Cholinergic interneurons in the rat striatum modulate substitution of habits

Abstract

Behavioural flexibility is crucial for adaptive behaviour, and recent evidence suggests that cholinergic interneurons of the striatum play a distinct role. Previous studies of cholinergic function have focused on strategy switching by the dorsomedial or ventral striatum. We here investigated whether cholinergic interneurons in the dorsolateral striatum play a similar role at the level of switching of habitual responses. Because the dorsolateral striatum is particularly involved in habitual responding, we developed a habit substitution task that involved switching habitual lever-press responses to one side to another. We first measured the effect of cholinergic activation in the dorsolateral striatum on this task. Chemogenetic activation of cholinergic interneurons caused an increase in the response rate for the substituted response that was significantly greater than the increase normally seen in control animals. The increase was due to burst-like responses with shorter inter-press intervals. However, there was no effect on inhibiting the old habit, or on habitual responding that did not require a switch. There was also no effect on lever-press performance and its reversal before lever-press responses became habitual. Conversely, neurochemically specific ablation of cholinergic interneurons did not significantly change habitual responding or response substitution. Thus, activation -but not ablation -of cholinergic interneurons in the dorsolateral striatum modulates expression of a new habit when an old habit is replaced by a new one. Together with previous work, this suggests that striatal cholinergic interneurons facilitate behavioural flexibility in both dorsolateral striatum in addition to dorsomedial and ventral striatum.

Keywords: acetylcholine; basal ganglia; behavioural flexibility; chemogenetics.

Figure 1

Specific DREADD (hM3Dq) activation of cholinergic interneurons in dorsolateral striatum. (A) Specific viral infection to CINs in DLS. AAV‐hsyn‐DIO‐hM3Dq‐mCherry was injected into bilateral DLS (Blue areas). This yields specific viral expression to CINs as ChAT and mCherry are co‐localized in fluorescent images. A representative DAB‐stained section shows the selective viral spread to DLS. Scale bars, Fluorescent images = 100 μm; Brightfield DAB = 500 μm. CC, corpus callosum. B, The extent of viral spread showing the largest (grey) and the smallest (black) viral spread in experimental groups. Distance from bregma is shown on the left‐hand. Note that the viral spread is restricted to DLS. (C) in vitro electrophysiological recordings from CINs showing an increase in firing rate under CNO application. (D) Time course of the change in firing rate of mCherry‐positive CINs (red, n = 5) and mCherry‐negative CINs (grey, n = 2). (E) A significant increase in firing rate of mCherry‐positive CINs under CNO. Scatter plots indicate individual data points. An asterisk indicates P < 0.05.

Figure 2

Chemogenetic activation of CINs in a habit substitution task. (A) A flow chart of experiment 1. Animals formed a habitual response under RI30 and RI60 (habit formation period). Next, a habit substitution task was commenced, during which CINs were manipulated. (B) Lever‐press rate (LPr) during habit formation. (C) Lever‐press rate during a 5‐min extinction test under the outcome devaluation procedure. Note that both groups showed insensitivity to the outcome devaluation, indicating that a habit has been formed successfully. (D and E) Lever‐press rate in a habit substitution task that involves reversal of responses. Animals needed to inhibit one habitual response and substitute a new response to an opposite lever. Both control rats (D) and rats with activation of CINs (E) show a successful reversal of lever‐press responses in the course of the session. Lever‐press rate is calculated by 2‐min bins. Lever‐press rate on the last day of RI60 is shown on the left‐hand of each panel (separated by a dashed line). (F) Evaluation of animal's reversal performance. Based on the number of lever‐presses in 2‐min bins (D and E), we compared the first time that animals scored the greater number of substituted responses than habitual ones. No statistical difference is seen in reversal performance. (G and H) Comparisons of habitual (G) and substituted (H) response rate between control rats and rats with activation of CINs. The neighbouring bar plots indicate mean response rate in the session. Note that in contrast to habitual responses, the number of substituted responses is significantly increased in rats with CIN activation than control rats. (I) Histogram of inter‐press intervals of substituted responses. Those responses are divided into 10 bins with 2‐s intervals (J and K) The number of rewards obtained (J) and head entry rate (K) during a habit substitution task. (L) Lever‐press rate in a following extinction test. Scatter plots indicate individual data points. Double asterisks indicate P < 0.01. Final group size is follows: control rats, n = 17 (virus‐control = 9; CNO‐control = 8); rats with CIN activation, n = 16.

