Clozapine Induces Neuronal Activation in the Medial Prefrontal Cortex in a Projection Target-Biased Manner

Abstract

The medial prefrontal cortex (mPFC) is associated with various behavioral controls via diverse projections to cortical and subcortical areas of the brain. Dysfunctions and modulations of this circuitry are related to the pathophysiology of schizophrenia and its pharmacotherapy, respectively. Clozapine is an atypical antipsychotic drug used for treatment-resistant schizophrenia and is known to modulate neuronal activity in the mPFC. However, it remains unclear which prefrontal cortical projections are activated by clozapine among the various projection targets. To identify the anatomical characteristics of neurons activated by clozapine at the mesoscale level, we investigated the brain-wide projection patterns of neurons with clozapine-induced c-Fos expression in the mPFC. Using a whole-brain imaging and virus-mediated genetic tagging of activated neurons, we found that clozapine-responsive neurons in the mPFC had a wide range of projections to the mesolimbic, amygdala and thalamic areas, especially the mediodorsal thalamus. These results may provide key insights into the neuronal basis of the therapeutic action of clozapine.

INTRODUCTION

The prefrontal cortex (PFC) plays a pivotal role in higher brain functions, including cognitive, executive, and decision-making processes.^1–3) Accumulating evidence suggests that dysregulation of the PFC contributes to the pathophysiology of schizophrenia, such as structural and functional alterations in the PFC, which correlate with hallmark cognitive and negative symptoms.^4–6) Thus, understanding the neuronal circuitry and activities of the PFC involved in higher brain functions and pathophysiology may offer insights into the neural basis of schizophrenia and its pharmacotherapy.

The atypical antipsychotic drug clozapine has shown promise for the treatment of schizophrenia, especially in treatment-resistant cases.^7–10) Unlike classical antipsychotics blocking the dopamine D2 receptor, clozapine has a complex pharmacological profile, with affinity for multiple neurotransmitter receptors including serotonin 5-HT2A and dopamine D4 receptors.¹¹⁾ Previous studies have reported that neurons expressing the 5-HT2A receptor have axonal projections to the ventral tegmental area and nucleus accumbens, and therefore, the pharmacological action of atypical antipsychotic drugs may be attributed to the inhibition of efferent projections from the PFC to the mesolimbic system through the blockade of the 5-HT2A receptor.^12,13) In contrast, several studies show clozapine-induced c-Fos expression in a subset of neurons in the PFC,^14–16) suggesting that activation of these neurons may have a role in clozapine action. However, it remains unclear whether neurons with clozapine-induced c-Fos expression in the medial prefrontal cortex (mPFC) have multiple projection targets across the whole brain and which brain regions are mainly innervated by these neurons. Thus, it is necessary to examine the mesoscopic projection patterns of clozapine-responsive neurons at the whole-brain level. Elucidating the anatomical and functional characteristics of the neurons that respond to clozapine is crucial for a precise understanding of the neural basis underlying the pharmacological actions of atypical antipsychotic drugs.

To investigate the projection patterns of neurons with clozapine-induced c-Fos expression, we employed activity-dependent genetic labeling based on c-Fos expression and viral tracing using Targeted Recombination in Active Population (TRAP2) mice.¹⁷⁾ By whole-brain imaging analysis using the block-FAce Serial microscopy Tomography (FAST) system,^18,19) we identified mesoscale projection patterns of formed by neuronal ensembles activated by clozapine, including axonal projection to the mediodorsal thalamus (MD).

