Habits are automated behaviors that are insensitive to changes in behavioral outcomes. Habitual responding is thought to be mediated by the striatum, with medial striatum guiding goal-directed action and lateral striatum promoting habits. However, interspersed throughout the striatum are neurochemically differing subcompartments known as patches, which are characterized by distinct molecular profiles relative to the surrounding matrix tissue. These structures have been thoroughly characterized neurochemically and anatomically, but little is known regarding their function. Patches have been shown to be selectively activated during inflexible motor stereotypies elicited by stimulants, suggesting that patches may subserve habitual behaviors. To explore this possibility, we utilized transgenic mice (Sepw1 NP67) preferentially expressing Cre recombinase in striatal patch neurons to target these neurons for ablation with a virus driving Cre-dependent expression of caspase 3. Mice were then trained to press a lever for sucrose rewards on a variable interval schedule to elicit habitual responding. Mice were not impaired on the acquisition of this task, but lesioning striatal patches disrupted behavioral stability across training, and lesioned mice utilized a more goal-directed behavioral strategy during training. Similarly, when mice were forced to omit responses to receive sucrose rewards, habitual responding was impaired in lesioned mice. To rule out effects of lesion on motor behaviors, mice were then tested for impairments in motor learning on a rotarod and locomotion in an open field. We found that patch lesions partially impaired initial performance on the rotarod without modifying locomotor behaviors in open field. This work indicates that patches promote behavioral stability and habitual responding, adding to a growing literature implicating striatal patches in stimulusresponse behaviors.

Habits are inflexible behaviors that develop after extensive repetition, and overreliance on habits is a hallmark of many pathological states. The striatum is involved in the transition from flexible to inflexible responding, and interspersed throughout the striatum are patches, or striosomes, which make up ~15% of the volume of the striatum relative to the surrounding matrix compartment. Previous studies have suggested that patches are necessary for normal habit formation, but it remains unknown exactly how patches contribute to habit formation and expression. Here, using optogenetics, we stimulated striatal patches in Sepw1-NP67 mice during variable interval training (VI60), which is used to establish habitual responding. We found that activation of patches at reward retrieval resulted in elevated responding during VI60 training by modifying the pattern of head entry and pressing. Further, this optogenetic manipulation reduced subsequent responding following reinforcer devaluation, suggesting modified habit formation. However, patch stimulation did not generally increase extinction rates during a subsequent extinction probe, but did result in a small ‘extinction burst’, further suggesting goal-directed behavior. On the other hand, this manipulation had no effect in omission trials, where mice had to withhold responses to obtain rewards. Finally, we utilized fast-scan cyclic voltammetry to investigate how patch activation modifies evoked striatal dopamine release and found that optogenetic activation of patch projections to the substantia nigra pars compacta (SNc) is sufficient to suppress dopamine release in the dorsal striatum. Overall, this work provides novel insight into the role of the patch compartment in habit formation, and provides a potential mechanism for how patches modify habitual behavior by exerting control over dopamine signaling.

Behavioural flexibility is key to survival in a dynamic environmentWhile flexible, goal‐directed behaviours are initially dependent on dorsomedial striatum, they become dependent on lateral striatum as behaviours become inflexible. Similarly, lesions of dopamine terminals in lateral striatum disrupt the development of inflexible habits. This work suggests that dopamine release in lateral striatum may drive inflexible behaviours, though few studies have investigated a causative role of subpopulations of striatal dopamine terminals in reversal learning, a measure of flexibility. Here, we performed two optogenetic experiments to activate dopamine terminals in dorsomedial (DMS), dorsolateral (DLS) or ventral (nucleus accumbens [NAc]) striatum in DAT‐Cre mice that expressed channelrhodopsin‐2 via viral injection (Experiment I) or through transgenic breeding with an Ai32 reporter line (Experiment II) to determine how specific dopamine subpopulations impact reversal learning. Mice performed a reversal task in which they self‐stimulated DMS, DLS, or NAc dopamine terminals by pressing one of two levers before action‐outcome lever contingencies were reversed. Largely consistent with presumed ventromedial/lateral striatal function, we found that mice self‐stimulating medial dopamine terminals reversed lever preference following contingency reversal, while mice self‐stimulating NAc showed parial flexibility, and DLS self‐stimulation resulted in impaired reversal. Impairments in DLS mice were characterized by more regressive errors and reliance on lose‐stay strategies following reversal, as well as reduced within‐session learning, suggesting reward insensitivity and overreliance on previously learned actions. This study supports a model of striatal function in which DMS and ventral dopamine facilitate goal‐directed responding, and DLS dopamine supports more inflexible responding.

Habits are inflexible behaviors that can be maladaptive in diseases including drug addiction. The striatum is integral to habit formation, and interspersed throughout the striatum are patches, or striosomes, which are characterized by unique gene expression relative to the surrounding matrix. Recent work has indicated that patches are necessary for habit formation, but how patches contribute to habits remains partially understood. Here, using optogenetics, we modulated striatal patches in Sepw1-NP67 mice during habit formation. We find that patch activation during operant training impairs habit formation, and conversely, that acute patch stimulation after reward devaluation can drive habitual reward seeking. Patch stimulation invigorates general locomotion but is not inherently rewarding. Finally, we use fast-scan cyclic voltammetry to demonstrate that patch stimulation suppresses dopamine release in dorsal striatum in vivo. Overall, this work provides novel insight into the role of the patch compartment in habit formation, and potential interactions with dopamine signaling.

14Habits are automated behaviors that are insensitive to changes in behavioral outcomes. Habitual responding is 15 thought to be mediated by striatum, with medial striatum guiding goal-directed action and lateral striatum promoting 16 habits. However, interspersed throughout the striatum are neurochemically differing subcompartments known as 17 patches, which are characterized by distinct molecular profiles relative to the surrounding matrix tissue. These 18 structures have been thoroughly characterized neurochemically and anatomically, but little is known regarding their 19 function. Patches have been shown to be selectively activated during inflexible motor stereotypies elicited by 20 stimulants, suggesting that patches may subserve habitual behaviors. To explore this possibility, we utilized 21 transgenic mice (Sepw1 NP67) expressing Cre recombinase in striatal patches to target these neurons for selective 22 ablation with a virus driving Cre-dependent expression of caspase 3. Mice were then trained to press a lever for 23 sucrose rewards on a variable interval schedule to elicit habitual responding. Mice were not impaired on the 24 acquisition of this task, but lesioning striatal patches disrupted behavioral stability across training and lesioned mice 25 utilized a more goal-directed behavioral strategy during training. Similarly, when mice were forced to omit 26 responses to receive sucrose rewards, habitual responding was impaired in lesioned mice. To rule out effects of 27 lesion on motor behaviors, mice were then tested for impairments in motor learning on a rotarod and locomotion in October 15, 2019 2/22 28 an open field. We found that patch lesions specifically impaired initial performance on the rotarod without 29 modifying locomotor behaviors in open field. This work indicates that patches promote behavioral stability and 30 habitual responding, adding to a growing literature implicating striatal patches in stimulus-response behaviors. 31 32 33