Abstract
The mammalian striatum is divided into two types of anatomical structures: the island-like, μ-opioid receptor (MOR)-rich striosome compartment and the surrounding matrix compartment. Both compartments have two types of spiny projection neurons (SPNs), dopamine receptor D1 (D1R)-expressing direct pathway SPNs (dSPNs) and dopamine receptor D2 (D2R)-expressing indirect pathway SPNs. These compartmentalized structures have distinct roles in the development of movement disorders, although the functional significance of the striosome compartment for motor control and dopamine regulation remains to be elucidated. The aim of this study was to explore the roles of striosome in locomotion and dopamine dynamics in freely moving mice. We targeted striosomal MOR-expressing neurons with male MOR-CreER mice, which express tamoxifen-inducible Cre recombinase under MOR promoter, and Cre-dependent adeno-associated virus vector. The targeted neuronal population consisted mainly of dSPNs. We found that the Gq-coupled designer receptor exclusively activated by designer drugs (DREADD)-based chemogenetic stimulation of striatal MOR-expressing neurons caused a decrease in the number of contralateral rotations and total distance traveled. Wireless fiber photometry with a genetically encoded dopamine sensor revealed that chemogenetic stimulation of striatal MOR-expressing neurons suppressed dopamine signals in the dorsal striatum of freely moving mice. Furthermore, the decrease in mean dopamine signal and the reduction of transients were associated with ipsilateral rotational shift and decrease of average speed, respectively. Thus, a subset of striosomal dSPNs inhibits contralateral rotation, locomotion, and dopamine release in contrast to the role of pan-dSPNs. Our results suggest that striatal MOR-expressing neurons have distinct roles in motor control and dopamine regulation.
Significance Statement
The striatum plays a crucial role in motor control, and it consists of two anatomical compartments: μ-opioid receptor (MOR)-rich striosomes and the surrounding matrix. The striosome sends efferent inhibitions to midbrain dopaminergic neurons, but it remains unknown whether striosomes are involved in motor control and dopamine regulation in behaving animals. We used MOR expression-based targeting, chemogenetics, and wireless fiber photometry with a genetically encoded dopamine sensor, and we found that chemogenetic stimulation of striosomal MOR-expressing neurons inhibits contralateral rotations, total distance traveled, and dopamine release in the dorsal striatum. The rotational shift and locomotion decrease might be differently associated with dopamine dynamics. This study provides compelling evidence that striosomal MOR-expressing neurons inhibit dopamine release and locomotion in freely moving animals.
Introduction
The striatum is the major input nucleus of the basal ganglia and plays an important role in motor control. The mammalian striatum is divided into two types of anatomical compartment structures: the island-like striosome (or patch) compartment and the surrounding matrix compartment (Pert et al., 1976; Graybiel and Ragsdale, 1978; Fujiyama et al., 2011). The striosome comprises roughly 15% of the area of the rostral striatum and is conserved in both rodents and humans (Johnston et al., 1990). The striosome compartment is classically defined by the strong expression of μ-opioid receptor (MOR; Pert et al., 1976; Herkenham and Pert, 1981). MOR and Oprm1 mRNA are highly expressed in the murine striosome compartment (Mansour et al., 1994; Minami et al., 1994; Arvidsson et al., 1995; Kaneko et al., 1995; Mansour et al., 1995). It has been reported that mouse models of Huntington's disease show characteristic changes in striosomal MOR expression (Morigaki et al., 2020). In patients with X-linked recessive dystonia-parkinsonism (XDP), selective neurodegeneration of the striosome compartment was reported in early stages of the disease (Goto et al., 2005). These studies suggest that the striosome may have distinct roles in motor function and that abnormalities in the striosome may be associated with the development of movement disorders (Crittenden and Graybiel, 2011).
Both striosomes and matrix compartments consist of two main types of projection neurons called spiny projection neurons (SPNs). One is known as direct pathway SPNs (dSPNs) that express dopamine D1 receptor (D1R) projecting to the internal segment of globus pallidus (GPi, entopeduncular nucleus in rodents) and the substantia nigra pars reticulata (SNr) and the other as indirect pathway SPNs (iSPNs) that express dopamine D2 receptor projecting to the external segment of globus pallidus (GPe). This dichotomy of the parallel processing model of motor control has been a mainstay in the understanding of basal ganglia function (Alexander and Crutcher, 1990, Gerfen and Surmeier, 2011), and it has been well-tested in transgenic mice by pan-direct or pan-indirect pathway manipulation (Lenz and Lobo, 2013). Optogenetic activation of dSPNs in the nucleus accumbens induces locomotion after cocaine exposure (Lobo et al., 2010). Likewise, optogenetic activation of pan-direct and pan-indirect pathways in the dorsal striatum have opposite roles in locomotor control (Kravitz et al., 2010). Chemogenetic manipulation of SPNs using designer receptors exclusively activated by designer drugs (DREADDs) produced biased rotational movements (Alcacer et al., 2017; Bay Kønig et al., 2019)––stimulation of pan-dSPNs caused contralateral rotations and suppression of pan-dSPNs resulted in ipsilateral rotational shifts.
dSPNs in the striosome project directly to the substantia nigra pars compacta (SNc; Gerfen, 1985; Fujiyama et al., 2011). A recent electrophysiological study using slice sections of genetically modified mice showed that striosomes send strong inhibitory projections to midbrain dopaminergic cells (Evans et al., 2020). Midbrain dopaminergic neurons send dense dopamine projections to the striatum (Matsuda et al., 2009) and are implicated in reinforcement learning (Jean-Richard-Dit-Bressel et al., 2018). A recent study suggested that a subset of dSPNs in the striosome negatively modulated reinforcement and locomotion (Xiao et al., 2020). Thus, it still remains to be elucidated whether striosome is involved in motor control and dopamine regulation in freely behaving animals.
The purpose of the present study was to examine whether the manipulation of genetically defined MOR-expressing striosomes controlled locomotion and dopamine dynamics in freely moving mice. We demonstrated that Gq-DREADD-based chemogenetic stimulation of striosomal MOR-expressing neurons reduced the number of contralateral rotations and locomotion with a marked decrease in dopamine signals. Furthermore, the rotational shift and average speed reduction were differently associated with dopamine dynamics. These results indicate that a subset of striosomal MOR-expressing cells modulate rotational movement with suppressed dopamine dynamics, quite in contrast to the prediction from the traditional dichotomy model of SPNs. This study therefore provides pathophysiological clues that abnormal activity of striosomal SPNs might account for the development of movement disorders via dopamine dysregulation.
Materials and Methods
Animals
All experiments conformed to the Japanese Act on Welfare and Management of Animals, the Standards relating to the Care and Keeping and Reducing Pain of Laboratory Animals (Notice of the Ministry of the Environment No. 88 of 2006), and were approved by the Animal Research Committee, Center for iPS Cell Research and Application, Kyoto University (approval numbers: 17-91-7 and 23-210-2). Male mice heterozygous for MOR-CreER allele (Okunomiya et al., 2020) maintained in a C57BL/6J background were used for this study (n = 50). Although tamoxifen treatment does not have persistent effects in adult open field behavior in both sexes (Rotheneichner et al., 2017), we intended to avoid the potential to confound effects of tamoxifen as an estrogen receptor agonist in adult females (Pinto et al., 2022). To prepare MOR-CreER mice heterozygous for the knock-in allele, in vitro fertilization using sperms of homozygous MOR-CreER males and oocytes of C57BL/6J females was performed, and embryos were transferred to pseudopregnant ICR female mice. Mice were housed with food and water ad libitum in light-controlled (12 h light/dark cycle) mouse facilities.
Adeno-associated virus (AAV) vector injection and Cre-dependent gene expression
MORDREADD mice and MORmCherry mice were prepared by stereotaxic injection of AAV2/9-hSyn-DIO-hM3Dq-mCherry generated from pAAV-hSyn-DIO-hM3D(Gq)-mCherry (gift from Bryan Roth, Addgene viral prep #44361-AAV9; http://n2t.net/addgene:44361; RRID: Addgene_44361; Krashes et al., 2011) and AAV2/9-hSyn-DIO-mCherry generated from pAAV-hSyn-DIO-mCherry (gift from Bryan Roth, Addgene viral prep #50459-AAV9; http://n2t.net/addgene:50459; RRID: Addgene_50459), respectively. For fiber photometry experiments, AAV2/9-hSyn-GRABDA2m generated from pAAV-hsyn-GRAB_DA2m (gift from Yulong Li, Addgene viral prep #140553-AAV9; http://n2t.net/addgene: 140553; RRID: Addgene_140553; Sun et al., 2020) was added to the viral solution. For stereotaxic injections into the striatum, male MOR-CreER mice aged 2–4 months were anesthetized with a mixture containing 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol. A glass pipette with a tip of ∼50 μm was stereotaxically placed into the striatum (0.7 mm anterior to the bregma, 2.2 mm lateral to the midline, and 2.8 mm deep from the brain surface), and 500 nl of viral solution diluted with PBS to a final concentration of 5.0 × 1012 viral particles/ml for AAV2/9-hSyn-DIO-hM3Dq-mCherry and AAV2/9-hSyn-DIO-mCherry or 2.0 × 1012 viral particles/ml for AAV2/9-hSyn-GRAB_DA2m was injected using a 25 μl Hamilton syringe connected to a microinfusion pump. Mice were allowed to recover from surgery for at least 2 weeks before tamoxifen treatment. To activate CreERT2 recombinase in adult MOR-CreER mice, 100 mg/kg of tamoxifen (T5648, Sigma-Aldrich) in corn oil (C8267, Sigma-Aldrich) was intragastrically administered on 5 consecutive days, followed by 1 week of recovery. This procedure was repeated for one more round.
