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Rapid signalling in distinct dopaminergic axons during locomotion and reward

Abstract

Dopaminergic projection axons from the midbrain to the striatum are crucial for motor control, as their degeneration in Parkinson disease results in profound movement deficits. Paradoxically, most recording methods report rapid phasic dopamine signalling (~100-ms bursts) in response to unpredicted rewards, with little evidence for movement-related signalling. The leading model posits that phasic signalling in striatum-targeting dopamine neurons drives reward-based learning, whereas slow variations in firing (tens of seconds to minutes) in these same neurons bias animals towards or away from movement. However, current methods have provided little evidence to support or refute this model. Here, using new optical recording methods, we report the discovery of rapid phasic signalling in striatum-targeting dopaminergic axons that is associated with, and capable of triggering, locomotion in mice. Axons expressing these signals were largely distinct from those that responded to unexpected rewards. These results suggest that dopaminergic neuromodulation can differentially impact motor control and reward learning with sub-second precision, and indicate that both precise signal timing and neuronal subtype are important parameters to consider in the treatment of dopamine-related disorders.

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Figure 1: Locomotion related signalling in dorsal-striatal-projecting dopamine axons.
Figure 2: Phasic dopamine signalling displays a sub-second timing preference with respect to acceleration bursts.
Figure 3: Pulsed optogenetic stimulation of dorsal-striatum-projecting dopamine axons can rapidly initiate locomotion and control acceleration frequency.
Figure 4: Functional heterogeneity and anatomical origin of dorsal-striatum-projecting dopamine axons.
Figure 5: Functional topography of reward and locomotion dopamine signalling across striatum dorsal-ventral axis.

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Acknowledgements

We thank A. Graybiel and members of the Dombeck laboratory for comments on the manuscript, and V. Jayaraman, R. Kerr, D. Kim, L. Looger and K. Svoboda from the GENIE Project for GCaMP6. This work was supported by Klingenstein Foundation, McKnight Foundation, Whitehall Foundation, Chicago Biomedical Consortium with support from the Searle Funds at Chicago Community Trust, Northwestern University, National Institutes of Health (T32 AG20506).

Author information

Authors and Affiliations

Authors

Contributions

M.W.H. performed the experiments, D.A.D. built the experimental apparatus, M.W.H. performed data analysis with strategy suggestions from D.A.D. Both authors conceived and designed the experiments, interpreted the data and wrote the paper.

Corresponding authors

Correspondence to M. W. Howe or D. A. Dombeck.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Synchronized dopamine projection axon dynamics across a single field in dorsal striatum.

a, Representative mean fluorescence image in dorsal striatum of a dense field of dopamine axons (compare to the sparse fields from sparse labelling in Fig. 4c, i) from one mouse (out of six) labelled with GCaMP6f. b, Coronal schematic showing approximate location and scale of region imaged at top. Arrow indicates the approximate range of medial/lateral positions used for two-photon imaging (see Methods). c, ΔF/F traces for the rectangular ROIs indicated in a. d, Correlation matrix for the ROIs indicated in a. Note high degree of transient co-activation across ROIs. e, Mean image of fluorescence during the transient indicated by the arrow in c minus mean image of fluorescence during non-transient periods for the field shown in a. Note that the morphology of active regions closely resembles the morphology of GCaMP6f-expressing axons in the whole field in a, indicating synchronous activation of large, dense regions of axons, likely belonging to several different parent neurons.

Extended Data Figure 2 Further characterization of two-photon imaging and analysis methods.

