Time locked inhibition of SPNs activity

To further evaluate the role of the neuronal activity of SPNs around seek lever retraction signaling that seeking behavior ended and reward will be available in the process of compulsion, a time locked optogenetic inhibition experiment was next performed. To this end, we injected AAV5-CamKII-eArchT3.0-eYFP in the cDS and placed optic fibers bilaterally (Figure 3.7A and B). In acute slices, orange light (wavelength; 585 nm) activated ArchT3.0 and effectively suppressed action potentials induced by a depolarizing current injection (Figure 3.7C). First mice underwent 5 sessions of punishment to identify perseverers which were used for the inhibition experiments. We then inhibited cDS SPNs for 4s starting at the first seek lever press following the end of RI (Figure 3.7D, Top). This dramatically reduced the compulsive seeking (Figure 3.7D and F). Importantly this inhibition at seek lever press did not affect seeking in baseline conditions (Figure S3.8).

Moreover, applying optogenetic inhibition at the first seek lever press within the RI (Figure 3.7E, top), when only small calcium transients were observed (Figure S3.4), had no impact on compulsive seeking behaviour (Figure 3.7 E and F). We conclude that brief cDS inhibition in not aversive per se, but can reduce seeking behavior of compulsive mice.

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Figure 3.7. Attenuation of compulsive reward seeking by time locked inhibition in the cDS. (A) Schematic for the preparation. (B) Image of a mouse brain infected with eArchT3.0-eYFP in the cDS and DIO-ChR2-eYFP in the VTA. (C) Orange light (593 nm) suppressed action potentials in eArchT3.0 expressing neurons. Action potentials were induced by current injection (300pA, 5s). (D) Schematic for the inhibition protocol. The first seek lever press after the end of RI triggered eArchT inhibition for 4 seconds (Top). Session duration, number of seek lever presses per trial, number of completed trials, delay to the first seek lever press, and delay to the last seek lever press over sessions (bottom). (E) Schematic for the inhibition protocol. The first seek lever press after the presentation of the seek lever triggered eArchT inhibition for 4 seconds (Top). Session duration, number of seek lever presses per trial, number of completed trials, delay to the first seek lever press, and delay to the last seek lever press over sessions (bottom). (F) Group data for panel (D) and (E). Behaviour was modified by the inhibition at the moment of the last seek LP but not first seek LP. Average of two sessions before inhibition (session -1 and 0) and last three days of inhibition (session 2 to 4) are shown as Ctrl and Test, respectively. Session duration; mixed two-way ANOVA: laser timing, F(1,12)=6.489, P=0.0256;

treatment, F(1,12)=7.876, P= 0.0159; interaction, F(1,12)=7.910, P=0.0157. Bonferroni post hoc analysis, **p<0.01. Number of seeking lever presses per trial; mixed two-way ANOVA: laser timing, F(1,13)=1.098, P=0.3138; treatment, F(1,13)=15.08, P=0.0019; interaction, F(1,13)=12.00, P=0.0042.

Bonferroni post hoc analysis, ***p<0.001. Number of completed trials; Wilcoxon matched-pairs signed rank test. P value was adjusted with Bonferroni-Dunn method. **P<0.01. Delay to the first seeking lever press; mixed two-way ANOVA: laser timing, F(1,13)=4.538 , P=0.0528; treatment, F(1,13)=3.696, P=0.0767; interaction, F(1,13)=5.469, P= 0.0360. Bonferroni post hoc analysis, ***p<0.05. Delay to the last seeking lever press; mixed two-way ANOVA: laser timing, F(1,13)=4.472, P=0.0543; treatment, F(1,13)=6.024, P=0.0290; interaction, F(1,13)=4.733,P= 0.0486. Bonferroni post hoc analysis,

**p<0.01.

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Figure S3.9. Time locked inhibition in the cDS did not modify reward seeking behavior not associated with punishment. (A) Schematic for the inhibition protocol. Inhibition was applied at the first seek lever press after the end of RI, as in figure 7D top. (B) First seek delay, number of seek lever presses per trial and last seek delay over session (left). Average of two sessions before inhibition (session -1 and 0) and last three days of inhibition (session 2 to 4) are shown as Ctrl and Test, respectively (bottom, right). Delay to the first seek LP, 𝑡₇=1.284, P= 0.2400. seek LP/trial, 𝑡₇=1.807, P= 0.1138. Delay to the last seek LP, 𝑡₇=0.6780, P= 0.5196. Error bars, s.e.m.

4.Discussion

In this study, we confirmed that the potentiation at the OFC to DS pathway is involved in compulsive reward seeking. Previously, it has been determined that this potentiation is crucial for the compulsive reward taking (Pascoli et al., 2018). However, in the seek-take chained schedule, the silencing of the dorsolateral striatum attenuated compulsive cocaine seeking (Jonkman et al., 2012). Our anatomical tracing data show that, to this part of the dorsal striatum, OFC sends only sparse projections. Moreover, punishments at the take lever presses are less efficient to suppress compulsive cocaine seeking than punishments at the seek lever presses (Pelloux et al., 2015). These results suggest that there might be differences in neuronal mechanisms between compulsive reward taking and seeking. In the present investigation, we demonstrated that the potentiation at OFC-DS is crucial for compulsive reward seeking, showing that the neuronal mechanisms underlying compulsive seeking and taking are highly overlapped. We also measured neuronal activity in two subregions of the dorsal striatum: the central part and the lateral part of the dorsal striatum (cDS and lDS, respectively). OFC mainly project to the cDS, and at this subregion, we observed a strong increase in neuronal activity at the end of the seeking behavior. Interestingly, the neuronal activity was stronger in perseverers than in renouncers, correlating with the synaptic plasticity. On the other hand, in the lDS, no difference was detected between the two groups of mice. This negative result supports the idea that the potentiation drives increase in neuronal activity in post synaptic neurons.

Chemogenetic inhibition of the OFC pyramidal neurons attenuated compulsive reward seeking and flattened the calcium signal at the end of the seeking behavior. Although the reduction of the peak amplitude of the calcium signal was small (~22%), the reductions of the signal and the compulsivity were highly correlated. It has been already shown that

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chemogenetic inhibition of OFC neurons suppressed compulsive reward taking (Pascoli et al., 2015), where around 85% of animals were perseverers before CNO injection, a ratio that dropped to 35%. In the present study, CNO injection was performed only in perseverers and 5 out of 9 mice fell into the criteria of renouncers with CNO injections, indicating that the efficiency of chemogenetic inhibition in attenuating compulsivity is quite similar between taking and seeking behaviour. In the end, to test the causality between the neuronal activity at the end of the seeking behavior and compulsive reward seeking, we silenced the cDS in a time locked way. This manipulation attenuated compulsive reward seeking, indicating that the cDS activity at the end of the seeking behavior promotes compulsive reward seeking. The silence was applied at the end of the seeking behavior, not before or during it. This means that the inhibition affected the seeking behavior in the next trials or sessions.