Open field test
We used 50 × 50 cm white plexiglass open field boxes. Mice were allowed to move freely to explore the open field arena. Spontaneous locomotor activity of MORDREADD and MORmCherry mice aged 4–9 months was observed with a video camera at 30 frames per second (HDR-CS470, SONY) from above the open field boxes in a dimly lit room. All experiments were performed during the light phase. Prior to the test, the mice were acclimated to the apparatus for 5 min daily for 3 d. For behavioral analysis without photometry, the mice were randomly allocated to Group 1 or Group 2. On open field test (OFT) Day 1, baseline locomotor activity was recorded for 60 min before DREADD activation. DREADD was activated using the high-affinity and selective agonist deschloroclozapine (DCZ; HY-42110, MedChemExpress; Nagai et al., 2020), and vehicle (normal saline containing 0.5% dimethyl sulfoxide) was used for control. DCZ (100 µg/kg) or vehicle was injected intraperitoneally, and locomotor activity was recorded for 90 min. On Day 2, the same OFT recording was performed using vehicle or DCZ treatment (DCZ on Day 1 and vehicle on Day 2 in Group 1 and vice versa in Group 2). To counterbalance possible bias of the order of drugs, OFT was repeated in reverse order of the DCZ/vehicle administration (vehicle on Day 1, DCZ on Day 2 in Group 1, and vice versa in Group 2) from 1 to 3 weeks later. Results of the two rounds of OFT were averaged for each animal and used for analysis. In the experiment for muscarinic acetylcholine receptor (mAChR) blockade, scopolamine (1 mg/kg; Sigma-Aldrich) was intraperitoneally injected immediately before the DCZ or vehicle treatment.
Behavioral assessment
For behavioral assessments without photometry, ipsilateral rotations, contralateral rotations, ipsilateral head-turn angles, contralateral head-turn angles, and total distance traveled were analyzed using ANY-maze ver 7.20 (Stoelting) in 10 min bins. A side of the square arena (50 cm) in the video images was used for calibrating the pixel in centimeters. Locomotion parameters within 30 min after treatment were used for statistical analysis. For behavioral assessment with simultaneous photometry recording, we used DeepLabCut v2.3.5 (Mathis et al., 2018: Nath et al., 2019) to identify the x–y coordinates of the body parts (left ear, right ear, left hip, right hip, tail base, and tail tip) to avoid possible effects of the head-mounted device. In the training dataset, body parts were manually labeled using 100 images per mouse. Coordinates were calibrated as seven pixels to 1 cm using the one side of the square arena (50 cm) in the video images. The body axis was defined as the angle of the line segment from the tail base to the midpoint between the left ear and right ear. The angular velocity was calculated as the body axis change between frames. Speed was defined as the x–y change in coordinates of the tail base between frames. Behavioral data from one mouse was excluded from behavioral analysis because the tail base could not be reliably detected probably due to shortening of the tail. Relative average speed was calculated for each mouse using average speed in the vehicle treatment as reference.
Wireless fiber photometry recording and analysis
After tamoxifen treatment, a 400-µm-diameter optic fiber was stereotaxically implanted in the dorsal striatum (0.7 mm anterior to the bregma, 2.2 mm lateral to the midline, and 2.2 mm deep from the brain surface) ipsilateral to the hemisphere of the viral injection. To minimize interference from the patch cable due to unilateral rotational shift, we utilized a wireless fiber photometry device (TeleFipho, BioResearch Center). The photometry signal was recorded at a sampling rate of 100 Hz using TeleFipho software (BioResearch Center) equipped with the first-order low-pass Butterworth filter with a cutoff frequency of 2 Hz for mean change analysis in Figure 7 and 10 Hz for dopamine transient analysis and simultaneous video recordings. Prior to recording, the mice were habituated to the OFT apparatus described above as well as a dummy head-mounted device for 5 min daily for 3 d. On the recording day, the head-mounted device was attached to the fiber ferrule after brief anesthesia with isoflurane, and the mice were allowed to move freely to explore the open field arena. Recording was started 20 min before the DCZ or vehicle treatment and stopped 40 min after the treatment. For simultaneous recording of dopamine photometry and spontaneous behavior, images from an overhead video camera (640 × 480 pixels; DMK33UP1300, Imaging Source) were simultaneously recorded from 5 min after the DCZ/vehicle treatment for 20 min at 20 frames per second. On the following day, the same recording was conducted using vehicle or DCZ treatment. Mice were randomly allocated to the DCZ-first group or the vehicle-first group. All recordings were made during the light phase. To correct for photobleaching, a raw photometry signal was fitted with an exponential curve using 20 min of data from 10 min before and from 30 min after the DCZ/vehicle treatment as references. Subtraction of the reference value from the raw signal divided by the reference value (dF/F) was used for further analysis. For analysis of the mean dopamine signal, dF/F was z-scored (zF) using the data from 10 min before the DCZ/vehicle treatment as baseline recording. zF scores were averaged for bins as indicated after DCZ/vehicle treatment and used for statistical analysis. For dopamine transient analysis, dF/F values were processed using a digital high-pass Butterworth filter with a cutoff frequency of 0.1 Hz for canceling baseline dopamine signal suppression. Dopamine transients were identified using the find_peaks function in SciPy (height, 1; distance, 250 ms; prominence, 1). The relative frequency of dopamine transients was calculated using the mean frequency of dopamine transients in the vehicle treatment during the video recording as a reference value. Postrecording analysis was performed on Python 3.9.4 with NumPy v1.26.4 and SciPy v1.11.4.
Histochemical analysis
Mice were deeply anesthetized with isoflurane and then transcardially perfused with 0.01 M PBS followed by 4% paraformaldehyde (PFA) in 0.1 M PB, pH 7.4. Brains were removed and postfixed with 4% PFA in 0.1 M PB overnight at room temperature for IHC or for 3 d at 4°C for ISH. After cryoprotection with 30% (wt/vol) sucrose in 0.01 M PBS, blocks of brains from adult mice were cut into 30-μm-thick sections with a cryostat. Free-floating brain sections were incubated overnight at room temperature with primary antibodies in PBS containing 0.3% (wt/vol) Triton X-100 (PBS-X) and 10% normal goat serum (NGS). Normal horse serum was used instead of NGS when using goat antibody. The primary antibodies used were 1:250 diluted guinea pig anti-MOR antibody (AB5509, Merck Millipore), 1:1,000 diluted mouse anti-tyrosine hydroxylase antibody (MAB318, Sigma-Aldrich), 1:500 diluted rat anti-mCherry antibody (M-11217, Thermo Fisher Scientific), 1:300 diluted rabbit anti-phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204) antibody (pERK1/2; #9101, Cell Signaling Technology), 1:500 diluted goat anti-choline acetyltransferase (ChAT) antibody (AB144P, Sigma-Aldrich), and 1:1,000 diluted mouse anti-neuronal nuclei (NeuN) antibody (MAB377, Merck Millipore). After three washes with PBS, the sections were incubated with secondary antibodies in PBS-X and 0.5% NGS. The secondary antibodies (8 μg/ml, Thermo Fisher Scientific) used were Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 488-conjugated goat anti-mouse IgG antibody, Alexa Fluor 488-conjugated donkey anti-goat IgG antibody, Alexa Fluor 546-conjugated goat anti-rat IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody, Alexa Fluor 647-conjugated goat anti-guinea pig IgG antibody, and Alexa Fluor 647-conjugated donkey anti-mouse IgG antibody.
Fluorescent labeling for mRNA signals
Relevant fragments of Drd1 cDNA (nucleotide 1,116–1,809; GenBank accession No. NM_010076.3) or Drd2 cDNA (1,636–2,723; NM_010077.3) were cloned into a pBluescript II SK(+) (Stratagene) plasmid vector. Digoxigenin (DIG)-labeled cRNA probes were synthesized with T3 RNA polymerase and DIG RNA Labeling Mix (11277073910, Sigma-Aldrich) using linearized plasmids as templates. Fluorescent ISH was combined with fluorescent IHC as reported previously (Hioki et al., 2010). Free-floating sections were treated with 2% (wt/vol) H2O2 followed by 0.3% Triton X-100 and then hybridized for 16–20 h at 60°C with 1 μg/ml DIG-labeled antisense cRNA probe in the hybridization buffer. After washing and ribonuclease A treatment, the sections were incubated overnight at room temperature with 1:2,000 diluted horseradish peroxidase-conjugated sheep anti-DIG antibody (11207733910, Sigma-Aldrich) and 1:500 diluted rat anti-mCherry antibody. After washing, the sections were incubated with 8 μg/ml Alexa Fluor 546-conjugated goat anti-rat IgG antibody (Thermo Fisher Scientific) for 1 h. The sections were then treated with a TSA-Plus Biotin Kit (Akoya Biosciences) for 20 min and finally incubated with 2 μg/ml Alexa Fluor 488-conjugated streptavidin (Thermo Fisher Scientific) for 2 h.
Image acquisition and cell counting
Sections were mounted on glass slides and coverslipped. Images were acquired under a confocal microscope (FV3000, Olympus) with a 4× or 10× objective lens for low-power field images and a 20× objective lens for cell counting. Whole section images were obtained with an inverted fluorescent microscope (APX100 digital imaging system, Olympus) with a 10× objective lens. For each mouse, three images were obtained from at least two separate sections for cell counting with the Multi-point Tool in ImageJ (National Institutes of Health). Areas with MOR-rich striosome were determined manually using the ROI Manager in ImageJ. NeuN-positive cells were counted using the Find Maxima function after denoising by Gaussian Blur (sigma = 2) filter in ImageJ. Cytoarchitectonic areas were determined according to the atlas (Paxinos and Franklin, 2012).