RESULTS

First, we conducted whole-brain mapping of the axonal projections from the mPFC in wild-type mice using the FAST imaging system, which we developed for whole-brain imaging at cellular and axonal spatial resolution using a spinning-disk confocal microscopy.^18,19) To achieve bright labeling of axons using a fluorescent protein, we used an adeno-associated virus (AAV) vector carrying the tetracycline transactivator (tTA) and the TET responsive element (TRE) system with a positive-feedback loop, which enables high levels of fluorescent protein expression,^20,21) and a membrane-targeted fluorescent protein to enhance axonal labeling^22,23) (Fig. 1A). Three-dimensional (3D) reconstruction of serial section images successfully helped visualize the long-range axonal pathways in the mPFC (Fig. 1B). Extensive axonal projections in subcortical areas were observed in the MD, ventromedial thalamus (VM), basolateral amygdala (BLA), ventral tegmental area (VTA), and periaqueductal gray (PAG) among all brain regions (Fig. 1C, Supplementary Fig. 1). Quantitative analysis of the axonal densities labeled with fluorescent protein in these brain regions indicated that neurons in the mPFC exhibited comparable and prominent levels of projections to the BLA, MD, and VM (Fig. 1D).

Fig. 1. Brain-Wide Axonal Distribution of Projection Neurons in Mice mPFC

(A) Schematic of the experimental paradigm. C57BL/6N mice received stereotaxic injection of the AAV cocktail into the unilateral mPFC (anteroposterior (AP) +1.9 mm, mediolateral (ML) +0.4 mm, and dorsoventral (DV) −2.3 mm from the bregma). (B) Representative 3D-rendered images of the whole brain. Dorsal view (left) and lateral view (right) are shown. White arrows indicate the brain regions. A, anterior; P, posterior; R, right; L, left; D, dorsal; V, ventral. (C) Representative images of local magnification of the mPFC and brain regions innervated by mPFC projection neurons. Optimal brightness and contrast settings were determined for each brain region. The numbers indicated at the bottom right of the coronal section diagrams show the estimated anteroposterior distances from the bregma. (D) Quantification of the axonal densities (pixels) in the five brain regions. Data are presented as mean ± S.E.M. (n = 4). Statistical analysis was performed using one-way repeated-measures ANOVA: brain region, F (4, 15) = 3.866, p = 0.024 followed by Tukey’s multiple comparisons test: * p < 0.05. Scale bars, 2 mm (B) and 200 µm (C). See also Supplementary Fig. 1.

Next, we explored the axonal projection targets of clozapine-activated neurons. To permanently label clozapine-activated neurons, we used Cre-loxP recombination-based genetic tagging of neurons using TRAP2 mice, in which tamoxifen-inducible Cre recombinase iCreERT2 is expressed together with endogenous c-Fos protein.¹⁷⁾ AAV vectors for Cre-dependent axonal labeling were injected into the mPFC of TRAP2 mice, and the active metabolite of tamoxifen, 4-hydroxytamoxifen (4-OHT), was administered 1 h after vehicle or clozapine administration to specifically identify activated neurons (Fig. 2A). In clozapine-treated mice, extensive axonal projections were labeled with fluorescent proteins across the whole brain, compared with vehicle-treated mice (Fig. 2B, Supplementary Figs. 2, 3), suggesting an increase in c-Fos expression in the mPFC, consistent with previous studies.^14–16) Whole-brain imaging analysis revealed that clozapine-responsive neurons have axonal projections to multiple brain regions with a biased distribution compared to activity-independent conditions (Figs. 1B, C, 2B, C). By quantitative analysis of the axonal densities in five brain regions, the MD, VM, BLA, VTA, and PAG, we found that clozapine-responsive neurons had denser projections to MD and VM compared to the other projection targets (Figs. 2C, D).

Fig. 2. Brain-Wide Axonal Projections of Neurons Activated by Clozapine in Mice mPFC