a, b, Top, example mean fluorescence images of a putative single SNc axon imaged over 2 consecutive days in one mouse. c, d, Mean images of fluorescence during locomotion periods minus mean image of fluorescence during reward periods for fields in a and b. White axonal regions indicate regions of elevated signalling during locomotion. Note the similar morphology and behaviour signalling of the identified axon (red arrow) over days. e, Acceleration (black) and ΔF/F (green) for the identified axon in ad across the two imaging days. Note the similar transient amplitudes and the elevated transient signalling during locomotion acceleration periods. f, Mean transient ΔF/F (mean of significant transients, excluding baseline periods) during locomotion and rest on days 1 and 2 for the axon shown in ad. g, Histograms of calcium transient duration times across all putative single axons imaged in dorsal striatum from SNc (n = 3,556 transients, 5 mice top) and VTA (n = 5140 transients, 5 mice bottom). Note the similar duration profile across the two populations (medians not significantly different, P > 0.05 Wilcoxon sign-rank test). h, Histograms of maximum calcium transient amplitudes across all putative single axons imaged in dorsal striatum from SNc (top) and VTA (bottom) (medians not significantly different, P > 0.05 Wilcoxon sign-rank test). i, Post-mortem image of a coronal section from a representative mouse showing the striatum imaging cannula window cortical lesion site. j, Post-mortem image from a different mouse than i that was used for fibre photometry recording (fibre track indicated by arrow). k, Similarity of raw (no baseline normalization) whole-field GCaMP6f F trace (top) with baseline normalized ΔF/F trace (bottom) for the example whole-field imaging session shown in Fig. 1e. In the top trace, note the lack of baseline change over the recording session and particularly the stability of the baseline level during locomotion periods. Bottom trace is duplicate of trace in Fig. 1e.

Extended Data Figure 3 Interpretation of mechanisms underlying calcium transients and characterization of putative single dopamine axon calcium transients.

a, Left, mock trace representing expected GCaMP6f calcium transient from a short millisecond timescale influx of calcium (arrows; for example, short burst of action potentials over tens of milliseconds; local modulation may also contribute to calcium influx). Right, three representative low-amplitude, short duration calcium transients (from putative single dopamine axons in dorsal striatum) (see Methods, Fig. 4) that display onset and decay kinetics consistent with mock transient (left). b, Left, mock trace representing expected GCaMP6f calcium transient from multiple calcium influx events separated by less than the indicator decay time (arrows; for example, longer burst of action potential firing over ~100s of milliseconds). Right, representative larger amplitude calcium transients (from putative single dopamine axons in dorsal striatum) with rapid rise times consistent with mock transient (left). c, Left, mock trace representing expected GCaMP6f calcium transient from a sustained increase in the rate of influx events separated by less than the decay time (for example, sustained increase in action potential firing). Right, representative trace of one of the longest duration calcium transients observed (from putative single dopamine axon in dorsal striatum). Note that no sustained increases (baseline shifts) similar to the mock trace (left) were observed in single axon recordings or whole-field ΔF/F measurements; however, the long duration transient shown (right) indicates that if such sustained increases had occurred, they would have been detected using our methods. Also, note that the mock traces shown in ac are for descriptive purposes and are not based on new data. These traces are based on two main assumptions: (1) ΔF/F is a monotonically increasing function of intracellular calcium concentration, which itself is a monotonically increasing function of the number of underlying action potentials (that is, a greater number of action potentials leads to a larger ΔF/F, but the relationship is not necessarily linear); and (2) ΔF/F transients summate (not necessarily linearly) when they overlap in time. d, Duration versus peak ΔF/F for all identified significant calcium transients in putative single SNc originating axons (see Methods, Fig. 4, n = 3,556 transients from 73 axons in 5 mice; Spearman’s Rho = 0.3 P < 10−10). e, Histogram of sustained locomotion period durations (from SNc injected mice, n = 5, top) and calcium transient durations for all putative single SNc axons (n = 5 mice, mid) and all whole-fields (n = 6 mice, bottom). Note that the median calcium transient duration (for either single axon or whole-field) is far less than the median locomotion duration, indicating that the increase in dopamine axon GCaMP6f ΔF/F observed during locomotion is due to an increase in relatively short duration calcium transients, rather than long-duration (sustained) increases in ΔF/F.