Experimental design and statistical analyses
Sample sizes for MORDREADD and MORmCherry were determined to detect the rotational differences after DCZ treatment based on preliminary observations. For simplicity, we injected the AAV vector into the right hemisphere of MOR-CreER mice for behavioral tests because left hemispheric stimulation of striosomal neurons showed the same tendency of rotational shift in the preliminary observation of the OFT. Descriptive statistics were presented using at least n = 3. Statistical analyses were performed using GraphPad Prism ver. 10 (GraphPad Software). Statistical comparisons were performed with a two-tailed paired Student's t test for cell counting. Repeated-measures (RM) two-way ANOVA was conducted with a 2 × 2 (Treatment [DCZ, vehicle] × Group [MORDREADD, MORmCherry]) design to estimate the effect of treatments and groups on behavioral metrics and dopamine transient metrics. Average z-scored signals (zF) of dopamine were analyzed by RM two-way ANOVA with treatment [DCZ, vehicle] and time as within-subject factors. The effect of striosomal stimulation on mean zF intensity was analyzed by comparing the effect of DCZ treatment between MORDREADD mice and MORmCherry mice using a RM two-way ANOVA with group as a between-subject factor and time as a within-subject factor. A two-way ANOVA was conducted with a 2 × 2 (Treatment [DCZ, vehicle] × Compartment [striosome, matrix]) design to estimate the effect of treatments and compartments on pERK1/2-immunoreactivity cell density. When the interaction was significant, post hoc Bonferroni's multiple-comparisons test was performed. Pearson's correlation coefficient within each test of a mouse was transformed by Fisher's z transformation and pooled between mice with a random-effects model (Corey et al., 1998; Borenstein et al., 2010). A regression analysis with linear mixed-effects models (Yu et al., 2022) used the average speeds as dependent variable, the relative frequency of dopamine transients as fixed effects, and the mouse as the grouping factor for random slope and random intercept. The random-effects models analysis and the linear mixed-effects models analysis were performed using rma function of the metafor package in R or statsmodels 0.14.1. The difference was considered significant at a p value <0.05.
Results
MOR-CreER mice target striosome compartment with Cre-dependent AAV vector
For genetic dissection of the striatum, new genetically modified mice have led to functional elucidation of the striatal compartment structure. We utilized a recently developed MOR-CreER mouse line (Okunomiya et al., 2020), which expressed inducible Cre recombinase under Oprm1 promoter. To achieve transgene expression in the striosome, we injected a Cre-dependent AAV vector expressing mCherry fluorescent protein under hSyn promoter (AAV2/9-hSyn-DIO-mCherry; Fig. 1A) into the unilateral dorsal striatum of MOR-CreER mice (referred to as MORmCherry mice; Fig. 1B). After two courses of tamoxifen treatment, mCherry-positive cell clusters corresponding to MOR-immunoreactivity were observed (Fig. 1C). The density of mCherry-positive cells, the percentage of mCherry-positive cells, and the percentage of NeuN-positive cells with mCherry fluorescence were significantly higher in the striosome than in the matrix as defined by strong signals for MOR-immunoreactivity (paired t test: n = 4, t(3) = 14.5, p = 0.0007; n = 4, t(3) = 11.06, p = 0.0016; n = 3, t(2) = 20.39, p = 0.0024, respectively; Fig. 1D–G). Striosomal projection neurons are known to form characteristic axon bundles in the substantia nigra pars reticulata (SNr; Crittenden et al., 2016). In MORmCherry mice, we observed mCherry-positive clear bundle-like fibers (Fig. 1H). These results indicate that the MOR-CreER mouse line combined with Cre-dependent AAV vectors can be used for highly striosome-preferential transgene expression.
MOR-CreER mice target striosome compartment with Cre-dependent AAV vector. A, Schematic illustration of Cre-dependent AAV construct (AAV2/9-hSyn-DIO-mCherry). B, Unilateral AAV injection in the dorsal striatum of MOR-CreER mice and tamoxifen administration schedule after AAV injection. C, mCherry fluorescence and MOR-immunoreactivity visualized with Alexa 647 (green color) in the dorsal striatum of MORmCherry. D, mCherry-positive cells are more densely distributed in the strong MOR-immunoreactivity area than in the surrounding weak MOR-immunoreactivity area (n = 4; t(3) = 14.5; p = 0.0007; paired t test). E, The percentage of mCherry-positive cells in the striosomes was higher than that in the matrix (n = 4; t(3) = 11.06; p = 0.0016; paired t test). F, mCherry fluorescence and immunoreactivity for MOR and neuronal nuclei (NeuN) in the dorsal striatum of MORmCherry. G, The percentage of NeuN-positive neurons with mCherry fluorescence in the striosome was higher than that in the matrix (n = 3; t(3) = 20.39; p = 0.0024; paired t test). H, mCherry fluorescence and tyrosine hydroxylase (TH)-immunoreactivity (green color) in a coronal section of MORmCherry mice. Dense bundles of mCherry-positive projections (arrowheads) are observed in the substantia nigra pars reticulata (SNr). Data are shown as mean. Error bars indicate SE. Scale bars: 100 µm in panels C, F and 200 µm in panel H. **p < 0.01; ***p < 0.001.
MOR-CreER mice mainly target striosomal dSPNs
To characterize the cell population targeted in the MORmCherry mouse line, mCherry fluorescence was examined in the output nuclei of the SPNs (n = 3). Dense axon terminals were observed in SN as described above, and less prominent axonal projection was found in GPe (Fig. 2A,B), suggesting that targeted projection neurons in the dorsal striatum of MORmCherry mice were dSPNs that mainly have axonal projections from the dorsal striatum to SNc dopaminergic neurons. To characterize the target cell populations of D1R-expressing direct and D2R-expressing indirect pathways, D1R and D2R mRNAs were visualized using in situ hybridization (Fig. 2C,E). Approximately 80% of the targeted cells were D1R-mRNA–positive direct pathway cells (Fig. 2D) and ∼26% of the targeted cells were D2R-mRNA positive (Fig. 2F). These proportions were consistent with previous studies (Banghart et al., 2015). Next, we questioned whether Cre recombination activity was present in the interneuron population, as MOR expression in the dorsal striatum has been reported in a subset of cholinergic interneurons not limited to SPNs (Jabourian et al., 2005). Consistent with the previous results, a small portion of the mCherry-positive cells was positive for choline acetyltransferase (ChAT)-immunoreactivity (Fig. 2G,H). The number was nonsignificantly higher than the proportion of ChAT-positive neurons among the total neurons of the dorsal striatum (mean ± SEM; 2.3 ± 0.79% among the targeted cells and 1.0 ± 0.09% among the total neurons; paired t test; t(4) = 1.772, p = 0.1511; Fig. 2I), compatible with high MOR expression in cholinergic interneurons (Jabourian et al., 2005). The percentage of ChAT-positive cells expressing mCherry was 14.8 ± 5.7% (Fig. 2J). These results indicate that the targeted neurons of MOR-CreER mice are mainly striosomal dSPNs.
MOR-CreER mice mainly target striosomal direct pathway spiny projection neurons. A, B, Representative images of the projection from dorsal striatum in MORmCherry mice. Dense axon terminals are observed in SNr and much less prominent axonal projection is found in GPe. C, Double fluorescent labeling for mCherry-immunoreactivity and Drd1a mRNA (D1R). D, The percentages of Drd1a positive cells among mCherry-immunoreactivity (IR) cells. Over 80% of mCherry-expressing cells express Drd1a (n = 3). E, Double fluorescent labeling for mCherry-immunoreactivity and Drd2 mRNA (D2R). Arrows indicate double positive for mCherry-immunoreactivity and Drd2. F, The percentages of Drd2-positive cells among mCherry-immunoreactivity cells (n = 3). G, mCherry fluorescence, ChAT-immunoreactivity visualized with Alexa 488, and NeuN-immunoreactivity visualized with Alexa 647. H, Percentage of mCherry fluorescence positive cells with ChAT-immunoreactivity (n = 5). I, Percentage of NeuN-immunoreactivity neurons with ChAT-immunoreactivity. J, Percentage of ChAT-expressing neurons with mCherry fluorescence. Data are shown as mean. Error bars indicate SE. Scale bars: 500 µm in panels A, B and 50 µm in panels C, E, G.
