Targeted Expression of OptoA 2A R and MAPK Signaling by OptoA 2A R Activation in the Striatopallidal Neurons

Two weeks after the injection of AAV5-EF1α-DIO-mCherry-optoA 2A R and its control vector into the striatum of the adora2a-Cre mice (Figure 1a), we verified the selective expression of optoA 2A R in the striatopallidal neurons. Quantitative analysis of double immunofluorescence staining result indicated that 88% of mCherry (optoA 2A R-mCherry)-positive cells were colocalized with encephalin (a marker for the striatopallidal neurons), whereas only 17% mCherry-positive cells were colocalized with substance-P (a marker for the striatonigral neurons) in the striatum (Figure 1b). Representative double-immunofluorescence staining images illustrated the colocalization of optoA 2A R-mCherry with enkephalin but not substance-P (Figure 1c). Furthermore, the red (mCherry) fluorescence was specifically expressed in the terminals of the striatopallidal neurons in the globus pallidus, but was absent in the terminals of striatonigral neurons in the substantia nigra pars reticularta where substance P are highly expressed (Figure 1d). These results confirmed the selective expression of optoA 2A R in the striatopallidal neurons. Moreover, optoA 2A R stimulation in the striatum for 5 min induced p-MAPK in the mCherry-positive cells underneath the optic fiber (Figure 1e) in a similar pattern as the A 2A R agonist CGS21680. Quantified analysis showed that light-induced p-MAPK activation was detected in 57% mCherry-optoA 2A R-positive cells (n=1218 from 4 mice). Thus, optoA 2A R and CGS21680 produced indistinguishable p-MAPK signaling in the striatum.

Figure 1 Targeted expression and phospho-MAPK (p-MAPK) signaling of optoA 2A R in striatopallidal neurons. (a) Schematic illustration of the optoA 2A R chimera construction by replacing the intracellular loops 1, 2, and 3 and C terminal of the bovine rhodopsin with that of the adenosine A 2A receptor (A 2A R) to achieve control of A 2A R signaling by 473 nm light (left panel). Representative fluorescent image shows the expression of mCherry-optoA 2A R in the striatum after injection of AAV5-DIO-mCherry-optoA 2A R to adora2a-cre mice for 2 weeks (right panel). (b) The quantitative data shows that 88% mCherry-positive cells (n=114, from four mice) were colocalized with enkephalin (ENK), whereas only 17% mCherry-positive cells (n=106, from four mice) were colocalized with substance P (SP). (c) Double immunostaining with the mCherry and the specific antibodies (ENK or SP) showed that optoA 2A Rs were specifically expressed in ENK-positive striatopallidal neurons (white arrows, upper panels) but not SP-positive striatonigral neurons (yellow arrows, lower panels). (d) Following injection of AAV-DIO-mCherry-optoA 2A R virus in the dorsomedial striatum (DMS) of adora2a-cre mice, the mCherry fluorescence of striatopallidal projection terminals was specifically expressed in the global pallidum (GP) but not in the substantia nigra pars reticulate (SNr). The green fluorescence of striatonigral projection terminals containing endogenous SP was specifically expressed in the SNr. (e) The expression of p-MAPK was induced by optoA 2A R stimulation (white arrows, left panels) or CGS21680 injection (white arrows, right panels). Quantified analysis showed that light-induced p-MAPK activation was detected in 57% mCherry-optoA 2A R-positive cells (n=1218 from four mice). Full size image Download PowerPoint slide

Optogenetic Activation of Striatopallidal A 2A R Signaling in the DMS, Precisely at (but not Randomly in Relation to) the Time of the Reward, Suppressed Goal-Directed Behavior

To determine the effect of optoA 2A R signaling in the DMS and DLS on goal-directed and habitual actions using a satiety-based instrumental learning paradigm, we first performed an devaluation time-course study to select specific RI training schedule that were most likely sensitive to bidirectional manipulation of the A 2A R activity in the DMS and DLS. Devaluation test revealed that after the CFRRI30RI60 training, mice showed a clear goal-directed behavior on the 3rd day, developed habitual behavior on the 4th day, and became a stable habitual behavior on the 5th day after RI60 training (Supplementary Figure 1). Since the mice on the 4th day of RI60 schedule were at the transition period from goal-directed to habitual behavior and were most sensitive to bidirectional manipulation of A 2A Rs in the DMS and DLS, we used the RI60 training for 4 days for the rest of the experiments.

