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Collectively, these findings support a key role for the aDLS in consolidating newly learned instrumental actions. MSNs that express dopamine D1 receptors comprise a direct pathway from the basal ganglia striatonigral cells , whereas MSNs that express D2 receptors comprise an indirect pathway striatopallidal cells.

D1 and D2-MSNs often have opposing roles in motor control, reward, and motivation 34 , 35 , 36 , but less is known about their contributions to striatal-dependent learning processes. To investigate the potential involvement of D1 and D2-MSNs in new instrumental learning, we assessed changes in the morphology of dendritic spines expressed by MSNs in the aDLS as a marker of learning-related structural plasticity. We found that spine head diameter Fig.

Also represented is the identification and processing of regions of interest ROIs from calcium imaging data, and representative calcium traces from ROIs collected during the post-learning consolidation period right panels. It was unclear if the learning-related structural plasticity in D1-MSNs reflected their involvement in the consolidation process or their more generalized recruitment during the training session when mice expressed the new instrumental response for the first time.

Therefore, to better understand the cellular mechanisms of consolidation, we used in vivo calcium imaging to monitor the activity of D1 and D2 cells in the aDLS during the critical period immediately after new instrumental learning.

After recovery, mice were habituated to the attachment of head-mounted microendoscopes miniscopes for 7 days 37 , then permitted to lever-press for 30 food rewards mice , according to the same procedure described above Supplementary Fig. For comparison, we also recorded neural activity immediately after the second magazine training session in these same animals, when food pellets were delivered noncontingently Fig.

We similarly recorded D1 and D2 MSN activity in these control mice immediately after magazine training and after the acquisition session. We detected a robust increase in D1 MSN activity in the mice during the post-learning consolidation period compared with their activity after magazine training Fig.

This is consistent with recent data suggesting that increases in D1 MSN activity throughout the entire dorsal striatum correlate with new instrumental leaning in mice when learning occurs across multiple training sessions Conversely, D2-MSNs showed a striking decrease in activity in the mice during this same period Fig.

In mice, we did not detect any changes in D1 MSN activity during the post-acquisition period compared with the post-magazine training baseline period Fig. However, D2 MSN activity was increased during the post-acquisition consolidation period compared with the baseline period in these mice Fig.

The fact that mice showed increased D2 MSN activity during the post-consolidation period, which is opposite to the decreased D2 MSN activity in mice during the same period, suggests that the failure of mice to consolidate the new lever-press response may not be a passive process that reflects poor learning because of a limited number of training opportunities.

Together, these findings suggest that consolidation of a new instrumental action is associated a dramatic shift in the balance of D1 and D2 MSN activity in the aDLS, with increased D1 MSN activity likely involved in consolidating the new response into long-term storage. Conversely, D2-MSNs may execute a quality control function, with post-learning decreases in their activity facilitating the consolidation of an advantageous new instrumental response and increases in their activity impeding the consolidation of non-beneficial action sequences.

The calcium imaging data described above suggest that D1-MSNs in aDLS encode new instrumental actions during the post-learning consolidation period and that a period of D2-MSN quiescence facilitates this process. To test these predictions, we investigated the effects of post-learning inhibition of D1 or D2-MSNs in the aDLS on consolidation of the new lever-press response.

Once again, the frequency of response bouts Fig. Cholinergic interneurons in the striatum also express dopamine D2 receptors 38 , raising the possibility that post-learning inhibition of cholinergic neurons in aDLS contributed to the increased consolidation observed in D2-Cre mice. The aDLS plays a well-established role in regulating the expression of reward-independent habitual actions Therefore, we next explored whether the same region of the aDLS targeted in our experiments, which consolidates newly learned instrumental actions, also regulates the expression of habitual responses.

This renders behavioral performance poorly correlated with reward delivery and thereby facilitates the emergence of goal-independent, habitual patterns of responding We found that vehicle-treated hM4Di-mCherry mice trained on the RI60 schedule responded in a habitual manner for food pellets, reflected by the fact that their lever-pressing behavior was unaffected by sensory-specific satiety-induced devaluation of the pellets Fig.

By contrast, CNO-treated hM4Di-mCherry mice reduced their responding only when food pellets were devalued but not when they were still valued Fig. This suggests that aDLS inhibition reduced the expression of habitual actions and restored the ability of mice to respond in a flexible reinforcer-dependent manner, confirming previous reports This suggests that response bouts and reward retrieval behaviors, which were closely linked when mice learned to respond for food rewards under an FR1 schedule see Supplementary Fig.

These findings confirm that the same region of the aDLS that consolidates new instrumental actions also controls the expression of habitual actions. Mice were pre-fed with chow valued condition or pellets devalued condition 1 , then permitted to lever-press under extinction conditions 2. Finally, we investigated whether the same cellular processes in aDLS that consolidate new instrumental actions also regulate the expression of habitual actions.