Striosomal neurons are chemogenetically activated with DCZ treatment of MORDREADD mice
To manipulate neuronal activity in the striosome with Gq-based DREADD, we stereotaxically injected a Cre-dependent AAV vector expressing hM3Dq-mCherry fusion protein under hSyn promoter (AAV2/9-hSyn-DIO-hM3Dq-mCherry) into the unilateral striatum of MOR-CreER mice (referred to as MORDREADD mice; Fig. 3A). After two courses of tamoxifen treatment, we administered DCZ, a highly selective DREADD agonist (Nagai et al., 2020), or vehicle intraperitoneally to MORDREADD mice and perfusion fixed them 10 min later to confirm Gq-DREADD-induced neuronal activation in the striosome for functional analysis. By immunohistochemical analysis, an intense hM3Dq-mCherry fluorescence was observed in the striosome (Fig. 3B). To visualize neuronal activation after DCZ treatment, pERK1/2 immunohistochemistry was performed (Fig. 3C,D) and the density of pERK1/2-positive cells was calculated. A two-way ANOVA analysis showed significant effects of treatments (F(1,10) = 19.18; p = 0.0014), compartments (F(1,10) = 9.697; p = 0.011), and treatment × compartment interaction (F(1,10) = 9.881; p = 0.0104). Post hoc Bonferroni's test showed that the pERK1/2 cell density was significantly higher in the striosome compartment of DCZ-treated MORDREADD mice than in the matrix compartment of DCZ-treated MORDREADD mice and both compartments of vehicle-treated MORDREADD mice (t(10) = 4.139, p = 0.0121 for striosome in DCZ treatment vs matrix in DCZ treatment; t(10) = 5.320, p = 0.0020 for striosome in DCZ treatment vs striosome in vehicle treatment; t(10) = 5.299, p = 0.0021 for striosome in DCZ treatment vs matrix in vehicle treatment; Fig. 3E). The difference in pERK1/2 positivity between DCZ-treated matrix and vehicle-treated matrix was not significant (t(10) = 0.8742; p > 0.99). Next, we carefully examined the possible extrastriatal hM3Dq-mCherry expression due to the potential retrograde infection capability of AAV serotype 9 (Castle et al., 2014). Although the native fluorescence of mCherry was hardly detected outside the striatum, we noticed a faint but reproducible signal for mCherry-immunoreactivity in the centrolateral and paracentral thalamic nuclei (CL/PC; Fig. 4A,B). This retrograde Cre-dependent expression in the thalamus was congruent with the previous findings that CL/PC nuclei send inputs to the dorsal striatum (Wall et al., 2013; Guo et al., 2015; Unzai et al., 2017) and CL/PC expresses Oprm1 mRNAs (Mansour et al., 1994; Okunomiya et al., 2020). After the DCZ treatment in MORDREADD, pERK1/2-immunoreactivity was not detected above the background level (Fig. 4C). These results indicate that DCZ causes neuronal activation predominantly in the striosome compartment of MORDREADD mice and that extrastriatal Gq-DREADD induction in DCZ-treated MORDREADD mice due to retrograde leaky expression is highly unlikely.
Striosomal neurons are chemogenetically activated with DCZ treatment of MORDREADD mice. A, Schematic illustration of the injection of AAV2/9-hSyn-DIO-hM3Dq-mCherry virus vector into the dorsal striatum of MOR-CreER mice. B, Double fluorescent images of mCherry fluorescence fused to hM3Dq-DREADD and MOR-immunoreactivity visualized with Alexa 647 in the dorsal striatum of MORDREADD mice. C, D, Representative confocal images of pERK1/2-immunoreactivity, mCherry fluorescence, and MOR-immunoreactivity in DCZ-treated MORDREADD mice (C) and vehicle-treated MORDREADD mice (D). E, pERK1/2-positive cells are more densely observed in the MOR-rich striosome compartment than in the matrix compartment (n = 3 for DCZ treatment and n = 4 for vehicle treatment). Two-way ANOVA indicates the effect of treatment (F(1,10) = 19.18; p = 0.0014), the effect of compartment (F(1,10) = 9.697; p = 0.011), and the treatment × compartment interaction (F(1,10) = 9.881; p = 0.0104). Bonferroni's multiple-comparisons test shows that pERK1/2-positive cell density in the DCZ-treated striosome is higher than in the DCZ-treated matrix (t(10) = 4.139; p = 0.0121), vehicle-treated striosome (t(10) = 5.320; p = 0.0020), and vehicle-treated matrix (t(10) = 5.299; p = 0.0021). A difference in pERK1/2 positivity between DCZ-treated matrix and vehicle-treated matrix was not detected (t(10) = 0.8742; p > 0.99). Scale bars: 500 µm in panel B and 50 µm in panels C, D. *p < 0.05; **p < 0.01.
Assessment of extrastriatal hM3Dq-mCherry expression, mCherry-immunoreactivity and neuronal activation after DCZ treatment. A, Representative confocal microscopic images of extrastriatal sites in MORDREADD mice. mCherry fluorescence was enhanced by immunolabeling for mCherry with Alexa 546. mCherry-immunoreactivity (IR) cells were noticed in centrolateral/paracentral thalamic nuclei (CL/PC, arrow) with minimal leakage along the injection route (arrowhead) whereas mCherry-IR cells were not reproducibly observed in the prelimbic (PrL), lateral orbitofrontal (LO), cingulate (Cg), primary motor (M1), secondary motor (M2), primary somatosensory (S1), and secondary somatosensory (S2) cortices. Images were acquired in the same laser power, high voltage (HV) and gain across regions and processed under the same brightness/contrast settings in ImageJ. B, Representative confocal microscopic images of dorsal striatum (DS) and thalamic nuclei with or without immunolabeling for mCherry. Note that native mCherry fluorescence is hardly seen in CL/PC. C, pERK1/2-immunoreactivity in DS and CL/PC of DCZ-treated MORDREADD mice and vehicle-treated MORDREADD mice. pERK1/2-positive signals above the background level are not detected in CL/PC. pERK1/2 images were acquired and processed under the same condition across mice and regions. Scale bars: 1 mm in panels A, B, 50 µm in panel C. CM, centromedian nucleus.
Chemogenetic stimulation of striosomal MOR-expressing neurons suppresses contralateral rotations and locomotion
To investigate the effect of MOR-expressing neurons in the dorsal striatum on locomotor control in freely moving mice, we chemogenetically stimulated striosomal MOR-expressing neurons using MORDREADD mice (n = 10) and treated MORmCherry mice as negative control (n = 10). We measured the locomotor activity of these mice in an open field area under the administration of DCZ or vehicle, analyzed mean values within 30 min after treatment, and estimated the effect of striosomal stimulation. Unexpectedly, despite the unilateral dSPNs being mainly stimulated, striosomal stimulation decreased the contralateral rotations (group, F(1,18) = 0.2596, p = 0.6166; treatment, F(1,18) = 9.704, p = 0.006; group × treatment interaction, F(1,18) = 10.55, p = 0.0045; post hoc test, DCZ vs vehicle; t(18) = 4.499, p = 0.0006 for MORDREADD mice; t(18) = 0.09373, p > 0.99 for MORmCherry mice; Fig. 5A,B). Ipsilateral rotations increased slightly in the DCZ-treated MORDREADD mice but no significant effect of striosomal stimulation was detected (group, F(1,18) = 3.369, p = 0.083; treatment, F(1,18) = 2.015, p = 0.1728; group × treatment interaction, F(1,18) = 2.638, p = 0.1217; Fig. 5C,D). Striosomal stimulation reduced cumulative contralateral head-turn angles (group, F(1,18) = 3.898, p = 0.0639; treatment, F(1,18) = 3.830, p = 0.066; group × treatment interaction, F(1,18) = 5.226, p = 0.0346; post hoc test, DCZ vs vehicle; t(18) = 3.0, p = 0.0154 for MORDREADD mice; t(18) = 0.2326, p > 0.99 for MORmCherry mice; Fig. 5E,F), whereas no significant effect of striosomal stimulation was detected on cumulative ipsilateral head-turn angles (group, F(1,18) = 0.0287, p = 0.8674; treatment, F(1,18) = 0.001763, p = 0.967; group × treatment interaction, F(1,18) = 0.2278, p = 0.6389; Fig. 5G,H). Striosomal stimulation reduced the total distance traveled (group, F(1,18) = 1.867, p = 0.1887; treatment, F(1,18) = 10.67, p = 0.0043; group × treatment interaction, F(1,18) = 10.45, p = 0.0046; post hoc test, DCZ vs vehicle; t(18) = 4.595, p = 0.0004 for MORDREADD mice; t(18) = 0.02387, p > 0.99 for MORmCherry mice; Fig. 5I,J). These results indicate that highly striosome-preferential Gq-DREADD stimulation reduces the number of contralateral rotations and locomotion.
Chemogenetic stimulation of striosomal MOR-expressing neurons inhibits contralateral rotations and locomotion. Rotational metrics and total distance traveled are shown for every 10 min between 60 min before and 90 min after treatment in MORDREADD mice and in MORmCherry mice as control. A, B, Rotations contralateral to the injection side. Striosomal stimulation decreased contralateral rotations during 30 min after treatment (group, F(1,18) = 0.2596, p = 0.6166; treatment, F(1,18) = 9.704, p = 0.006; group × treatment interaction, F(1,18) = 10.55, p = 0.0045; post hoc test, DCZ vs vehicle; t(18) = 4.499, p = 0.0006 for MORDREADD mice; t(18) = 0.09373, p > 0.99 for MORmCherry mice). C, D, Rotations ipsilateral to the injection side. No significant effect of striosomal stimulation was detected on ipsilateral rotations during 30 min after treatment (group, F(1,18) = 3.369, p = 0.083; treatment, F(1,18) = 2.015, p = 0.1728; group × treatment interaction, F(1,18) = 2.638, p = 0.1217). E, F, Cumulative head-turn angles contralateral to the injection side. Striosomal stimulation decreased contralateral head-turn angles during 30 min after DCZ administration (group, F(1,18) = 3.898, p = 0.0639; treatment, F(1,18) = 3.830, p = 0.066; group × treatment interaction, F(1,18) = 5.226, p = 0.0346; post hoc test, DCZ vs vehicle; t(18) = 3.0, p = 0.0154 for MORDREADD mice; t(18) = 0.2326, p > 0.99 for MORmCherry mice). G, H, Cumulative head-turn angles ipsilateral to the injection side. No significant effect of striosomal stimulation was detected on ipsilateral head-turn angles during 30 min after treatment (group, F(1,18) = 0.0287, p = 0.8674; treatment, F(1,18) = 0.001763, p = 0.967; group × treatment interaction, F(1,18) = 0.2278, p = 0.6389). I, J, Total distance traveled. Striosomal stimulation decreased the total distance traveled during 30 min after treatment (group, F(1,18) = 1.867, p = 0.1887; treatment, F(1,18) = 10.67, p = 0.0043; group × treatment interaction, F(1,18) = 10.45, p = 0.0046; post hoc test, DCZ vs vehicle; t(18) = 4.595, p = 0.0004 for MORDREADD mice; t(18) = 0.02387, p > 0.99 for MORmCherry mice). Data are shown as mean. Error bars indicate SE. Statistical comparisons were performed using data during 30 min after the DCZ/vehicle treatment. *p < 0.05; ***p < 0.001.