Extended Data Figure 4 Further characterization of acceleration-associated dopamine signalling.

a, Representative whole-field ΔF/F fluorescence trace (one field from one out of six mice, green) aligned to treadmill acceleration (black) during a locomotion onset (dashed line: onset). b, Video frames of mouse for time points shown in a. c, d, Same as a and b but for a period of continuous locomotion. e, Top, normalized spectral power of treadmill acceleration trace during continuous locomotion periods for each two-photon imaging session (each row represents a session, n = 6 mice). Bottom, normalized mean power from all sessions shown in e, top. f, Top, mean whole-field ΔF/F trace triggered on reward delivery time for all fields; each row is mean for each field/session (n = 22 fields, 6 mice). Bottom, mean treadmill velocity (black), mean whole-field ΔF/F (green), and spout licking (light blue) all triggered on reward time (mean across all 22 fields/sessions in 6 mice). g, Mean whole-field ΔF/F (top), velocity (mid), and acceleration (bottom) for trials in which reward was delivered mid-locomotion (n = 13 sessions, 4 mice, red) or when animals were at rest (n = 12 sessions, 4 mice, blue). Note the sharp decrease in ΔF/F relative to baseline when animals decelerated from locomotion to consume the reward and the relative absence of phasic reward signalling when animals were given reward from rest. Reward responses were also not observed in single SNc axons when animals received reward from rest (see Extended Data Fig. 9). h, Comparison of mean whole-field fluorescence change from significant calcium transients (excluding baseline periods) between locomotion and resting periods; each point represents mean ΔF/F for running or resting over one session for each field (lines connect same field/session). All fields included here were imaged before mice ever receiving any rewards on the treadmill (n = 14 fields, 4 mice). *P < 10−5 (Wilcoxon rank-sum test). i, top, Mean acceleration (black) and whole-field ΔF/F (green) triggered on acceleration onsets (mean across all fields) during continuous locomotion. Bottom, mean acceleration (black) triggered on all short duration calcium transients (green, mean of transients) during continuous locomotion across all fields. All fields included here were imaged before mice ever receiving any rewards on the treadmill (n = 14 fields, 4 mice). j, Top, whole-field ΔF/F from all locomotion initiations in a representative single session (single imaging field, single session, one out of 6 mice); each row represents a single locomotion initiation time period (sorted by peak ΔF/F time). Mid, treadmill accelerations corresponding to locomotion initiations shown in j, top. Bottom, average of acceleration (black) and ΔF/F (green) across all locomotion onset traces displayed in Top and Mid panels. k, same as j, but for continuous locomotion periods. l, Reproduction of Fig. 2a with zoomed-in time axes to show the timing of the mean DF/F in relation to the first acceleration at locomotion initiations from rest. Shaded red region indicates bins that were significantly (*P < 0.01, Wilcoxon sign-rank, n = 15 fields in 6 mice) elevated relative to rest baseline. Shaded region covers ~107 ms before acceleration onset. m, Reproduction of Fig. 2b with zoomed-in time axes to show the timing of the mean dopamine transient in relation to the accelerations during continuous locomotion (n = 18 fields, 6 mice). n, ROC curves for each two-photon whole-field ΔF/F trace (n = 22 fields from 6 mice, grey; mean, black line) assessed for ability to discriminate locomotion versus resting periods (21 out of 22 exhibited significant discriminability, P < 0.01). Area under the curve (AUC) = 0.76 ± 0.02 (mean ± s.e.m.). o, ROC curves for each two-photon whole-field ΔF/F trace (n = 17 fields in 6 mice, grey; mean, black line) assessed for ability to discriminate pre-locomotion onset rest periods (250 ms before onset) from other rest periods (10 out of 17 exhibited significant discriminability, P < 0.01, two sessions included did not meet onset criteria for Fig. 2a, Methods). AUC = 0.58 ± 0.02 (mean ± s.e.m.). Dashed red lines indicate the line of no discrimination. Shaded regions in f, g, i, l, m denotes mean ± s.e.m.

Extended Data Figure 5 Dopamine axon calcium transients are temporally associated with preceding acceleration bursts and their amplitude is correlated with both preceding and subsequent acceleration bursts.