Similarly, chemogenetic inhibition of D2-MSNs had no effects on responding under the RI schedule when food pellets were still valued Fig. This is consistent with recent observations suggesting that D2-MSNs in aDLS undergo synaptic remodeling as habitual patterns of responding emerge 40 and that D2 receptor antagonists attenuate the expression of habit-like actions Together, these data suggest that D1-MSNs in aDLS regulate the consolidation of newly learned instrumental actions whereas D2-MSNs in the aDLS oppose this consolidation process and instead promote the expression of previously learned habitual actions.

In comparison to our considerable understanding of the molecular, cellular and circuit mechanisms of Pavlovian and other forms of learned associations 42 , 43 , 44 , 45 , much less is known about how new instrumental associations are encoded in the brain. Here, we show that the aDLS region of the striatum plays a critical role in consolidating new instrumental actions.

The aDLS is known to regulate the acquisition and expression of stimulus-response associations, in which conditioned stimuli in the environment come to elicit a behavioral response independent of any representation of the reward that originally reinforced that response Such value-free stimulus-response learning contributes to the development and persistence of habitual actions However, new instrumental learning often coincides with new sensorimotor learning, suggesting that some of the same brain systems may participate in both types of behavioral plasticity Our data support this hypothesis by establishing an important role for the aDLS in regulating new instrumental conditioning.

Our data also reveal a striking partitioning of cellular function in the aDLS in which D1-MSNs consolidate newly acquired instrumental actions and D2-MSNs oppose this consolidation process and instead promote the expression of habitual actions.

Finally, our data suggest that the aDLS acts in concert with the NAc during new instrumental conditioning to encode information relevant to reinforcer magnitude and effort requirements, respectively. In vivo cellular recordings have established that neurons in the aDLS are active during the earliest stages of new instrumental learning in rodents 16 , 18 , 32 , 47 , The putamen region of the human brain, considered equivalent to the rodent DLS, is similarly active during new instrumental learning 10 , Based on these findings, it was hypothesized that the DLS may participate in learning new instrumental responses in addition to its well-established role in stimulus-response learning Similarly, chemogenetic inactivation of the aDLS soon after instrumental learning, or inhibition of only those ensembles of aDLS neurons active soon after instrumental learning, impeded the ability of mice to consolidate the new instrumental action.

The frequency of response bouts was the feature of behavior that was most reliably impacted in rats and mice by post-learning aDLS manipulations. Propensity to engage in bouts of responding is thought to be influenced by the value of the reinforcer delivered by that same instrumental response 24 , 25 , 26 , Consistent with this link, we found that the propensity to engage in response bouts during a retention test was related to the value of the reinforcer available during the training session sucrose versus chow pellets.

Hence, the aDLS likely encodes information related to the perceived value of a new response during instrumental conditioning. This is surprising in light of the well-established role of the aDLS in regulating habitual actions that are executed in a value-independent manner As described in more detail below, this apparent discrepancy likely reflects the dissociable contributions of D1 and D2-MSNs in the aDLS to influence value-dependent and -independent behaviors, respectively.

Most previous studies investigating striatal mechanisms of instrumental conditioning have focused on the role of the NAc in this process 12 , 32 , 50 , 51 , Similarly, pharmacological blockade of NMDA or AMPA glutamate receptors 53 , 54 , 55 , 56 , D1 dopamine receptors 54 , 55 or muscarinic acetylcholine receptors 57 in the NAc prevented the acquisition of a new instrumental response in rats, reflected by low levels of lever-pressing for food rewards across training sessions compared with vehicle-treated rats.

Excitotoxic lesion of the NAc core also disrupted the acquisition of a lever-press response in rats 8. However, recent observations support a more nuanced reinterpretation of these data. For example, anisomycin infused into the NAc can trigger aversion to food items consumed soon afterwards 11 , suggesting that this manipulation may not block consolidation per se but instead decrease the perceived value of recently consumed food rewards.

This, in turn, would be expected to reduce the willingness of animals to engage in a new instrumental action that delivered a devalued food reward independent of any abnormalities in the consolidation process. Consistent with this interpretation, intra-NAc anisomycin infusion had no effects on consolidation of a new instrumental response when its inhibitory effects on food reinforcer valuation were prevented using a sucrose protection procedure Furthermore, glutamatergic, dopaminergic and muscarinic receptor antagonists disrupted the execution of a new instrumental action only when infused into the NAc before each daily training sessions but had no effects when infused after training This suggests that neurotransmission in the NAc regulates the performance of behaviors necessary to learn new instrumental actions but not the post-learning consolidation processes.