Muscarinic acetylcholine receptor antagonist does not abolish the behavioral effects of chemogenetic stimulation of striosomal MOR-expressing neurons
Using these mice, we tested whether cholinergic modulation that might be exerted by a small population of ChAT-positive MOR-expressing neurons primarily inhibited contralateral rotations and locomotion. To this end, scopolamine, a nonselective mAChR antagonist, was coadministered with the DCZ/vehicle treatment to MORDREADD mice (n = 10). The contralateral rotations and total distance traveled were decreased in the DCZ-treated MORDREADD mice compared with vehicle-treated MORDREADD mice even under blockade of mAChRs (t(9) = 12.82, p < 0.0001 for contralateral rotations; t(9) = 5.879, p = 0.0002 for total distance traveled; paired t test; Fig. 6A,C). The ipsilateral rotations were increased in DCZ-treated MORDREADD mice compared with vehicle-treated MORDREADD mice with scopolamine cotreatment (t(9) = 3.866, p = 0.0038, paired t test; Fig. 6B), probably due to induced overall hyperlocomotion by scopolamine. These results indicate that suppression of contralateral rotations and locomotion in the DCZ-treated MORDREADD mice were not abolished by mAChR blockade. Thus, our data suggest that the suppression of contralateral rotations and locomotion is a result of Gq-DREADD stimulation in the striosomal MOR-expressing neurons and is not mainly caused by elevated cholinergic synaptic transmission through mAChR.
Effects of chemogenetic stimulation of striosomal MOR-expressing neurons are not abolished by systemic cholinergic receptor blockade. Rotations contralateral to the injection side (A), rotations ipsilateral to the injection side (B), and total distance traveled (C) are shown for every 10 min between 60 min before and 90 min after simultaneous treatment with scopolamine (SCP) and DCZ or SCP and vehicle in MORDREADD mice. A, Contralateral rotations after SCP and DCZ treatment decreased compared with SCP and vehicle treatment in MORDREADD mice (t(9) = 12.82; p < 0.0001; paired t test). B, Ipsilateral rotations increased compared with SCP and vehicle treatment in MORDREADD mice (t(9) = 3.866; p = 0.0038; paired t test). C, The total distance traveled after treatment was significantly decreased in SCP and DCZ cotreated MORDREADD mice compared with SCP and vehicle cotreated MORDREADD mice (t(9) = 5.879; p = 0.0002; paired t test). Data are shown as mean. Error bars indicate SE. Statistical comparisons were performed using rotation or locomotor data during 30 min after the DCZ/vehicle treatment. **p < 0.01; ***p < 0.001.
Chemogenetic stimulation of striosomal MOR-expressing neurons suppresses dopamine signals in the dorsal striatum
GABAergic striosomal SPNs form characteristic bundles of axon terminals called “dendron bouquets” on midbrain dopaminergic neurons (Crittenden et al., 2016). It has been reported that turning behavior is correlated with ipsilateral depression and contralateral elevation of dopamine signals (Jørgensen et al., 2023). To test whether the stimulation of striosomal SPNs modulates dopamine dynamics in the dorsal striatum of freely moving animals, we injected AAV2/9-hSyn-GRABDA2m, which expresses the genetically encoded dopamine sensor GRABDA2m (Sun et al., 2020; Fig. 7A) and AAV2/9-hSyn-DIO-hM3Dq-mCherry into the dorsal striatum of MOR-CreER mice (n = 3) and performed wireless fiber photometry (Fig. 7B,C). After habituation to the OFT apparatus and the head-mounted wireless device, we recorded GRABDA2m dopamine signals from the dorsal striatum with DCZ (Fig. 7D) or vehicle treatment for 2 consecutive days. After injection of DCZ, z-scored signals (zF) of GRABDA2m fluorescence intensity decreased in the dorsal striatum of MORDREADD mice compared with zF after vehicle treatment (Fig. 7E,F). Average zF signals for every 5 min were significantly decreased in the DCZ treatment compared with average zF signals after vehicle treatment (treatment, F(1,2) = 1390, p = 0.0007; time, F(1.974,3.947) = 30.76, p = 0.0039; treatment × time interaction, F(1.781,3.562) = 32.45, p = 0.0054; post hoc test, DCZ vs vehicle; t(2) = 28.27, p = 0.0075 for 0–5 min, t(2) = 31.48, p = 0.0060 for 5–10 min, t(2) = 14.37, p = 0.0288 for 10–15 min, t(2) = 6.035, p = 0.1583 for 15–20 min, t(2) = 11.6, p = 0.0441 for 20–25 min, t(2) = 3.533, p = 0.4297 for 25–30 min; Fig. 7G). In the control experiments with MORmCherry mice (Fig. 7H,I), no effect of DCZ treatment on zF of GRABDA2m fluorescence intensity was detected in the dorsal striatum (treatment, F(1,3) = 2.055, p = 0.2472; time, F(2.643,7.929) = 0.4506, p = 0.7024; treatment × time interaction, F(1.349,4.046) = 1.235, p = 0.3527; Fig. 7J), consistent with a previous report using DCZ treatment with GRABDA2m (X. Liu et al., 2022). The effect of striosomal stimulation on mean zF intensity was detected by comparing the contrasts of DCZ treatment (DCZ vs vehicle) between MORDREADD mice and MORmCherry mice (group, F(1,5) = 34.39, p = 0.0020; time, F(2.267,11.34) = 15.61, p = 0.0004; group × time interaction, F(5,25) = 13.18, p < 0.0001; post hoc test, MORDREADD vs MORmCherry; t(3.228) = 7.884, p = 0.0195 for 0–5 min, t(3.213) = 6.745, p = 0.032 for 5–10 min, t(4.053) = 6.375, p = 0.0178 for 10–15 min, t(5) = 4.402, p = 0.0421 for 15–20 min, t(3.557) = 5.561, p = 0.0431 for 20–25 min, t(4.613) = 3.187, p = 0.1635 for 25–30 min; Fig. 7K). Given that the fluctuation of dopamine transients is related to the onset of spontaneous behavior (Howe and Dombeck 2016; da Silva et al., 2018; Markowitz et al., 2023), we assessed dopamine transients with high-pass filtered photometry data by canceling baseline suppression (n = 9 MORDREADD mice; n = 4 MORmCherry mice; Fig. 8A–D). Striosomal stimulation reduced the frequency of dopamine transients (group, F(1,11) = 0.3703, p = 0.5552; treatment, F(1,11) = 12.73, p = 0.0044; group × treatment interaction, F(1,11) = 5.016, p = 0.0467; post hoc test, DCZ vs vehicle; t(11) = 5.235, p = 0.0006 for MORDREADD mice; t(11) = 0.7982, p = 0.8833 for MORmCherry; Fig. 8E,F) and the mean peak amplitude (group, F(1,11) = 2.879, p = 0.1178; treatment, F(1,11) = 10.97, p = 0.0069; group × treatment interaction, F(1,11) = 5.108, p = 0.0451; post hoc test, DCZ vs vehicle; t(11) = 5.022, p = 0.0008 for MORDREADD mice; t(11) = 0.6317, p > 0.99 for MORmCherry; Fig. 8G,H). These results demonstrate that chemogenetic stimulation of striosomal MOR-expressing neurons suppresses mean dopamine levels and dopamine transients in the dorsal striatum.
Suppression of striatal dopamine signal after chemogenetic stimulation of striosomal MOR-expressing neurons using wireless fiber photometry in freely moving mice. A, A representative image of dual expression of hM3Dq-mCherry and GRABDA2m in the dorsal striatum. B, A schematic illustration of optic fiber insertion. C, Head-mounted device for wireless fiber photometry used in this study. D, A raw trace of GRABDA2m fluorescence signal in photometry recording. At the injection, a large signal change was observed due to restraining the mouse for intraperitoneal injection. To cancel photobleaching, an exponentially fitted curve was used for reference. E, Representative z-scored dopamine trace in DCZ or vehicle treatment in a MORDREADD mouse. Photometry signals were corrected using the fitted curve as reference, and z-score (zF) was calculated using data for 10 min before treatment as baseline. Traces are drawn as rolling averages for the last 10 s. F, Average zF traces of DCZ-treated MORDREADD mice and vehicle-treated MORDREADD mice (n = 3). G, Mean dopamine signals after DCZ or vehicle treatment in MORDREADD mice in 5 min bins (treatment, F(1,2) = 1390, p = 0.0007; time, F(1.974,3.947) = 30.76, p = 0.0039; treatment × group interaction, F(1.781,3.562) = 32.45, p = 0.0054; post hoc test, DCZ vs vehicle; t(2) = 28.27, p = 0.0075 for 0–5 min, t(2) = 31.48, p = 0.0060 for 5–10 min, t(2) = 14.37, p = 0.0288 for 10–15 min, t(2) = 6.035, p = 0.1583 for 15–20 min, t(2) = 11.6, p = 0.0441 for 20–25 min, t(2) = 3.533, p = 0.4297 for 25–30 min). H, Representative z-scored dopamine trace in DCZ or vehicle treatment in a MORmCherry mouse. I, Average zF traces of DCZ-treated MORmCherry mice and vehicle-treated MORmCherry mice (n = 4). J, Mean dopamine signals after DCZ or vehicle treatment in MORmCherry mice in 5 min bins (treatment, F(1,3) = 2.055, p = 0.2472; time, F(2.643,7.929) = 0.4506, p = 0.7024; treatment × time interaction, F(1.349,4.046) = 1.235, p = 0.3527). K, Difference between DCZ and vehicle treatment in mean dopamine signals (group, F(1,5) = 34.39, p = 0.0020; time, F(2.267,11.34) = 15.61, p = 0.0004; group × time interaction, F(5,25) = 13.18, p < 0.0001; post hoc test, MORDREADD vs MORmCherry; t(3.228) = 7.884, p = 0.0195 for 0–5 min, t(3.213) = 6.745, p = 0.032 for 5–10 min, t(4.053) = 6.375, p = 0.0178 for 10–15 min, t(5) = 4.402, p = 0.0421 for 15–20 min, t(3.557) = 5.561, p = 0.0431 for 20–25 min, t(4.613) = 3.187, p = 0.1635 for 25–30 min). Scale bar: 1 mm in panel A. Data are shown as mean traces (line) and SE (shaded area) in panels F and I. Data are shown as mean and error bars indicate SE in panels G, J, and K. *p < 0.05; **p < 0.01.