We verified that the locations of the optical fiber implantation sites and expression of optoA 2A R were restricted to the DMS by immunofluorescence (Figure 2a). At the RI sessions, we used the ‘time-locked’ method to deliver optoA 2A R stimulation (for 2 s per reward) precisely at the time of reward delivery (Figure 2b). Mice with ‘light off’ serviced as controls. All mice gradually increased their lever pressing rates to obtain reward and reached the lever pressing plateau at the second day of RI training. There was no main effect of optoA 2A R stimulation (F 1,14 =0.371, p>0.05) nor optoA 2A R stimulation × RI training course interaction effect (F 5,70 =0.098, p>0.05) by repeated-measures ANOVA. Thus, optogenetic activation of the striatopallidal A 2A R signaling in the DMS did neither impair lever pressing performance nor affect acquisition of instrumental learning (Figure 2c).

Figure 2 ‘Time-locked’ but not random optogenetic activation of striatopallidal adenosine A 2A receptor (A 2A R) signaling in the dorsomedial striatum (DMS) suppresses goal-directed behavior. (a) Left panel: Schematic illustration of the locations of the fiber tips for each animal in the ‘light-off’ group (the red triangles) and ‘time-locked’ activation group (the blue circles). Right panel: Typical coronal section of mCherry-optoA 2A R expression in the DMS of adora2a-cre(+) mice. The white arrow indicates the optical fiber tip. (b) Schematic illustration of timing of lever pressing, sucrose reward delivery, and optical stimulation. Light stimulation (the blue flash) was delivered to the DMS during a 2-s period in ‘time-locked’ manner with (the flashes between the two red dotted vertical lines) or in ‘random’ manner with (the flashes in the random interval periods) reward delivery (the liquid drops). (c) Two groups of mice expressing optoA 2A R in the DMS were subjected to either ‘time-locked’ light stimulation or ‘light off’ (n=8 per group) during the random interval (RI) training session (as indicated by the blue bar). The two groups performed indistinguishably in the acquisition phase of instrumental learning by repeated-measures analysis of variance (ANOVA)—RI period × optoA 2A R stimulation interaction effect: F 5,70 =0.098, p>0.05; optoA 2A R stimulation main effect: F 1,14 =0.371, p>0.05. (d) Following the RI training sessions, a 2-day devaluation test without any experimental (optoA 2A R activation) manipulation was conducted as described in the Materials and Methods section. Mice without optoA 2A R activation during the RI training sessions significantly reduced their lever presses in devalued condition compared with valued condition (normalized devaluation: t 1,7 =6.861, ***p<0.001, preplanned t-test). By contrast, mice with optoA 2A R ‘time-locked’ stimulation showed no significant devaluation effect (normalized devaluation: t 1,7 =0.709, p>0.05, preplanned t-test). However, there was no normalized devaluation × optoA 2A R interaction effect by repeated-measures ANOVA analysis (F 1,14 =0.429, P=0.523). (e) We further performed instrumental behavioral analyses of a separate set of four experimental groups: mice expressing mCherry with ‘time-locked’ light stimulation (n=7), mice expressing optoA 2A R with ‘light off’ (n=9), mice expressing optoA 2A R with ‘time-locked’ light stimulation (n=8), and mice expressing optoA 2A R with random light stimulation (n=8). Consistent with the result in (c) repeated-measures ANOVA analysis indicated that there was neither between-subject effect (F 3,28 =1.481, p=0.241) nor RI training sessions × manipulation groups interaction effect (F 15,140 =1.284, p=0.220) in the acquisition phase. (f) Repeated-measures ANOVA analyses of the devaluation test revealed that there was significant effect of optogenetic manipulation × (normalized) devaluation interaction effect: F 3,28 =3.258, p=0.036. Similarly, the simple main-effect analyses of the devaluation test in four groups indicated that only mice with optoA 2A R expression in the DMS and time-locked light stimulation performed habitually, whereas other groups displayed goal-directed behavior (simple effect analyses: F 1,8 =7.141, *p<0.05 for ‘light off’ and F 1,7 =6.074, *p<0.05 for ‘random’ stimulation groups, and F 1,6 =16.050, **p<0.01 for mCherry group). Data are presented as the mean±SEM. The color reproduction of this figure is available on the Neuropsychopharmacology journal online. Full size image Download PowerPoint slide