Similarly, lesions of the NAc disrupted new instrumental learning only when a delay was imposed between execution of the new action and delivery of the reward 8 , with lesioned animals learning at normal rates when food rewards were delivered without delay 8. This is consistent with proposals that the NAc, and dopaminergic transmission in this site, regulates effort-based decision-making According to these proposals, the NAc regulates willingness to engage in actions depending on the costs associated with obtaining associated rewards, such as the amount of effort that must be invested or the length of a delay that must be endured after executing an action but before reward delivery occurs 8 , 58 , 59 , Consistent with these observations, we found that disrupting protein synthesis in the NAc soon after new instrumental learning did not block the subsequent expression of the new response, although there was a non-statistically significant trend for decreased total lever-pressing during the retention test.

Instead, post-learning NAc infusions of anisomycin reduced the vigor of executing the new response, reflected by decreased density of response bouts. As noted above, bout density is thought to depend on the amount of effort required to obtain a reward, with increases in bout density occurring when greater effort is required Consistent with this interpretation, we found that increasing the amount of effort necessary to earn a food reward during the training session FR2 instead of FR1 schedule increased bout density during the subsequent retention test.

Hence, our findings support an important role for the NAc in encoding performance-relevant information during instrumental conditioning. This contrasts with the effects of post-learning infusions of anisomycin into the aDLS, which specifically decreased the frequency of response bouts during the retention test, suggesting that the aDLS encodes information relevant to reinforcer value during instrumental conditioning.

As the frequency and density of response bouts are dissociable aspects of the same instrumental response, this implies that the aDLS and NAc must act in a coordinated fashion during instrumental conditioning to encode discrete but closely related aspects of the same action sequence.

Whatever the underlying mechanisms, our findings support key roles for the aDLS and NAc in consolidating new instrumental actions and suggest that close interactions between these striatal regions is necessary for successful consolidation to occur. Using in vivo calcium imaging, we detected increased activity of D1-MSNs in the aDLS during the post-learning phase when a new instrumental response was consolidated, whereas D2-MSN activity was decreased during this same period.

Chemogenetic inhibition of D1-MSNs in aDLS during this period almost completely ablated the ability of mice to encode the new action, whereas post-learning inhibition of D2-MSNs dramatically strengthened this process. This portioning of function between D1 and D2-MSNs provides a parsimonious explanation for how the aDLS can regulate both value-dependent and -independent behaviors. Considering that inhibition of D2-MSNs in the aDLS dramatically strengthened the consolidation of new instrumental learning while also blocking habitual responding, it is likely that this cell population is the ultimate arbiters of whether new instrumental actions are encoded or previously learned habitual actions are expressed.

Finally, it is noteworthy that both D1 and D2-MSNs in the DLS regulated the propensity to engage in bouts of responding, with each cell type assuming control over bout frequency in different behavioral contexts. D1-MSNs regulated bout frequency when animals first learned a new instrumental response that delivered an unexpected reward, whereas D2-MSNs regulated bout frequency when animals expressed a previously learned action in a habitual manner.

As noted above, the frequency of response bouts appears to be related to the relative value of the action 24 , 25 , 26 , If so, this would suggest that MSNs in the aDLS serve to attribute value to actions when there is some ambiguity about the precise relationship between the action and its outcome. More specifically, D1-MSNs may attribute value to novel action sequences that unexpectedly deliver rewards to promote new instrumental learning, whereas D2-MSNs may attribute value to previously learned actions expressed autonomously because they deliver rewards in an unpredictable manner.

Consistent with this possibility, neural activity related to reward, action, and choice all converge in the DLS when the consequences of an action are ambiguous at the time of its execution, most prominently when there is a delay between action and reinforcer delivery In summary, our data support an important role for D1-MSNs in the aDLS in consolidating newly learned instrumental actions and suggest that D2-MSNs in aDLS oppose this new learning and instead promote the expression of previously learned habitual actions.

These findings have important implications for understanding how actions are acquired, stored, and expressed by the striatum. Animals were housed in groups 2 per cage for rats or per cage for mice in an environmentally controlled vivarium on a hh reversed light:dark cycle, with food and water available ad libitum until behavioral training commenced.

San Diego, CA. See Supplemental Table 2 for all primers used. Anisomycin Sigma was dissolved in an equimolar concentration of HCl, adjusted to pH 7. To test the effect of intracranial infusion into domains of the striatum of anisomycin or other agents on consolidation or expression of a new instrumental response by rats, we implanted bilateral gauge, stainless-steel guide cannula Plastics One, Wallingford, CT 1.

Standard stereotaxic techniques were used, and the cannula was secured to the skull using bone screws and dental cement. Twenty-nine-gauge obturators flush with the end of the guide cannula Plastics One, Roanoke, VA were inserted in the guide cannula. After surgery, rats were allowed a recovery period of at least 7 days before behavioral testing. Before the start of experimental infusions, the rats were habituated to the infusion procedure with a surrogate infusion, which consisted of the removal and replacement of the obturator during gentle restraint within a time course identical to that of drug infusion.

