Reward-Predictive Neural Activities in Striatal Striosome Compartments

Mice showed odor-induced reward-predictive licking behavior proportional to expected reward size. A, Schematic illustration of the behavioral apparatus. Mice were restricted, head and body, by the metal frame and tube. The odor mask, water spout, and air-puff tube were set in front of their noses, mouths, and eyes. Spout-licking behaviors were monitored using an infrared sensor. The miniature microscope was mounted on their heads. B, Time sequence of a classical conditioning task. C, An example of reward-predictive spout-licking behaviors after sufficient learning. In trials of reward conditions, spout-licking behaviors started during odor presentation periods. Black dots indicate spout-licking behaviors. Yellow areas show CS-delay periods. D, Daily changes of spout-licking frequency during CS-delay periods of the mouse illustrated in C. Early and late stages were defined based on the appearance of reward-predictive licking. Error bars indicate SEs. E, Average spout-licking frequencies during CS-delay periods of all eight mice. Error bars indicate SEs.

An endoscopic microscope was used for selective in vivo calcium imaging of striosomal neurons in the striata of Sepw1-NP67 mice expressing Cre-dependent GCaMP6s. A, Striosome group. To express GCaMP6s selectively in striosomal neurons, AAV.Syn.Flex.GCaMP6s was injected into the DMS. B, GCaMP6s (green) was selectively expressed in striosomes (red) three weeks after virus injection. Scale bar: 50 µm. C, Control group. To express GCaMP6s in both striosomes and matrix, AAV.Syn.GCaMP6s was injected to DMS. D, GCaMP6s expressed in both striosomes and matrix three weeks after virus injection. E, Schematic illustration of endoscopic in vivo calcium imaging. F, Averaged fluorescence images recorded by miniature microscope. White dots indicate neurons. The same neurons in striosomes were stably observed over two weeks. G, Images showing endoscope placement and Cre-dependent GCaMP6s-expressing neurons within the striatum. The focal plane in tissue is 250–300 µm from the bottom of the endoscope, as indicated by the white arrow heads. Scale bar: 200 µm.

Reward-associated odors activated striosomal neurons in a specific learning stage. A, Normalized ΔF/F of a striosomal neuron showing reward-predictive activity specifically in the early stage. Black dots indicate detected Ca²⁺ events. B, C, Averaged ΔF/F and Ca²⁺ events of the striosomal neuron illustrated in A. Yellow areas show the CS-delay period. D, Amplitudes of CS-delay period Ca²⁺ events of the striosomal neuron illustrated in A were averaged over trials and plotted against reward size. In the early stage, Ca²⁺ events show a positive correlation with reward size (r = 0.25, p = 1.6e-04). On the other hand, this correlation disappeared in the late stage (r = -0.038, p = 0.58). Error bars and lines indicate SEs and regression lines. E, Normalized ΔF/F of another striosomal neuron showing reward-predictive activity specifically in the late stage. F, G, Averaged ΔF/F and Ca²⁺ events of the striosomal neuron illustrated in E. H, Amplitudes of CS-delay period Ca²⁺ events of the striosomal neuron illustrated in E were averaged over trials and plotted against the reward size. In the early stage, Ca²⁺ events show no significant correlation with reward size (r = −0.035, p = 0.60). However, a positive correlation was observed in the late stage (r = 0.31, p = 1.7e-06).

During each learning stage, different neural ensembles participated in reward prediction and population coding of expected reward differed between two groups. A, To remove effects of motor behavior on neural activities, we first performed a regression analysis of the sum of amplitudes of Ca²⁺ events during the CS-delay period with frequencies of licking. Then we analyzed the residual component using prediction of reward (Vr). Scatter plots of t-values for regression coefficients of Vr in each learning stage. Dashed lines indicate levels of significant Vr slope at p = 0.05. Letters A and E indicate the example neurons in Figure 3A,E. B, Proportions of reward-predictive neurons in each learning stage. Numbers in bars indicate actual counts of reward-predictive neurons; **p < 0.01, n.s.: p ≥ 0.05, χ² test. C, Schematic illustration of neural decoding analysis. Forthcoming reward size was estimated from the sum of weighted neuronal activities. x_j: sum of amplitudes of Ca²⁺ events during the CS-delay period. w_j: weight for j-th neuron out of n neurons. D, MSEs between actual and decoded reward sizes at each number of neurons used for analyses; **p < 0.01, paired t test.

During each learning stage, different neural ensembles in the striosome predicted air-puff stimuli. A, Normalized ΔF/F of a striosomal neuron showing air-puff-predictive activities specifically in the early stage. Black dots indicate detected Ca²⁺ events. B, C, Averaged ΔF/F and Ca²⁺ events of the striosomal neuron illustrated in A. Yellow areas show the CS-delay period; **p < 0.01, n.s.: p ≥ 0.05, two-sample t test. D, Normalized ΔF/F of another striosomal neuron showing air-puff-predictive activities specifically in the late stage. E, F, Averaged ΔF/F and Ca²⁺ events of the striosomal neuron illustrated in D; **p < 0.01, n.s.: p ≥ 0.05. G, Scatter plots of t-values for regression coefficients of prediction of air puff (Va) in each learning stage. Dashed lines indicate levels of significant Va slope at p = 0.05. Letters A and D indicate the example neurons in A, D. H, Proportions of air-puff-predictive neurons in each learning stage. Numbers in bars indicate actual counts of air-puff-predictive neurons. **p < 0.01, *p < 0.05, χ² test. I, MSEs between actual and decoded air-puff values at each number of neurons used for analyses; **p < 0.01, paired t test.