Striatal dopamine signals are region specific and temporally stable across action-sequence habit formation

Abstract

Habits are automatic, inflexible behaviors that develop slowly with repeated performance. Striatal dopamine signaling instantiates this habit-formation process, presumably region specifically and via ventral-to-dorsal and medial-to-lateral signal shifts. Here, we quantify dopamine release in regions implicated in these presumed shifts (ventromedial striatum [VMS], dorsomedial striatum [DMS], and dorsolateral striatum [DLS]) in rats performing an action-sequence task and characterize habit development throughout a 10-week training. Surprisingly, all regions exhibited stable dopamine dynamics throughout habit development. VMS and DLS signals did not differ between habitual and non-habitual animals, but DMS dopamine release increased during action-sequence initiation and decreased during action-sequence completion in habitual rats, whereas non-habitual rats showed opposite effects. Consistently, optogenetic stimulation of DMS dopamine release accelerated habit formation. Thus, we demonstrate that dopamine signals do not shift regionally during habit formation and that dopamine in DMS, but not VMS or DLS, determines habit bias, attributing "habit functions" to a region previously associated exclusively with non-habitual behavior.

Keywords: action repetition; automated behavior; basal ganglia; behavior; dopamine; goal-directed behavior; habit formation; habits; rat; striatum.

Graphical abstract

Figure 1

A seeking-taking chain task to monitor dopamine signaling during habit formation (A) A VI60:FR1 trial begins with seeking-lever extension. Seeking-lever presses (“distal”) are registered until the final VI60 press (“intermediate”), which triggers seeking-lever retraction, followed by taking-lever extension 3 s later. Approximately 1 s later, animals press the taking lever (“proximal”), it is retracted, and the food-pellet reward is delivered 3 s later, marking the start of the variable inter-trial interval (ITI). (B) Number of seeking-lever presses (left) and food-magazine head entries (middle) during the VI60 segment. The probability of a food-magazine head entry during the 3-s period following seeking-lever retraction and prior to taking-lever extension (right) decreases rapidly with training, demonstrating task acquisition. Data are mean ± SEM (black) with individual animals (gray). (C) Rats that were trained for 10 weeks underwent outcome devaluation via sensory-specific satiety (pre-feeding), in which, on two separate days, rats had 1-h ad libitum access to either regular pellets (bottom) or alternative (grain-based) pellets (top) before they were exposed to the seeking-lever (extinction) test. Seeking was unaffected by outcome devaluation, demonstrating that a habit was induced. Subsequently, rats were exposed to both pellets (“choice test”) to demonstrate that outcome-specific devaluation was successful (non-pre-fed pellets preferred; p < 0.001). (D) Coronal brain sections with recording sites in VMS (blue), DMS (green), and DLS (red), relative to bregma. (E) Representative verification of electrode-tip placement in VMS (blue circle), DMS (green circle), and DLS (red circle) and corresponding electrode tracks (arrows). (F) Dopamine release to an unpredicted food pellet demonstrates stable electrode sensitivity. Data are median with range in quartiles and mean (+); ns, not significant. (G) DMS dopamine following seeking-lever extension (distal) was modestly diminished after 10 weeks. No significant differences (ns) were observed in VMS or DLS. (H) During proximal actions, no significant differences (ns) were detected in dopamine release between weeks 1 and 10. ^∗p < 0.05, ^∗∗∗p < 0.001. See also Figure S1.

Figure 2

A novel preference test to measure the development of habits (A) Habit formation was tested by presenting both levers simultaneously for 1 min under extinction conditions (left). In week 1 of VI60:FR1 seeking-taking training, rats preferentially pressed the taking lever, but by week 10, preference had shifted to the seeking lever (right). (B) In the habit group, the number of food-magazine head entries remained stable (top) across weeks, but rats switched from a taking- to a strong seeking-lever preference (middle) and exhibited more seeking than taking presses in week 10 (bottom). (C) Food-magazine head entries remained stable in the no-habit group (top), but rats switched from a taking-lever preference to no preference (middle) and exhibited no significant difference between seeking and taking presses in week 10 (bottom). (D and E) Habitual rats performed more seeking-lever presses (D) and fewer taking-lever presses (E) than non-habitual rats in week 10, but not in week 1. (F) The seeking-taking preference index demonstrates a higher seeking-lever preference in habitual rats compared to non-habitual rats in week 10, but not in week 1. (G) The conditioning “logic” translated from training to preference test: food-magazine head entries were executed more frequently after taking-lever presses than after seeking-lever presses, both in habitual and non-habitual rats. Histogrammed data with mean ± SEM shown separately. (H and I) Linear regression analyses demonstrate associations of the seeking-lever index with other habit-like behavior during the preference test, i.e., (H) a positive correlation with seeking-lever stickiness and (I) a negative correlation with food-magazine head-entry probability. Data are mean ± SEM, in some panels combined with individual animals (open circles). ^∗p < 0.05, ^∗∗∗p < 0.001. See also Figures S2 and S3.