The devaluation test (Figure 2d) revealed that there was no normalized devaluation × optoA 2A R interaction effect (F 1,14 =0.429, p=0.523) by repeated-measures ANOVA. However, preplanned t-test showed that the optoA 2A R mice with ‘light off’ displayed a goal-directed behavior with sensitivity to devalued reward (t 1,7 =6.861, ***p<0.001, n=8). The goal-directed behavior in the ‘light-off’ group probably reflects unstable (transient) nature of instrumental behavior for the 4-day RI60 training schedule and might be partially attributed to the relatively low level of lever pressing in this group (and the total rewards received) when the optical fiber implanted in the DMS compared with other experimental groups. Importantly the optoA 2A R with ‘time-locked’ stimulation during the RI sessions failed to show sensitivity to outcome devaluation (preplanned t-test, t 1,7 =0.709, p>0.05, n=8), indicating that their responding was habitual.

To better define the temporal importance of optoA 2A R signaling precisely at the time of reward and to exclude the nonspecific effect caused by light, we have performed behavioral analyses with separate set of four experimental groups: mice expressing mCherry with ‘time-locked’ light stimulation (n=7), mice expressing optoA 2A R with ‘light off’ (n=9), mice expressing optoA 2A R with ‘time-locked’ light stimulation (n=8), and mice expressing optoA 2A R with ‘random’ (n=8) light stimulation. The light stimulation scheme was illustrated in Figure 2b. Consistent with the result in Figure 2c, there was neither between-subject effect (F 3,28 =1.481, p=0.241) nor RI training sessions × manipulation groups interaction effect (F 15,140 =1.284, p=0.220) in the acquisition phase by repeated-measures ANOVA (Figure 2e). However, analyses of the devaluation test (Figure 2f) revealed that there was a significant effect of optogenetic manipulation × (normalized) devaluation interaction effect (repeated-measures ANOVA, F 3,28 =3.258, p=0.036). The simple main-effect analyses of the devaluation test, respectively, in each group confirmed that only mice with optoA 2A R expression in the DMS and time-locked light stimulation performed habitually (F 1,8 =7.141, *p<0.05 for light off and F 1,7 =6.074, *p<0.05 for random stimulation groups, F 1,6 =16.050, **p<0.01 for mCherry group). Taken together, statistical analyses of both sets of the experiments (Figure 2d by the preplanned t-test and Figure 2f by the repeated-measures ANOVA) support that optogenetic activation of striatopallidal A 2A R signaling in the DMS modulated the mode of instrumental behaviors by acting precisely at the time of the reward.

