Environmental cues, through Pavlovian learning, become conditioned stimuli that guide animals toward the acquisition of rewards (for example, food) that are necessary for survival. We tested the fundamental role of midbrain dopamine neurons in conferring predictive and motivational properties to cues, independent of external rewards. We found that brief phasic optogenetic excitation of dopamine neurons, when presented in temporal association with discrete sensory cues, was sufficient to instantiate those cues as conditioned stimuli that subsequently both evoked dopamine neuron activity on their own and elicited cue-locked conditioned behavior. Notably, we identified highly parcellated functions for dopamine neuron subpopulations projecting to different regions of striatum, revealing dissociable dopamine systems for the generation of incentive value and conditioned movement invigoration. Our results indicate that dopamine neurons orchestrate Pavlovian conditioning via functionally heterogeneous, circuit-specific motivational signals to create, gate, and shape cue-controlled behaviors.
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We thank R. Keiflin and all members of the Janak laboratory for discussion and comments on the manuscript; A. Haimbaugh, D. Acs, H. Pribut, K. Lineback, N. Pettas, B. Persaud, and L. Kinny for assistance with histology and behavioral video scoring; P. Fong for conducting surgical procedures for ex vivo physiology studies; K. Deisseroth (Stanford) for the ChR2 construct; E. Boyden (MIT) for the ChrimsonR construct; and the Janelia Research Campus GENIE Project and Stanford Gene Vector and Virus Core for the GCaMP6f construct. This work was supported by National Institutes of Health grants DA036996 (B.T.S.), DA042895 (B.T.S.), AA022290 (J.M.R.), AA025384 (J.M.R.), DA030529 (E.B.M.), and DA035943 (P.H.J.), as well as grants from the Brain and Behavior Research Foundation (B.T.S. and J.M.R.).
The authors declare no competing interests.
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Integrated supplementary information
(a) Injection of a cre-dependent ChR2-eYFP AAV vector resulted in targeting of ChR2-eYFP to TH+ neurons in the (b) SNc and (c) VTA. (d) Targeting specificity was high (96.9%; 1027 TH+/1060 eYFP+ neurons counted) across medial/lateral and anterior/posterior sections of the midbrain.
Coronal plates showing the location of optic fiber tips relative to Bregma for TH-cre+ and cre- control rats in the (a) ChR2 experiments, (b) fiber photometry experiments, (c) ChrimsonR intracranial self-stimulation, and (d) Projection-specific ChR2 experiments.
(a) VTA and SNc cre+ paired rats learned conditioned responses during the cue period at the same rate (2-way repeated measures ANOVA, no interaction, F(3,42) = 0.691, p = 0.563). (b) Neither VTA nor SNc cre+ unpaired rats developed conditioned responses during the laser period. (c) Learning curves for individual rats in all groups during the cue period. (d) Learning curves for individual rats in all groups during the laser period. (e) The average cumulative probability of CR occurrence for VTA and SNc paired rats within 7-sec cue periods across training. VTA and SNc rats acquire CRs rapidly, and CRs emerged earlier in the cue period as training progressed. Data expressed as mean ± SEM.
(a) TH-cre+ rats (n = 5; 3 SNc targeted, 2 VTA targeted) underwent optogenetic Pavlovian conditioning where they received 1-sec long laser presentations, delivered during the final 1 sec of each 7-sec cue. (b) Across training, these rats developed conditioned behavior (i.e., locomotion) in response to cue presentations, relative to unpaired controls, that did not differ in likelihood when compared with 5-sec laser paired conditioning groups (2-way repeated measures (RM) ANOVA, session X group interaction, F(2,32) = 66.44, p < 0.0001; post hoc comparisons with paired and unpaired groups). (c) The onset latency of conditioned behavior during cue presentations decreased across training for 1 and 5-sec paired stimulation groups (main effect of session F(1,19) = 60.96, p < 0.0001; no effect of stimulation group, F(1,19) = .0446, p = .835). Data expressed as mean ± SEM. ***p < 0.0001.
