do not measure at the injection site. The GCaMP expression becomes more uniform and less intense when moving away from the injection site
		The GCaMP fluorescence is from damaged cells	Move further away from the injection site
		The targeted cell type may not show transient activation in freely moving animals. They may only activate in response to specific stimuli that are not present	Use the specific stimuli that are known to activate the cells of interest
		Isoflurane anesthesia may not inhibit the neural activity of the targeted cells	Normal phenomenon, no solution needed
76	Broken fibers	Occasionally, some animals may display turning behavior strongly biased toward one direction after the fiber probe has been implanted, especially if they are not pre-trained with tethering. The jacketed fibers can usually tolerate twisting very well when the twisting force is evenly distributed through the whole length of fibers. However, strong twisting of fibers may cause a small diameter local loop to form when there is extra slack, which may break the fiber	Pre-train the animals with dummy fibers in later stages of operant training to accustom them to being tethered
			Avoid extra slack after the mouse has been implanted with the fiber probe
			Closely supervise the fiber-tethered mouse after surgery and manually rotate the housing container to release the twisting when necessary
			Use connectors between the implanted fibers and the fibers connected to the laser and the detector (Fig. 4g,h and supplementary Fig. 1). Disconnect the mice from the rest of the system after each experiment
			Select thicker furcation tube for the fibers, such as 1/16-inch-diameter polyolefin heat-shrink tubing
81	Timing error between the recorded optical data, behavior time stamps and video files	Multiple independent systems with different clocks, streaming and buffering causing variable delays in different systems	Send synchronizing TTL pulses to all the systems at the beginning of the recording and periodically throughout the recording. Later, align these reference marks in different files to calibrate for time. (e.g., supplementary Fig. 3)