Optogenetic Activation of Striatopallidal A 2A R Signaling in the DLS had Relatively Limited Effects on Habitual Formation

Next, we examined the effect of optoA 2A R signaling in the DLS on instrumental behaviors. Similarly, we confirmed the optical fiber implantation sites and expression of optoA 2A R to be restricted to DLS by immunofluorescence (Figure 3a). Following the RI training sessions, optoA 2A R mice with ‘light off’ (n=10) or with ‘time-locked’ stimulation (n=13) gradually increased lever presses. There was no main effect of optoA 2A R stimulation (F 1,21 =0.156, p>0.05) and no interaction effect of training session × optoA 2A R stimulation in the RI sessions (F 5,105 =0.916, p>0.05) by repeated-measures ANOVA (Figure 3b). After the 4th day of RI60 training, repeated-measures ANOVA analyses of the devaluation test revealed that there was no optogenetic manipulations × normalized devaluation interaction effect (F 1,21 =0.022, p=0.884). However, the preplanned t-test showed that optoA 2A R mice with ‘time-locked’ stimulation tended to perform goal-directed behavior (normalized devaluation test, t 1,12 =3.725, **p<0.01 (Figure 3c); devaluation test, t 1,12 =2.030, p>0.05 (Supplementary Figure 2c)). Conversely, optoA 2A R mice with ‘light off’ displayed habitual behavior (normalized devaluation test, t 1,9 =1.270, p>0.05 (Figure 3c); devaluation test, t 1,9 =1.868, p>0.05 (Supplementary Figure 2c)). Thus, optogenetic activation of striatopallidal A 2A R signaling in the DLS tended to promote goal-directed behavior, but its effect was relatively limited.

Figure 3 Optogenetic activation of striatopallidal adenosine A 2A receptor (A 2A R) signaling in the dorsolateral striatum (DLS) exerts relatively limited and possibly opposite control over habitual action compared with the optoA 2A R in the dorsomedial striatum (DMS). (a) Left: Schematic illustration of the sites of optical fibers implantation. Right: A representative image of mCherry-optoA 2A R expression and fiber implantation. (b) Mice were under continuous reinforcement (CRF) training followed by RI30 and then RI60 training with or without optoA 2A R stimulation as described in the Materials and Methods section. The performances of optoA 2A R mice with ‘time-locked’ stimulation (n=13) or with ‘light off’ (n=10) during the acquisition phase were indistinguishable (repeated-measures analysis of variance (ANOVA), random interval (RI) training course × optogenetic stimulation interaction: F 5,105 =0.916, p>0.05; optoA 2A R stimulation main effect: F 1,21 =0.156, p>0.05). (c) OptoA 2A R mice with ‘time-locked’ stimulation or ‘light off’ during the RI training sessions were subjected to devaluation test as described in the Materials and Methods section. Repeated-measures ANOVA analyses revealed that there was no normalized devaluation × optogenetic stimulation interaction effect (F 1,21 =0.022, p=0.884). However, preplanned t-test analysis revealed that optoA 2A R mice receiving ‘time-locked’ stimulation tended to perform goal-directed behavior (only for the normalized devaluation test: t 1,12 =3.725, **p<0.01; but not for devaluation test: t 1,12 =2.030, p>0.05; Supplementary Figure 2c). Whereas optoA 2A R mice with ‘light off’ displayed habitual behavior (normalized devaluation test: t 1,9 =1.270, p>0.05; devaluation test: t 1,9 =1.868, p>0.05; Supplementary Figure 2c). Data are presented as the mean±SEM. The color reproduction of this figure is available on the Neuropsychopharmacology journal online. Full size image Download PowerPoint slide

Knockdown of A 2A Rs in the DMS Enhanced Goal-Directed Behavior, Whereas Knockdown of the A 2A Rs in the DLS had a Limited Effect on Habitual Behavior

We further evaluated the effects of focal knockdown of the A 2A Rs in the DMS and DLS on instrumental learning. Figures 4a and 5a provided representative outline of the AAV transfection and A 2A R focal knockdown areas of the DMS and DLS. Fluorescent images showed that A 2A Rs expression (the red fluorescence) was reduced selectively in the Cre-expressing regions (indicated by green fluorescence). Quantitative analysis of the A 2A R immunoreactivity (Figures 4b and 5b) confirmed selective knockdown of A 2A Rs in the DMS (by 91%) and DLS (by 94%) after transfection with AAV-Cre-zsGreen only in A 2A Rflox/flox mice but not in WT mice (A 2A R+/+).