a, b, Distribution of latencies from each significant calcium transient onset (mean whole-field fluorescence; 6 mice) to the first acceleration burst onset within 1 s preceding (n = 1,087, 6 mice, a) or following (n = 990, 6 mice, b) during continuous locomotion. Latencies are less variable (F-test for difference between variance of latencies, P = 7.1 × 10−5) and shorter (Wilcoxon test for difference between latency means, P = 1.2 × 10−5) to the preceding acceleration onsets, indicating more precise relative timing between the GCamp6f transients and the preceding acceleration burst versus the following acceleration burst. c, d, Mean acceleration traces from the first acceleration (within 1 s) preceding (c) or following (d) all short duration (<0.5 s) large amplitude (>75th percentile, n = 149 transients, grey) and small amplitude (<25th percentile, n = 149 transients, bronze) calcium transients occurring during continuous locomotion; aligned on acceleration onsets. Insets are schematics of the GCaMP6f transients. A significant correlation is present between the transient amplitudes and the immediately preceding acceleration amplitudes (Spearman’s Rho = 0.16, P = 1.2 × 10−4, from all transient-acceleration pairs; binned data from this plot shown in c). A significant correlation is also present between the transient amplitudes and the immediately following acceleration amplitudes (Spearman’s Rho = 0.13, P = 0.006, from all transient-acceleration pairs; binned data from this plot shown in d). e, Schematic summarizing relationship between the timing and amplitude of dopamine axon calcium transients and acceleration bursts during continuous locomotion. f, Mean acceleration (black) and whole-field ΔF/F (green) all triggered on all accelerations during continuous locomotion that were less than 1.7 m s−2 in amplitude (n = 596 accelerations, n = 6 mice); this demonstrates that dopamine axon GCaMP6f signalling displays a timing preference with respect to small amplitude accelerations, with similar timing and amplitude to that shown in Fig. 2b (which includes both large and small amplitude accelerations).

Extended Data Figure 6 Pulsed optogenetic stimulation of dorsal-striatum-projecting and ventral-striatum-projecting dopamine axons.

ad, Pulsed optogenetic stimulation of dorsal-striatum-projecting dopamine axons can entrain accelerations during locomotion. eh, Pulsed optogenetic stimulation of ventral-striatum-projecting dopamine axons leads to little effect on locomotion. a, b, Representative acceleration traces from continuous locomotion periods during (and initiated by) 6 Hz (a) and 3 Hz (b) laser stimulation trains in the same mouse. Blue, laser stimulation train, one mouse out of seven. c, d, Mean accelerations triggered on individual laser burst onsets during continuous locomotion periods for 6 Hz (c) and 3 Hz (d) across all laser bursts in all mice and sessions (n = 7 mice). e, Mean absolute value of mouse accelerations aligned on onset of laser stimulation train applied to mice at rest (mean across all stimulation onsets, n = 55 and 91 for ventral and dorsal respectively, in all sessions and mice, 3 and 6 Hz stimulation included). Dorsal and ventral striatum stimulations are from same ChR2-expressing mice (n = 4). Three mice were not stimulated in ventral striatum and thus not included in this figure. Mean acceleration elicited by ventral stimulation was significantly (P < 0.01, Wilcoxon rank-sum test) less than that elicited by dorsal axon stimulation. However, acceleration from ventral stimulation was significantly greater than chance (P < 0.01 shuffle test). This small effect in the ventral striatum could be due to activation of fibres, which also project to the dorsal striatum or to an increase in arousal. Although note that acceleration frequency during locomotion was not altered for stimulation in ventral striatum (see f, h). f, g, Centre of mass of acceleration power spectra for each mouse for locomotion periods initiated during 3 or 6 Hz stimulations (n = 4 mice). Horizontal bars indicate means, lines connect same mouse. h, Mean difference between the centre of mass of the acceleration power spectra computed for locomotion periods initiated during 3 Hz or 6 Hz axon stimulations in ventral (f) or dorsal (g) striatum. Positive values indicate a shift towards higher frequency accelerations for 6 Hz stimulations. *P < 0.05, Wilcoxon rank-sum test.

Extended Data Figure 7 Histology and response distributions from each sparsely injected mouse.

a, GCaMP6f expression (green) and immunofluorescence (red) from all VTA-targeted mice (n = 5). b, Reward response versus locomotion index (as in Fig. 4n) for each axon recorded from the corresponding mice in a. Green, significant locomotion; red, significant reward; blue both significant; neither significant not shown. c, d, Same as a and b, except from all SNc-injected mice (n = 5). Six out of ten mice were not stained for tyrosine hydroxylase. Scale bars, 500 μm.