Figure 3

Chemogenetic activation of CINs in responses under a RI60 schedule without habit substitution. (A) A flow chart of experiment 3. Similar to the Experiment 1, animals formed a habit under the random‐interval schedule. In a test day, animals with or without CIN activation perform the same RI60 schedule without a switch of habitual responses. This procedure controls the effect of activating CINs on lever‐press behaviour and its reinforcement in general. (B) Lever‐press rate during a habit formation period under RI schedules. (C) Lever‐press rate during an outcome devaluation test. Note that both groups successfully formed a habit as confirmed by insensitivity to the outcome devaluation. (D) Lever‐press rate on both active and inactive levers in a test day under the same RI60 schedule. (E) Inter‐press intervals of habitual responses to an active side on the same testing day. Note that CIN activation had no effect on responses. Scatter plots indicate individual data points. Final group size is follows: control rats, n = 13 (virus‐control = 5; CNO‐control = 8); CINs manipulated rats, n = 10.

Figure 4

Chemogenetic activation of CINs in an early phase of learning. (A) A flow chart of experiment 4. Animals underwent a continuous reinforcement schedule followed by RI30. Unlike the other experiments, chemogenetic activation was performed 2 days after the initiation of RI30 schedule. After the one‐day activation, the devaluation test was performed to examine whether the prior treatment accelerated habit formation. This procedure controlled for the facilitative effect of activating CINs on new lever‐press learning or habit formation, regardless of switching habits. (B) Lever‐press rate before cholinergic manipulation. Experiment and control groups showed similar baseline lever‐press rates. (C) Performance of RI30 under cholinergic manipulation. Cholinergic activation does not affect lever‐press performance in the early phase of learning. (D) A devaluation test following cholinergic activation showed that both control and rats with cholinergic interneurons that had been activated are sensitive to outcome devaluation, indicating that there is no effect of the cholinergic manipulation on lever‐press rate or habit learning before the habit has been formed. Scatter plots indicate individual data points. Final group size is follows: control rats, n = 13 (virus‐control = 7; CNO‐control = 6); CINs manipulated rats, n = 10.

Figure 5

Chemogenetic activation of CINs in a substitution (reversal) task before a habit has been developed. (A) A flow chart of experiment 5. As in Experiment 4, animals underwent continuous reinforcement schedule followed by 2 days of RI30. On the third day of RI30, chemogenetic activation was applied when rats performed a substitution task in which response contingency was reversed. After the one‐day activation, an extinction test was conducted to evaluate learning of response contingency. (B) Lever‐press rate during training before cholinergic activation. Experiment and control groups showed similar baseline lever‐press rates on the last day. (C and D) Lever‐press rate during reversal under RI30. Both control rats (C) and rats with activation of CINs (D) showed a successful reversal of lever‐press responses in the course of the session. Lever‐press rate of the last day of RI30 is shown on the left‐hand of each panel (separated by a dashed line). (E) Animal's reversal performance. Based on the number of lever‐presses in 2‐min bins (C and D), we compared the first time that animals scored a greater number of substituted responses with presses on the previously correct lever. F and G, Comparison of previously correct (F) and substituted (G) response rates between control and experimental groups. The neighbouring bar plots indicate mean response rate in the session. (H) Lever‐press rate in the subsequent extinction test. Scatter plots indicate individual data points. Final group size is follows: control rats, n = 12 (virus‐control = 6; CNO‐control = 6); rats with CIN activation, n = 10.

Figure 6

Effect of DTA‐mediated cholinergic ablation on a habit substitution task. (A) Injection of AAV‐mCherry‐flex‐DTA into DLS causes specific ablation of cholinergic interneurons. Scale = 1 mm. Absence of labelled neurons is only evident in ChAT staining after the lesion and it is selective to DLS. (B) A flow chart of experiment 6. Animals formed a habitual response under RI30 and RI60 (habit formation period). Next, those animals are divided into two groups based on their lever‐press performance (Lesion or Control). After the 4‐day recovery period, additional RI60 was continued and later a habit substitution task was commenced. An extinction test was conducted after the habit substitution. (C) Lever‐press rate (LPr) in a habit formation phase. (D) Lever‐press rate during a 5‐min extinction test under the outcome devaluation procedure. Note that both groups showed insensitivity to the outcome devaluation, indicating that a habit has been formed successfully. (E and F) Lever‐press rate in a habit substitution task that involves reversal of responses. Both control rats (E) and rats with ablation of CINs (F) successfully reversed their responses in the course of the session. Lever‐press rate of the last day of RI60 is shown on the left‐hand of each panel. (G) Animal's reversal performance. This comparison is based on the first time that animals scored a greater number of substituted responses than habitual ones in 2‐min bins. (H and I) Comparisons of habitual (H) and substituted (I) response rate between control and cholinergic ablated rats. The corresponding bar plots indicate the mean response rate across a session. (J) Histogram of inter‐press intervals of substituted responses. (K and L) The number of obtained rewards (K) and head entry rate (L) during a habit substitution task. (M) Lever‐press rate in the subsequent extinction test. Scatter plots indicate individual data points. Final group size is follows: control rats, n = 18; lesioned rats, n = 18.