Chemogenetic stimulation of striosomal MOR-expressing neurons suppresses dopamine fluctuations in freely moving mice. A, A representative trace of z-scored GRABDA2m fluorescence in a DCZ-treated MORDREADD mouse. B, For detecting dopamine transients, the dF/F traces were high-pass filtered and z scored. C, D, Dopamine transients were identified by peak detection as indicated by green circles. Representative traces from a vehicle-treated MORDREADD mouse (C) and a DCZ-treated MORDREADD mouse (D) are shown. E, F, The number of dopamine transients in 5 min bins and statistical analysis between 5 and 15 min after the DCZ/vehicle treatment in MORDREADD mice (E) and in MORmCherry mice (F). Striosomal stimulation reduced the frequency of dopamine transients (group, F(1,11) = 0.3703, p = 0.5552; treatment, F(1,11) = 12.73, p = 0.0044; group × treatment interaction, F(1,11) = 5.016, p = 0.0467; post hoc test, DCZ vs vehicle; t(11) = 5.235, p = 0.0006 for MORDREADD mice; t(11) = 0.7982, p = 0.8833 for MORmCherry mice). G, H, The mean peak amplitude in 5 min bins and statistical analysis between 5 and 15 min after the DCZ/vehicle treatment in MORDREADD mice (G) and in MORmCherry mice (H). Striosomal stimulation reduced the mean peak amplitude (group, F(1,11) = 2.879, p = 0.1178; treatment, F(1,11) = 10.97, p = 0.0069; group × treatment interaction, F(1,11) = 5.108, p = 0.0451; post hoc test, DCZ vs vehicle; t(11) = 5.022, p = 0.0008 for MORDREADD mice; t(11) = 0.6317, p > 0.99 for MORmCherry mice). Data are shown as mean in bar graphs and as mean traces (line) and SE (shaded area). ***p < 0.001.
Suppression of mean dopamine signal levels is positively correlated with ipsilateral turn shift but is negatively correlated with average speed reduction
We then examined the relationship between dopamine suppression and locomotion change after striosomal stimulation. To this end, we analyzed video recordings by overhead camera during wireless dopamine photometry from 5 min after DCZ or vehicle treatment for 20 min (Fig. 9A,B) and identified the body parts of mice using DeepLabCut (Fig. 9C; Mathis et al., 2018: Nath et al., 2019). The mean angular velocity between frames was calculated as positive value indicating contralateral direction, and the average speed between frames was calculated using the positional changes of the tail base of the mouse (Fig. 9C). In the video recording sessions, cumulative turns were biased to the ipsilateral side and average speed was decreased in DCZ-treated MORDREADD compared with vehicle-treated MORDREADD (t(7) = 2.808, p = 0.0262; t(7) = 2.838, p = 0.0251; paired t test; Fig. 9D,E), indicating that wireless photometry attachment with GRABDA2m coexpression did not obscure behavioral changes caused by the striosome activation. To investigate the association between dopamine fluctuations and locomotion, we calculated the cross-correlation between mean dopamine signals and angular velocity or average speed. Cross-correlation between dopamine signal level and mean angular velocity showed a positive peak in vehicle-treated MORDREADD and DCZ-treated MORDREADD (Fig. 9F). Cross-correlation between mean dopamine signal level and average speed showed a negative peak in vehicle-treated MORDREADD and DCZ-treated MORDREADD (Fig. 9G). We first analyzed the correlation between dopamine signal levels and angular velocity in 1 s bins. The mean angular velocity was positively correlated with mean dopamine signal levels (zF) in vehicle-treated MORDREADD and DCZ-treated MORDREADD (average correlation coefficient, r = 0.11, p = 0.0003; r = 0.11, p < 0.0001; n = 9,600 bins from 8 mice; Fig. 9H–J). Considering that the effect of DCZ persists at a timescale of minutes, we analyzed the correlation in the bins from 0.5 to 60 s after DCZ treatment. After striosomal stimulation, the mean angular velocity was positively correlated with mean dopamine levels in a relatively longer timescale (up to 30 s bin; Fig. 9K), indicating that baseline suppression of dopamine correlated with the ipsilateral shift of turns.
Suppression of mean dopamine signal levels is positively correlated with ipsilateral turn shift but negatively correlated with average speed reduction. A, A schematic illustration of simultaneous recording for wireless fiber photometry and video images. B, Time course of photometry and video recording. C, A representative image of marker-less feature detection using DeepLabCut. Positive angular velocity indicates contralateral turn between video frames. D, E, Cumulative angles and average speed were decreased in DCZ-treated MORDREADD mice (t(7) = 2.808, p = 0.0262; t(7) = 2.838, p = 0.0251: paired t test) with simultaneous recording for dopamine photometry. F, Cross-correlation analysis between mean angular velocity and mean dopamine signals in vehicle-treated MORDREADD mice and DCZ-treated MORDREADD mice. G, Cross-correlation analysis between average speeds and mean dopamine signals in vehicle-treated MORDREADD mice and DCZ-treated MORDREADD mice. H, I, Average correlations between mean angular velocity and mean dopamine signal levels in vehicle-treated MORDREADD mice (H) and DCZ-treated MORDREADD mice (I; n = 9,600 bins from 8 mice; vehicle, r = 0.11, p = 0.0003; DCZ, r = 0.11, p < 0.0001). J, Overlap drawing of panels H and I. K, Average correlation coefficients between mean dopamine signal levels and mean angular velocity in different time bins (from 0.5 to 60 s). L, M, Average correlations between mean dopamine signal levels and relative average speed in vehicle-treated MORDREADD mice (L) and DCZ-treated MORDREADD mice (M; n = 9,600 bins from 8 mice; vehicle, r = −0.18, p < 0.0001; DCZ, r = −0.098, p < 0.0001). N, Overlap drawing of panels L and M. O, Average correlation coefficients between mean dopamine signal levels and relative average speed in different time bins (from 0.5 to 60 s). Shaded areas in panels F, G, K, and O indicate 95% confidence intervals. Regression lines for each mouse are drawn in panels H, I, L, and M. *p < 0.05.
For average speeds, vehicle-treated MORDREADD data showed negative correlation between mean dopamine signal levels and average speeds in freely moving animals exploring the arena (average correlation coefficient, r = −0.18, p < 0.0001, n = 9,600 bins from 8 mice; Fig. 9L), consistent with a recent report (Markowitz et al., 2023). In the DCZ-treated MORDREADD mice, the mean dopamine signal also correlated negatively with average speeds (average correlation coefficient, r = −0.098, p < 0.0001, n = 9,600 bins from eight mice; Fig. 9M,N). Negative correlations in the vehicle-treated MORDREADD and DCZ-treated MORDREADD mice were observed when bins varied from 0.5 to 60 s (Fig. 9O). These results suggest that the decrease of mean dopamine signal levels after striosomal stimulation does not positively correlate with locomotor suppression within the 20 min test period.
Dopamine transients are positively correlated with future average speed under chemogenetic stimulation of striosomal MOR-expressing neurons
Given that dopaminergic neuron activity in the striatum is associated with movement onset (Howe and Dombeck 2016; da Silva et al., 2018; C. Liu et al., 2022), we investigated the frequency of dopamine transients. In vehicle-treated controls, the relative frequency of dopamine transients was not significantly correlated with average speeds (average correlation coefficient, r = 0.034, p = 0.6628; n = 320 bins from eight mice; Fig. 10A). In DCZ-treated animals, the relative frequency of dopamine transients was not significantly correlated with average speeds in 30 s bin (average correlation coefficient, r = 0.10, p = 0.1426, n = 320 bins from eight mice; Fig. 10B,C). The positive correlation was present with longer bins (Fig. 10D), suggesting that the association between average speed and the frequency of dopamine transients was detectable when striosomal dSPNs were activated. In the cross-correlation analysis using 30 s bin, the peak positive cross-correlation between the relative frequency of transients and average speed at 36 s lag was observed (Fig. 10E,F). Mean dopamine signal levels were negatively cross-correlated with the average speed at lags of 0–60 s (Fig. 10G). With a 36 s lag, the correlation between the relative frequency of dopamine transients and the average speed in the next 36 s bin was not detected in vehicle-treated MORDREADD mice but was detected under striosomal stimulation (average correlation coefficient, r = 0.21, p = 0.0007, n = 304 bins from eight mice with DCZ treatment; r = 0.033, p = 0.6490, n = 304 bins from eight mice with vehicle treatment; Fig. 10H–J). Linear mixed model analysis using the relative dopamine transient frequency as fixed effects with mouse as a group factor to estimate future average speeds showed the association of the frequency of dopamine transients with future average speed in DCZ-treated MORDREADD mice (β = 2.290; p = 0.007; Fig. 10K, Extended Data Table 10-1), but not in vehicle-treated MORDREADD mice (β = 0.236; p = 0.790; Fig. 10K, Extended Data Table 10-1). These results indicated that suppression of the dopamine transient frequency was associated with a reduction of future average speeds under striosomal stimulation within a 20 min test period. These results suggest that the mean dopamine signal levels and dopamine transients might be associated differently with rotational shifts and locomotion speed under the modulation of striosome neuronal activity. Our working hypothesis of striosomal modulation of locomotion and dopamine release is illustrated in Figure 11.