(a) AAVs containing cre-driven GCaMP6f and ChrimsonR were co-injected into the midbrain in TH-cre rats. (b and c) This led high GCaMP6/ChrimsonR co-expression and specificity to TH+ neurons within the recording area below optic fiber placements. (d) Delivery of 20 or 100 5-ms 590-nm laser light pulses resulted in rapid increases in GCaMP6f fluorescence (depicted as ΔF/F, the change in fluorescence during the stimulation period over baseline, n = 5 rats) that showed stable peak levels, and rapid offset. (e) Intracranial self-stimulation was used to assess the effectiveness of ChrimsonR activ ation to support behavior. (f) ChrimsonR activation via 590-nm laser delivery to dopamine neurons in the midbrain supported robust self-stimulation behavior (n = 7), measured as nose pokes, that rapidly extinguished when a 473-nm laser was substituted (2-way repeated measures ANOVA, session X response type interaction, F(2,12) = 37.27, p < 0.0001; post hoc comparisons with inactive responses). (g) Optogenetic activation of dopamine neurons was compared to activation produced by sucrose (n = 5). Delivery of 100 5-ms 590-nm laser pulses produced an overall pattern and duration of increased fluorescence that was similar to that produced by consumption of 0.1 ml of a 10% sucrose solution. (h) Example whole session trace of the demodulated 465-nm LED signal. (i) Example whole session trace of the demodulated 405-nm LED signal. (j) Trace shown in (i) after applying a least-squares fit. (k) Normalized 465 signal (ΔF/F) = (465-nm signal – fitted 405-nm signal)/(fitted 405-nm signal). Laser-evoked fluorescence is denoted by the orange bars. Data expressed as mean ± SEM. ***p < 0.0001.
VTA cre+ paired rats developed (a) cue approach conditioned behavior, relative to cre+ unpaired and cre- control groups. (b) No SNc cre+ paired rats developed cue approach.
Supplementary Figure 7 Proximity to cue location at cue onset and stimulation hemisphere are not related to behavioral results.
(a) The location of each rat in the experimental chamber was recorded at the onset of each cue presentation. The average location at cue onset across training was determined by assigning a value of “1” to a trial if the rat was located on the side of the chamber containing the cue light, or a value of “2” to a trial if the rat was located in the back half of the chamber at cue onset. (b) The average location at cue onset for cre+ paired VTA and SNC rats did not differ, nor did it change across training (2-way RM ANOVA, no effect of group, F(1,14) = 3.178, p = 0.0963; no interaction, F(3,42) = 0.706, p = 0.554). (c-f) The probability of locomotion, approach, and rotation during cue presentations did not differ across rats depending on the hemisphere in which they received optogenetic stimulation. Data expressed as mean ± SEM.
(a) Behavioral videos were recorded during optogenetic Pavlovian conditioning. (b-e) Videos from the final day of conditioning were analyzed using Ethovision software to localize the frame-by-frame position of each rat’s head within the experimental chambers. Depicted are screenshots of an example rat from each experimental group, superimposed with the behavior tracking data for three cue-laser trials for that subject. Rats in the paired groups exhibited movement during the cue period (cue directed for the VTA rats, rotational for the SNc rats), while unpaired rats were immobile.
Supplementary Figure 9 Similar light-evoked responses in ventral and dorsal striatal projecting dopamine neurons.
(a) Among quiescent neurons, both NAc projectors and DS projectors showed high fidelity up to 50-Hz stimulation. Fidelity was observed in response to both 1-ms and 5-ms pulse trains. Each marker represents a cell, but some cells were tested with more than one frequency. (b) Summary of the locations of the recorded neurons in the horizontal slice. DS projectors were localized in the substantia nigra pars compacta (SNc) and NAc projectors were located in the VTA. (c) Example recording in a ChR2 expressing VTA neuron that was also firing spontaneously during recording. Although the LED light stimulation did increase the firing rate of the cell (lower panel), the increase in firing was not due to AP firing time-locked to the light pulses (upper panel). (d) Example spontaneous firing in the same cell, without light stimulation. (e) Summary of the impact of light pulses on spontaneously firing, ChR2 expressing neurons. While fidelity was moderate, stimulation did increase the firing rate in these cells.
Supplementary Figure 10 Retrograde targeting of dopamine neurons reveals projection-specific expression patterns in the midbrain.
(a) Transfection in striatum of TH-cre rats with a retrogradely-transported DIO-ChR2-eYFP construct resulted in robust expression of ChR2-eYFP in TH+ cells in the midbrain, but the expression pattern varied according to striatal target. (b) Summary of expression patterns for NAc shell (n = 5 rats, 6-10 slices per rat), NAc core (n = 4), and dorsal striatum (n = 5) projecting dopamine neurons. Shell projections were concentrated in the ventromedial VTA, while core projections were concentrated in the laterodorsal VTA. Projections to the dorsal striatum were localized throughout the SNc.
Supplementary Figures 1–10
Cue approach in a VTA Cre+ paired rat.
Cue presentation for a Cre+ unpaired rat.
Laser delivery for a Cre+ unpaired rat.
Cue presentation for a Cre– paired rat.
Cue-evoked rotation in an SNc Cre+ paired rat.
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Saunders, B.T., Richard, J.M., Margolis, E.B. et al. Dopamine neurons create Pavlovian conditioned stimuli with circuit-defined motivational properties. Nat Neurosci 21, 1072–1083 (2018) doi:10.1038/s41593-018-0191-4
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