Extended Data Figure 8 Distribution of reward and locomotion indexes and fraction of reward and locomotion signalling axons from VTA and SNc are highly similar using different correlation thresholds for clustering axon segments (a, b); single axon signaling timing during locomotion onset versus continuous locomotion (c–e).

a, Reward response versus locomotion index for putative single axons from SNc (n = 5 mice, top row) and VTA (n = 5 mice, bottom row) using different correlation thresholds (no clustering, 0.5, and 0.2) for hierarchical clustering of activity patterns (see Methods). Axons are colour-coded by significant responses to locomotion (green), reward (red), or both (blue). Note that despite the total number of putative axons decreasing with correlation threshold, the inverse relationship between locomotion and reward signalling across the population remains the same. b, Table showing the total numbers and fractions of responsive axons across the VTA and SNc populations for different clustering thresholds. Note that despite the total number of putative axons decreasing with correlation threshold, the fraction of axons signalling either reward, locomotion, both or neither is highly similar. c, Correlations (Pearson’s) between acceleration and selected putative single SNc axon ΔF/F traces at different relative time-lags (that is, cross-correlations) during locomotion initiation periods; each row is mean for each axon for a single session (axons from n = 3 out of 5 mice). d, Same as c, but during continuous locomotion periods; same axons during same sessions as in c. e, Peak cross-correlation times for data shown in c and d (lines connect same axons during same sessions).

Extended Data Figure 9 Further characterization of putative single dopamine axons in relation to reward and licking.

a, Mean ΔF/F trace for VTA reward responsive axons (Methods, n = 23 axons, n = 4 mice with variable reward sessions) (top), velocity (mid), and licking (bottom) triggered on large volume (red), small volume (blue) and omission (black, solenoid click was present, but no reward delivered; Methods, n = 17 axons, 3 mice with omission sessions) reward deliveries. b, Same as a except for SNc locomotion responsive axons (n = 62 and 18 axons for reward and omission traces respectively). c, Mean VTA reward axon ΔF/F trace (top) and velocity (bottom) triggered on reward deliveries during continuous locomotion (red, n = 25 axons) or rest periods (blue, n = 37 axons). d, Same as c except for SNc locomotion responsive axons (n = 25 and 62 axons for locomotion and rest respectively). e, Mean VTA reward axon ΔF/F (top) and mouse licking (bottom) triggered on spontaneous, non-reward licking onsets (n = 15 axons, 3 mice). f, Same as e except for SNc locomotion responsive axons (n = 15 axons, 3 mice). Mice that did not lick outside reward periods were excluded. Shaded regions in af denote mean ± s.e.m.

Extended Data Figure 10 Dopamine axon locomotion signalling measured by fibre photometry from different striatal sub-regions.

a, Top, comparison of mean photometry fluorescence ΔF/F (mean of significant transients, excluding baseline periods) recorded from dorsal striatum between locomotion and resting periods; each point represents mean ΔF/F for running or resting over one session for recording from a single dorsal striatum location (lines connect same recording location/session; n = 5 mice). Bottom, comparison of mean baseline (periods with no significant calcium transients) photometry ΔF/F recorded from dorsal striatum between locomotion and resting periods; each point represents mean baseline ΔF/F for running or resting over one session for recording from a single dorsal striatum location (lines connect same recording location/session, n = 5 mice). b, c, Same as a, except for recordings from central and ventral striatum, respectively. d, Mean photometry ΔF/F recorded from dorsal striatum triggered on locomotion initiations (mean across all initiations, n = 5 mice). e, f, Same as d, except for recordings from central and ventral striatum, respectively. g, Locomotion index (top) and reward response (bottom) versus striatum recording depth (from data presented in Fig. 5). h, Schematic of prominent current model for dopamine signalling dynamics in the striatum. i, Schematic of our new model for dopamine signalling dynamics based on data presented here. j, k, Saggital schematics illustrating current homogenous dopamine signalling model (j) and our new model incorporating functional heterogeneity (k). **P < 0.01, Wilcoxon rank-sum test; n.s., not significant. Shaded regions in df, mean ± s.e.m across initiations (n = 20, 28 and 56 from dorsal, central and ventral respectively in 5 mice).

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Howe, M., Dombeck, D. Rapid signalling in distinct dopaminergic axons during locomotion and reward. Nature 535, 505–510 (2016). https://doi.org/10.1038/nature18942

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