Dopamine transients are positively correlated with future average speed under chemogenetic stimulation of striosomal MOR-expressing neurons. A, B, Correlations between the relative frequency of dopamine transients and relative average speed in vehicle-treated MORDREADD mice (A) and DCZ-treated MORDREADD mice (B; n = 320 bins from 8 mice; vehicle, r = 0.034, p = 0.6628; DCZ, r = 0.10, p = 0.1426). C, Overlap drawing of panels A and B. D, Average correlation coefficients between the relative frequency of dopamine transients and relative average speed in different time bins (from 0.5 to 60 s). E, Schematic illustration of cross-correlation analysis between the relative frequency of dopamine transients and relative average speed. F, Cross-correlation between the relative frequency of dopamine transients and the relative average speed in DCZ-treated MORDREADD mice. The maximum point of cross-correlation function delays at 36 s in DCZ-treated MORDREADD mice. G, Cross-correlation between the mean dopamine signal levels (zF) and the relative average speed in DCZ-treated MORDREADD mice. H, I, Correlation between the relative frequency of dopamine transients and relative average speed in vehicle-treated MORDREADD mice (H) and DCZ-treated MORDREADD mice (I) with a 36 s lag (n = 304 bins, from 8 mice; vehicle, r = 0.033, p = 0.6490; DCZ, r = 0.21, p = 0.0007). J, Overlap drawing of panels H and I. K, A linear mixed-effects model analysis with mouse identity as a group factor confirmed the association of the relative frequency of dopamine transients in 30 s bin with future average speeds at 36 s lag (p = 0.007) in DCZ-treated MORDREADD mice. Error bars indicate 95% confidence intervals in panel K. Shaded areas indicate 95% confidence intervals in panels D, F, and G. Regression lines for each mouse are drawn in panels A, B, H, and I. See Extended Data Table 10-1 for more details.
Table 10-1
Summary statistics of linear mixed-effects models for average speed estimation. Download Table 10-1, DOCX file.
Summary of findings and a working hypothesis of locomotor modulating mechanisms under chemogenetic stimulation of striosomal MOR-expressing neurons. Our data shows that chemogenetic stimulation of striosomal MOR-expressing neurons causes suppression of mean dopamine signals and the frequency of dopamine peaks. Decrease of mean dopamine signal level is associated with reduction of contralateral rotations whereas decrease of the dopamine transient frequency is associated with decrease of average speed within test sessions. We hypothesize that tonic dopamine release and phasic dopamine release modulate the direction of rotations and locomotion vigor, respectively, in freely moving mice under activation of the striosomal direct pathway spiny projection neurons (dSPNs). MOR, μ-opioid receptor; SNc, substantia nigra pars compacta.
Discussion
In this study, we demonstrated that chemogenetic stimulation of striosomal MOR-expressing cells caused a decrease in contralateral turns and overall locomotor activity, quite in contrast to the traditional understanding of dSPNs enhancing motor activity. With wireless fiber photometry, we found that the mean dopamine signals and transients of genetically encoded dopamine sensor GRABDA2m decreased under chemogenetic stimulation of striosomal MOR-expressing neurons in the dorsal striatum of freely moving mice. Furthermore, with simultaneous analysis of dopamine photometry and behavior, we found that ipsilateral turn shift was associated with mean dopamine signal suppression while a decrease of average speed was associated with a reduced frequency of dopamine transients.
Functions of the striosome in motor control and dopamine regulation
Our results indicated an inhibitory effect of striosomal MOR-expressing neurons on contralateral rotation and locomotion. We demonstrated that chemogenetic stimulation of unilateral, MOR-expressing striosomal neurons resulted in an ipsilateral rotational shift even though the targeted cells were predominantly D1R-positive striosomal dSPNs. In contrast, a contralateral rotational shift was observed by the stimulation of unilateral pan-dSPNs optogenetically (Kravitz et al., 2010) or chemogenetically (Alcacer et al., 2017) using D1-Cre transgenic mice. Thus, our results reveal a unique role of the striosomal MOR-expressing neurons in motor control, quite in contrast to the role of dSPNs predicted by the traditional model (Alexander and Crutcher, 1990). However, no clear consensus regarding the function of striosomal neurons on locomotion has yet been attained. A study of genetic ablation of striosomal neurons reported that a lesion of striosomes slightly impaired motor learning initially, but it did not affect locomotion (Nadel et al., 2020). Optogenetic bilateral stimulation of striosomes was reported to acutely induce elevated locomotion (Nadel et al., 2021). The discrepancy between our results and those reports might be explained by a possible difference between targeted SPNs in the MOR-CreER mouse and those in the Sepw1-Cre NP67 line, which were used in those studies. Recently, an inhibitory effect on locomotion was also reported in Tshz1-expressing SPNs, a subset of striosomal dSPNs (Xiao et al., 2020). Together, this study provides functional evidence of the distinct roles of striosomal MOR-expressing dSPNs in motor control.
The question then is which neural circuit contributes to motor inhibition by striosome activation? To answer this, we utilized genetically encoded dopamine sensor GRABDA2m. Striosomal dSPNs send axon terminals to SNc (Gerfen, 1985; Fujiyama et al., 2011) and form characteristic bundles of axon terminals called “dendron bouquets” on midbrain dopaminergic neurons (Crittenden et al., 2016). Optogenetic stimulation of GABAergic striosome projections suppresses the activity of dopaminergic neurons in acute brain slices (Evans et al., 2020) and optogenetic stimulation of striosome suppresses dopamine release in the dorsal striatum of anesthetized mice in vivo (Nadel et al., 2021). Similar to those studies, our targeted striosomal neurons in MORmCherry mice also formed dendron bouquets (Fig. 1H). Thus, we provide in vivo experimental evidence for the long-standing anatomical predictions that striosome inhibits SNc dopaminergic neurons. Recent preprint reports found that optogenetic stimulation of striosomal dSPNs causes strong inhibition of striatal dopamine release in behaving animals, consistent with our findings with chemogenetic manipulation (Dong et al., 2024; Lazaridis et al., 2024).
We sought to address whether the locomotor changes induced by striosomal activation were correlated with the modulation of dopamine release. To this end, we performed simultaneous recordings of dopamine photometry and behavior. For turning behavior, we found that the mean angular velocity was positively correlated with the mean dopamine signal levels in vehicle-treated and DCZ-treated mice, indicating that suppression of mean signal levels of dopamine in striosomal stimulation correlated with the ipsilateral shift of turns. Our results are consistent with recent studies revealing transient dopamine activity with contralateral elevation and ipsilateral depression in dopamine signals in the dorsal striatum during turns (C. Liu et al., 2022: Jørgensen et al., 2023; Markowitz et al., 2023), along with the classic findings that 6-OHDA-treated dopamine-depleted animals show suppression of contralateral rotatory movements (Ungerstedt, 1968).
For average speeds, association with dopamine fluctuation has been reported in self-initiated moving behavior (Howe and Dombeck 2016; da Silva et al., 2018; C. Liu et al., 2022; Markowitz et al., 2023). Curiously, our data showed that mean dopamine signals were negatively correlated with average speeds in vehicle-treated and DCZ-treated MORDREADD mice. This negative correlation might contradict the facts that drug-induced dopamine elevation causes hyperlocomotion and dopamine depletion causes hypolocomotion. However, our data is consistent with a recent study using fluorescent dopamine sensor in self-paced, freely moving, and physiologic condition (Markowitz et al., 2023). Under striosome stimulation, we found that the frequency of transients was correlated with future average speed, suggesting that suppression of dopamine transients might decrease future vigor for locomotion in the striosomal stimulation. Our results are in agreement with the previous report that the dopamine neuron activity facilitates future vigor for locomotion (da Silva et al., 2018). We could not identify the circuit mechanism underlying the correlation between dopamine transient and future average speed during striosomal stimulation. Recent evidence suggests that striosomes preferentially inhibit the ventral tier of SNc dopaminergic neurons in brain slices (Evans et al., 2020) and that the activity of the ventral portion of SNc dopaminergic neurons correlates positively with acceleration and speed (Azcorra et al., 2023). Given that the contrast between phasic and tonic dopamine signals has been proposed as a key functional feature of dopamine release (L. Zhang et al., 2009), one possible explanation is that striosome-induced inhibition of the dopaminergic neurons located in ventral SNc might alter the contrast between acceleration-related phasic and tonic dopamine signals and enhance the correlation between the dopamine activity and locomotion. Further studies will be needed to elucidate the downstream circuit mechanisms that contribute to the behavioral changes under striosomal activation. Together, our data suggests that the dopamine release dynamics during striosomal activation might be distinctly associated with turns direction and speeds. We propose a working hypothesis that striosomal dSPNs modulate motor behavior associated with distinct dopamine modulating mechanisms (Fig. 11).