During infusions, the rats were gently restrained while the obturators were removed and a gauge bilateral injector, which protruded 1. The injector was then carefully removed, and the obturator replaced.

All infusions were delivered in the behavioral testing room. Bilateral injections 0. Following injection, mice were allowed to recover for at least 2 weeks before experimentation. Mice and rats were deeply anesthetized with an isoflurane and perfused through the ascending aorta with 0. The brains were removed and post-fixed in paraformaldehyde. Cannula locations were mapped onto standardized sections of the rat brain Paxinos and Watson, , and investigated for any signs of tissue damage.

Following additional washing, brains were dehydrated and cleared in methanol and dichloromethane, then refractive index matching occurred in dibenzyl ether. Brains were imaged at 1. The ClearMap Python package www.

Cells were detected with a peak intensity threshold of Cell objects were painted via a watershed until reaching this threshold, and only cells with sizes between 8 and continuous pixels were included. The Transformix module of the Elastix toolbox was then used to apply transformation vectors from the registration step to cellular coordinates, and cell counts for each region were calculated.

Custom scripts, as well as example parameter and process scripts containing all detailed cell detection parameters and thresholds, are available at www. Free-floating sections were permeabilized in 0. Cells were counted using ImageJ software. Tungsten particles 1.

Optimal sampling frequency was calculated using the Nyquist-Shannon sampling theorem. One segment from each neuron was quantified, and the minimum spine head diameter was set at 0. Between 6 and 12 neurons were imaged in each animal. Miniature fluorescent microscopes and data acquisition electronics were built using parts lists, schematics, and instructions available at miniscope.

D1-Cre and D2-Cre mice were single housed for these experiments in order to prevent cage-mates from interfering with lens implants. Mice underwent two surgical procedures. Artificial cerebrospinal fluid was continuously applied to prevent drying of exposed tissue. In the case of prolonged bleeding, a blunted gauge needle was used to remove blood with minimal additional tissue loss.

Cortical tissue was aspirated until horizontal striations of the corpus callosum were clearly visible. A set screw was then implanted into the contralateral skull and the GRIN lens was slowly lowered and fixed in place using cyanoacrylate dental cement. Mice were injected with the glucocorticosteroid dexamethasone 0. Two weeks later, a metal baseplate with a miniscope attached was positioned over the GRIN lens until optimal focal plane was observed.

The baseplate was then fixed in place using cyanoacrylate dental cement, and the miniscope was removed. A magnetic plastic cover was placed over the baseplate at all times except for during imaging. After habituation to the miniscope, mice were placed into the operant chamber and magazine training occurred, as described below. Experiments were carried out in sound-attenuated operant conditioning chambers Med Associates, St.

Albans, VT. The metal grid flooring was cleaned with a paper towel and 0. They were placed into operant conditioning chambers with the house-light turned off, the levers retracted, and underwent magazine training sessions on consecutive days.

After criterion numbers of pellets were earned 50 pellets for rats, 30 pellets for mice the acquisition session was ended, and animals returned to their home-cage. Consumption of all noncontingently delivered or earned pellets was visually confirmed for each animal after every session.

At the beginning of each RI60 session, the lever was extended in an inactive state. Each second there was a 1 in 60 chance that the lever would become active. When active, the next lever press response resulted in reinforcer delivery and the lever returned to an inactive state.

Mice that responded for food pellets under a RI60 schedule of reinforcement were allocated to vehicle and CNO groups, counter-balanced based on treatment history, cage-mate such that at least one mouse from each cage was in each condition , and the number of rewards earned during RI60 training. They were then injected with vehicle or CNO according to the experimental design see below and Main text and placed back into their home-cage. During the test session, mice lever-pressed for food rewards under extinction conditions under the or RI60 schedule i.

Each animal was tested twice, once in the valued and once in the devalued condition in an order counterbalanced by RI responding , but both times under the same treatment condition vehicle or CNO injection. Bilaterally cannulated rats underwent new instrumental conditioning as described above. With some slight modifications. The assignment of rats to these treatment groups was counterbalanced for time required to earn 50 pellets during the acquisition session.

After intra-striatal infusion, rats were returned to their home-cage and left undisturbed. Sucrose consumption was again recorded by weighing the bottle before and after the free consumption session.

In this manner we could determine whether intra-striatal anisomycin infusion caused aversion to of the sucrose solution. The next day, rats were placed in the operant conditioning chamber with the house-light off, and 80 food pellets were placed in the magazine.

In this manner we could determine whether intra-striatal anisomycin infusion caused any avoidance of the food pellets. Fos 2A-iCreER i. The next day mice were permitted to lever-press under a FR1 schedule until they earned 30 food pellets. Fos-Cre ERT2 i.