Figure 4 Focal knockdown of adenosine A 2A receptors (A 2A Rs) in the dorsomedial striatum (DMS) enhances goal-directed behavior. (a) Left: Schematic illustration of the maximal (black) and minimal (gray) A 2A R knockdown areas in the DMS. Right: Representative immunofluorescent photomicrographs show focal knockdown expression of A 2A Rs in the DMS after injection of AAV-Cre-zsGreen into the A 2A R(flox/flox) (right panels) and A 2A R(+/+) mice (left panels). Intensity of A 2A Rs (red) were significantly deceased in the overlapping area with zsGreen expression (the yellow circle) in A 2A R(flox/flox) mice but not in A 2A R(+/+) mice. (b) Quantitative analysis showed that A 2A R expression were markedly reduced in the virus-transfected regions of A 2A R(flox/flox) mice compared with A 2A R(+/+) mice. (c) Two–three weeks after bilateral injection of AAV-Cre-zsGreen into the DMS, A 2A R(flox/flox) mice and A 2A R(+/+) mice (n=8 per group) were under CRF-RI30-RI60 training paradigm as described in the Materials and Methods section. Both groups similarly increased their lever pressing rate during the acquisition phases (repeated-measures analysis of variance (ANOVA) revealed no random interval (RI) period × genotype interaction effect: F 5,65 =0.859, p>0.05; and no genotype main effect: F 1,13 <0.001, p>0.05). (d) Mice with DMS A 2A R knockdown significantly reduced their lever pressing in the devalued condition compared with that of the valued condition, but the A 2A R(+/+) mice responded insensitively to the selective satiety devaluation treatment (normalized devaluation × genotype interaction effect: F 1,13 =9.161, p=0.01; simple effect analysis: F 1,6 =35.683, **p<0.01 for A 2A R focal knockdown mice by repeated-measures ANOVA). Data are presented as the mean±SEM. CRF, continuous reinforcement. The color reproduction of this figure is available on the Neuropsychopharmacology journal online. Full size image Download PowerPoint slide

Figure 5 Focal knockdown of the adenosine A 2A receptors (A 2A Rs) in the dorsolateral striatum (DLS) exerts relatively limited effects on habitual behaviors. (a) Representative image shows that A 2A Rs were knocked down selectively in the area with the AAV-Cre-zsGreen expression in the DLS of A 2A R(flox/flox) mice but not in A 2A R(+/+) mice. The yellow circle depicted the boundary of the AAV-Cre-zsGreen expression and A 2A R knockdown area. (b) Quantitative analysis shows that A 2A R expression was markedly reduced in the AAV-Cre-zsGreen transfected regions of A 2A R(flox/flox) mice compared with A 2A R(+/+) mice. (c) Focal A 2A R knockdown in the DLS (n=7) did not affect lever pressing during the acquisition phase compared with their A 2A R(+/+) controls (n=6) (repeated-measures analysis of variance (ANOVA) revealed no random interval (RI) period × genotype interaction effect: F 5,55 =1.234, p>0.05; and no genotype main effect: F 1,11 =0.534, p>0.05). (d) There was no genotype × devaluation interaction effect (F 1,11 =1.993, p=0.186, repeated-measures ANOVA) for the normalized devaluation test. Both groups similarly showed insensitivity to outcome devaluation (DLS A 2A R knockdown mice: normalized devaluation; t 1,6 =0.646, p>0.05; wild-type (WT) mice: normalized devaluation; t 1,5 =2.017, p>0.05). Data are presented as the mean±SEM. CRF, continuous reinforcement. Full size image Download PowerPoint slide