Figure 3

Diametrically opposing DMS dopamine signals in habitual and non-habitual rats during distal and proximal reward seeking (A) Habitual animals showed a higher frequency of distal seeking-lever presses during the VI60 trial segment compared to non-habitual rats (middle left). Conversely, habitual animals exhibited fewer food-magazine head entries during VI60 (middle right). However, this difference in head entries was not present outside VI60, when no levers were extended (left). Similarly, no group differences were observed for the head-entry probability during the 3-s period following seeking-lever retraction and prior to taking-lever extension (right). Data are mean ± SEM. (B) Dopamine release to an unpredicted food-pellet delivery (proxy for electrode sensitivity) did not differ between groups. Data are median with range in quartiles and mean (+); ns, not signifcant. (C) During distal actions (start of VI60), no significant dopamine group differences were observed in VMS or DLS. However, DMS dopamine was greater in habitual rats in both weeks 1 and 10. Data are mean + SEM; traces are aligned to seeking-lever extension. (D) During proximal actions, no significant group differences in dopamine were observed in VMS or DLS. However, again, DMS dopamine differed between habitual and non-habitual rats, but in contrast to distal actions, proximal events significantly decreased dopamine in habitual animals in both weeks 1 and 10. Data are mean + SEM; gray-shaded areas indicate approximate final seeking-press timing. (E) To evaluate the association of dopamine with reward seeking (distal actions: lever presses early in the VI60 segment), trial-by-trial correlations were calculated for week 1 (top) and week 10 (bottom). Gray bars display the animals’ R values distributed over given histogram brackets. Each circle represents a single animal (habitual in orange, non-habitual in brown), and significant correlations are depicted as filled circles and non-significant correlations as empty circles. VMS demonstrated the closest relationship between dopamine and seeking-lever presses. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. See also Figure S4.

Figure 4

Optogenetic stimulation of DMS dopamine during seeking accelerates habit formation (A) AAV injection into substantia-nigra pars-compacta (SNpc) of Th::Cre rats induced ChR2-EYFP or EYFP (control) expression in dopamine neurons. DMS dopamine terminals were optogenetically stimulated bilaterally. (B) Immunostaining for tyrosine hydroxylase (TH) and EYFP indicates selective virus expression in midbrain dopamine neurons (bottom) and their striatal terminals (top). SNR, substantia nigra pars reticulata; VTA, ventral tegmental area. (C) Percentage of time spent in the DMS-photo-stimulated (active) quadrant of an open-field arena. No significant difference (ns) was observed between ChR2 and EYFP rats. (D) Nose pokes into the active port triggered DMS dopamine photo stimulation. We observed no significant differences (ns) within or between ChR2 and EYFP animals in the number of active and inactive nose pokes. (E) During seeking-taking training, seeking presses triggered DMS dopamine photo stimulation. The conditioning “logic” translated from training to preference test: food-magazine head entries were executed more frequently after taking-lever presses than after seeking-lever presses, both in habitual and non-habitual rats. Histogrammed data with mean ± SEM shown separately. (F) No significant differences were found in frequency of food-magazine head entries outside VI60, when no levers were extended. (G) ChR2 animals made more distal VI60 seeking-lever presses (left). No significant difference was found for food-magazine head entries during VI60 (right). (H) Similarly, no group differences were observed for the head-entry probability during the 3-s period following seeking-lever retraction and prior to taking-lever extension (right). (I) In week 1, both ChR2 and EYFP rats preferentially pressed the taking lever during the habit test. By week 3, this preference had shifted to the seeking lever for the ChR2 rats, but not the EYFP rats. (J) The ChR2 group made significantly more seeking-lever responses, but groups did not differ in taking-lever presses. Data in (E) and (F) are mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01.