A remaining question concerns the functional role of striosomal iSPNs (Fujiyama et al., 2011) in dopamine regulation. D1R-SPNs were significantly preferred in MOR-CreER mice, although with a substantial portion being D2R-SPNs (Fig. 2F). Striosomal iSPNs inhibit dSPNs within the striosome microcircuit (Banghart et al., 2015), suggesting that striosomal iSPNs might indirectly influence dopamine release and locomotion. For greater understanding of the striosomal neural circuits, genetic dissection of iSPNs in the striosome will be an important target. The NTS-Cre driver line has recently been shown useful for dissecting the roles of striosomal iSPNs in increasing dopamine release (Lazaridis et al., 2024).
DCZ is a recently identified highly specific DREADD agonist (Nagai et al., 2020). Clozapine N-oxide has been widely used for DREADD induction. The effect of clozapine N-oxide appears first at 5–10 min after its systemic administration and peaks at 45 min or later (Alexander et al., 2009). Our data provide the time course of Gq-DREADD-induced neural circuit manipulation in vivo and behavioral response after intraperitoneal DCZ administration showing a rapid onset (∼5 min) and a peak (10∼20 min) of the effect followed by a gradual decrease in 30 min∼1.5 h, a time course similar to that reported previously (Nagai et al., 2020; X. Liu et al., 2022). Thus, DCZ may be useful for behavioral studies that require a short response time.
Of note, we could not detect rebound DA signals, which have been reported after cessation of the optogenetic stimulation of striosomes (Evans et al., 2020; Nadel et al., 2021). This discrepancy might be partly due to the stimulation method, as our chemogenetic manipulation has a relatively longer timescale and gradual termination compared with optogenetic short-time stimulation and abrupt termination. Dopamine response to striosomal activation at different durations will be an important topic for future research.
Molecular-based striosome-targeting and limitations of the study
Several Cre recombinase-driver mouse lines are used for genetic labeling of the striosome or its specific genetic subgroup. The Sepw1-Cre mouse NP67 line can be used to selectively express channelrhodopsins (Gerfen et al., 2013; Smith et al., 2016; Evans et al., 2020) or genetically encoded calcium indicators (Yoshizawa et al., 2018) in the striosomes. The Mash1-CreER mouse line is also used for the induction of Cre recombinase in the developmental stage (Bloem et al., 2017; Bloem et al., 2022). In combination with Mash1-CreER and LSL-FLPo mice, FLPo-dependent AAV vectors can be used to express DREADD in a striosome-predominant manner (Friedman et al., 2020). The hs599CreER mouse line is used for targeting striosome projection neurons (McGregor et al., 2019). The Tshz1-2A-FlpO line is used to target a genetically defined subpopulation of dSPNs enriched in the striosome (Xiao et al., 2020). Importantly, our striosome-targeting strategy is based on the MOR expression, which is a robust marker of the striosome consistently reported by ligand binding assay (Pert et al., 1976; Herkenham and Pert, 1981), mRNA distribution (Mansour et al., 1994; Minami et al., 1994; Arvidsson et al., 1995), and immunohistochemistry (Kaneko et al., 1995; Mansour et al., 1995). The knock-in strategy has also been reported in the development of conditional or constitutional Cre driver lines under the Oprm1 promoter (Märtin et al., 2019; Bailly et al., 2020; X. Y. Zhang et al., 2020) and is used to target striosomes for gene expression analysis and calcium imaging (Märtin et al., 2019; Weglage et al., 2021). The advantage of these Orpm1 driver lines including the MOR-CreER line is their highly specific recombination (>95%) in Oprm1 expressed cells (Okunomiya et al., 2020; Weglage et al., 2021), which makes transgene expression in Oprm1-negative cells highly unlikely. This property is suitable for neuronal manipulation functional analysis. Moreover, we provided quantification for the striosome-preferential recombination in the MOR-CreER line when using the Cre-dependent AAV vector as a gene delivery, not using the Rosa26 reporter that was used in the previous report (Weglage et al., 2021). Thus, the MOR-CreER line is considered the most appropriate tool for functional analysis of the striosome compartment in this study.
Interpretation of our results may be limited by a concomitant transgene expression in the matrix, MOR-expressing interneurons within the striatum, and MOR-expressing cells outside the striatum. First, a substantial portion of targeted cells was located in the matrix (Fig. 1E). However, the scattered extrastriosomal labeling is commonly seen in most of the striosome-preferential mouse lines mentioned above, in part because the definition of striosomes by MOR immunohistochemistry does not reach cellular-level resolution. The genetically defined cells in the matrix, or “exo-patch” cells, express MOR and have molecular, anatomical, and functional properties similar to striosomal neurons (Smith et al., 2016) in spite of specific molecular markers being proposed (Märtin et al., 2019). Second, a small population of targeted cells consisted of ChAT-positive cholinergic interneurons, in agreement with previous observations (Jabourian et al., 2005; Ponterio et al., 2013). The ChAT-positive proportion of targeted cells was numerically higher than that of total neurons (Fig. 2H,I), although the difference was not significant. Consequently, chemogenetic stimulation on MOR-expressing cells resulted in a Gq stimulation in a subset of cholinergic interneurons. Cholinergic interneurons in the striatum control motor function especially in dopamine-depleted parkinsonism models (Kaneko et al., 2000). To exclude the effect of striatal cholinergic stimulation, we showed that suppression of locomotor activity in DCZ-treated MORDREADD mice was not abolished by nonspecific mAChR blockade with scopolamine. Unlike toxin- or viral-mediated ablation studies, recent works reported that bilateral optogenetic stimulation of striatal cholinergic interneurons does not alter spontaneous locomotion (Maurice et al., 2015) and that unilateral optogenetic stimulation of striatal cholinergic interneurons does not bias movement direction preference (Kondabolu et al., 2016), both of which are in agreement with our findings. Nicotinic AChRs (nAChRs) have gained much attention in the modulation of presynaptic dopamine release. Although potential involvement of the cholinergic transmission through nAChRs was not tested in the present study, unilateral nAChR blockade in the dorsal striatum suppresses contralateral movement initiations (C. Liu et al., 2022) and bilateral nAChR blockade does not affect locomotion in the open field (Y. F. Zhang et al., 2024). Recently, studies revealed opposing modulation of evoked dopamine response with mAChRs (Collins et al., 2016) and nAChR (Collins et al., 2016; Y. F. Zhang et al., 2024) antagonism and highlighted the complexities of interactions between acetylcholine signaling and dopamine release under physiological conditions (Chantranupong et al., 2023; Krok et al., 2023). Nevertheless, striatal dopamine release is generally facilitated rather than suppressed as a net result of cholinergic interneuron stimulation (Cachope et al., 2012; Threlfell et al., 2012), in contrast to the dopamine suppression observed in our data. In addition, nAChR blockade in the dorsal striatum suppresses dopamine release and fluctuations in freely moving mice (C. Liu et al., 2022). Thus, it is likely that the decrease of locomotion and dopamine release in MORDREADD mice is explained at least partly by noncholinergic neural circuit manipulation, including the inhibitory striosome→SNc projection. Third, MOR-expressing cells are widely distributed across the central nervous system. We used AAV serotype 9 capsid for its high transgene expression, although AAV might infect the afferent axon terminals from areas outside the injection site (Castle et al., 2014). To address concern of the extrastriatal DREADD manipulation, we carefully examined mCherry fluorescence and immunoreactivity outside the striatum of MORDREADD mice and noticed a weak mCherry-immunoreactivity in the CL/PC thalamic nuclei. The extrastriatal mCherry-immunoreactivity was presumably due to the retrograde infection and subsequent MOR-CreER mediated Cre recombination, as CL/PC densely innervate the SPNs (Guo et al., 2015; Unzai et al., 2017) and expresses Oprm1 mRNAs (Mansour et al., 1994; Okunomiya et al., 2020). However, the DREADD-mCherry signals in the extrastriatal sites were much weaker than in the targeted striosomes, and pERK1/2-immunoreactivity was not above the background level. Thus, we concluded that extrastriatal DREADD induction in MORDREADD mice with DCZ treatment was highly unlikely and did not influence the interpretation of our striosome-preferential stimulation.
Our behavioral study used male mice only, which limits the applicability of our results in regard to female mice. Sex-dependent differences in the functional roles of striosomal MOR-expressing neurons will be an important topic for future investigations.
In conclusion, we demonstrated that chemogenetic stimulation of unilateral striosomes resulted in suppressed dopamine release, ipsilateral rotational shifts, and decreased locomotion in freely moving mice. Our results suggest that a subset of direct pathway striosomal MOR-expressing neurons have distinct roles in motor control and dopamine regulation. This study provides a pathophysiological clue regarding the possibility that abnormal neural activity in the striosome compartments might be related to the development of movement disorders.
Footnotes
This study was supported in part by Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI (JP20K16494, JP23K14776, JP21H02807, and JP23H03886), by Japan Agency for Medical Research and Development (JP23bm1323001, JP23bm1423012, and JP24wm0625501), by the Canon Foundation Grant Program, and by a grant from the invited project at iACT, Kyoto University Hospital (0709992110). We thank Akito Tanaka for performing in vitro fertilization. We also thank Kayoko Tsukita, Ayako Nagahashi, Ikuyo Inoue, Takako Enami, Mako Takiguchi, Rina Shimizu, Aya Okusa, Yoko Ishida, and Yuma Okazaki for their technical assistance; Fumika Enomoto, Noriko Ito, Tomomi Urai, Yuki Ueda, Motoko Sugimoto, and Chikako Masuda for their administrative support; as well as other members of the Watanabe, Inoue, and Takahashi laboratories for their valuable advice.
The authors declare no competing financial interests.
- Correspondence should be addressed to Haruhisa Inoue at haruhisa{at}cira.kyoto-u.ac.jp or Ryosuke Takahashi at ryosuket{at}kuhp.kyoto-u.ac.jp.