1. Nigrotectal Stimulation Stops Interval Timing in Mice
Koji Toda, Nicholas A. Lusk, Glenn D.R. Watson, Namsoo Kim, Dongye Lu, Haofang E. Li, Warren H. Meck, Henry H. Yin 
Current Biology 
Volume 27, Issue 24, Pages e3 (December 2017)
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2. Figure 1 Experimental Setup and Behavioral Tasks
(A) Head-fixed setup. Mice were placed on a raised platform and secured in place by head bars attached to chronic implants. A blunt-tipped needle is placed within licking distance, allowing for administration of a 10% sucrose reward using a solenoid under the control of an Arduino and MATLAB Psychtoolbox.
(B) Schematic diagram of the behavioral tasks. In the fixed-time-schedule task (left), a drop of water was delivered every 10 s throughout the session. Once mice demonstrated anticipatory licking, they were transitioned to a peak procedure (right), where 20% of rewards were omitted on probe trials in order to estimate their internal representation of expected time of reward delivery.
(C) Examples of behavioral data. Individual trial (top) and cumulative Peri-stimulus time histogram (PSTH) (bottom) of licking behavior during rewarded (left) and peak (right) trials.
See also Figure S1.
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3. Figure 2 Demonstration of Scalar Property
(A) PSTHs for 7.5-, 10-, and 15-s fixed-time-schedule tasks (n = 3–5 sessions for each condition for three mice). Vertical dashed lines represent the expected reward time.
(B) Accuracy of timing behavior across durations determined by average peak time for individual sessions (n = 13 sessions for each condition).
(C) Overlay of the behavior from the three fixed-interval durations normalized by max lick rate (y axis) and time of peak lick rate (x axis), demonstrating superimposition (n = 3–5 sessions for each condition for three mice).
(D) Relative precision of timing behavior as indicated by Weber fraction (n = 13 sessions for each condition). n.s., not significant (n = 13 sessions; p value > 0.05, one-way ANOVA).
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4. Figure 3 Licking Behavior in Peak Trials
(A) Lick rate from single session aligned to time of previous reward (left; n = 36 trials) or start time of peak trial licking (right; n = 36 trials).
(B) Same analysis as in (A) except related to the end of licking on peak trials (left, n = 36 trials; right, n = 36 trials).
(C) Demonstration of motivation effect during a single session. Licking raster (top) is plotted with start (beige squares) and end (orange squares) times of peak licking activity calculated using single-trial analysis (see STAR Methods).
(D) Comparison of within-session licking behavior between the first (first) and last (last) quartiles. Both individual data (light blue; n = 10 mice) and group averages (blue) are shown. ∗p value < 0.05; n.s., not significant (n = 10; paired t test, p value > 0.05).
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5. Figure 4
Optogenetic Stimulation of SNr-SC Pathway Cancels Licking Behavior
(A) Schematic of surgical and optogenetic procedures.
(B) Diagram of licking-related circuitry: dorsolateral striatum (DLS); substantia nigra pars reticulata (SNr); superior colliculus (SC); and central pattern generators (CPGs).
(C) Image of axons within the SC expressing ChR2 after injections of AAV5-EF1α-DIO-hChR2(H134R)-eYFP into the SNr.
(D) Schematic of stimulation conditions relative to presentation of reward (top). Examples (left) and summary (right) of optogenetic stimulation effects on anticipatory and consummatory licking behavior with Vgat::ChR2SNr-SC mice showing frequency-dependent modulation of licking behavior by optogenetic stimulation. ∗p value < 0.05 (n = 6 mice; one-way ANOVA, post hoc Tukey test).
See also Figures S2 and S3A.
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6. Figure 5 Photo-Stimulation Reliably Generates Spiking in SNr Neurons
(A) Whole-cell current clamp experiments in brain slices show that SNr projection neurons can follow pulse trains in a frequency-dependent manner. Representative traces at frequencies of 10 Hz, 25 Hz, 50 Hz, and 100 Hz are shown.
(B) Summary of evoked spiking to photo-stimulation. Neurons demonstrated increased spiking as stimulation frequency increased with each increment in stimulation frequency, inducing a significantly greater number of spikes (n = 6; p < 0.05, one-way ANOVA, post hoc Tukey test) and near perfect fidelity up to 50 Hz.
(C) The evoked firing rate can be used to estimate the in vivo SNr output. A linear regression analysis shows that the estimated SNr output can account for most of the variance in licking behavior from Figure 4 (r2 > 0.90; p < 0.05 in each).
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7. Figure 6
SNr-SC Stimulation Stops Timing of Licking Behavior in Vgat::ChR2SNr-SC Mice
(A) Example of the resetting effect by photo-stimulation (100 Hz) of the SNr-SC GABAergic pathway during 1 s after the reward delivery.
(B) Effect of photo-stimulation during 1 s after the reward delivery on start time, end time, bout length, and peak time. Photo-stimulation significantly delayed start time, end time, and peak, but not bout length.
(C) Effect of photo-stimulation during 1 s before the reward delivery on start time, end time, bout length, and peak time. Photo-stimulation significantly delayed start time and peak, but not end time and bout length.
(D) Effect of photo-stimulation during 2 s to 1 s prior to the reward delivery on start time, end time, bout length, and peak time. Photo-stimulation significantly advanced end and peak time, but not start time and bout length. ∗p value < 0.05 (n = 5; unpaired t test); n.s, not significant (n = 5 mice, unpaired t test).
See also Figures S3B and S4–S6.
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