(A) Schematic of the experimental paradigm. TRAP2 mice received stereotaxic injection of the AAV cocktail into the unilateral mPFC. For the specific labeling of neurons activated by clozapine, 4-OHT (25 mg/kg) was administered 1 h after vehicle or clozapine (5 mg/kg) administration. (B) Representative 3D-rendered images of whole brains of clozapine- and vehicle-treated mice. Dorsal (left) and lateral (right) views are shown. White arrows indicate brain regions of interest. A, anterior; P, posterior; R, right; L, left; D, dorsal; V, ventral. (C) Representative images of local magnification of the mPFC and brain regions innervated by mPFC projection neurons. Optimal brightness and contrast settings were determined for each brain region. The numbers indicated at the bottom right of the coronal section diagrams show the estimated anteroposterior distances from the bregma. (D) Quantification of axonal densities (pixels) in five brain regions of clozapine- and vehicle-treated mice. Data are presented as mean ± S.E.M. (n = 5). Statistical analysis was performed using two-way repeated-measures ANOVA: brain region, F (4, 32) = 15.31, p < 0.0001; interaction, F (4, 32) = 5.684, p = 0.0014, followed by the Bonferroni multiple comparisons test: * p < 0.05. Scale bars, 2 mm (B) and 200 µm (C). See also Supplementary Figs. 2 and 3.

To determine the extent to which neuronal population projecting to the MD was activated by clozapine, we performed retrograde labeling of these neurons and immunohistochemical staining of clozapine-induced c-Fos (Fig. 3A). The MD-projecting neurons were predominantly localized in the deep layers of the mPFC (Fig. 3B). The number of c-Fos-positive cells was higher in clozapine-treated mice than in vehicle-treated mice, and approximately 6.2% of the MD-projecting neurons were c-Fos-positive (Figs. 3C, D). Although anterograde tracing using viral vectors revealed axonal fibers in the VM, retrogradely labeled VM-projecting neurons were rarely observed in the mPFC (Supplementary Fig. 4). The reactivity of MD-projecting neurons to clozapine was observed not only in wild-type mice but also in a mouse model of human 3q29 deletion syndrome caused by microdeletion of the 3q29 genomic locus associated with schizophrenia^24,25) (Supplementary Fig. 5), suggesting that the reactivity of these neurons to clozapine is maintained across healthy and disease-like states.

Fig. 3. Retrograde Characterization of Neurons Expressing Clozapine-Induced c-Fos in the mPFC in Wild-Type Mice

(A) Schematic of the experimental paradigm. C57BL/6N mice received stereotaxic injections of retrobeads into the unilateral MD (AP −1.2 mm, ML +0.3 mm, and DV −3.2 mm from the bregma). For c-Fos protein induction, mice were fixed for 150 min after clozapine of vehicle administration. (B) Representative images of the mPFC in the mice treated with vehicle or clozapine. (C) Images of local magnification indicated by white boxes in B. Arrowheads indicate cells that are double positive for c-Fos and retrobeads. (D) Quantification of c-Fos- and retrobead-positive cells in the mPFC. The density of total c-Fos-positive cells (left), total retrobead-positive cells (center), and the percentage of cells that are double positive for c-Fos and retrobeads, relative to retrobead-positive cells (right) are indicated. Data are presented as mean ± S.E.M. (n = 4). Statistical analysis was performed the using unpaired Student’s t-test. ** p < 0.01, *** p < 0.001. Scale bars, 200 µm (A and B), and 50 µm (C). See also Supplementary Fig. 4.

DISCUSSION

In the present study, we identified the projection target of neurons activated by clozapine in the mPFC, using genetic tagging of activated neurons, viral and retrograde tracing approaches, and whole-brain imaging. Among the cortical and subcortical areas innervated by the mPFC, we found that the thalamic nuclei, including the MD and VM, received dense axonal projections from clozapine-activated neurons. We further demonstrated that these clozapine-responsive neurons were a small subset of MD-projecting neurons in the mPFC (approximately 6.2% of MD-projecting neurons). These results suggest that a subset of the corticothalamic pathway may contribute to the pharmacological action of atypical antipsychotic drugs.

Identification of the projection targets of drug-responsive neurons is considered important for understanding their pharmacological mechanisms at the systems level, because optimal control of emotion, cognition, and behavioral flexibility is orchestrated by specific neuronal ensembles in the mPFC with different projection targets.^26–28) Indeed, the mPFC to MD pathway has been reported to be involved in cognitive controls.^27–29) A previous study revealed that the cortical layer 6 neurons, characterized by synaptotagmin 6 (Syt6) expression in the mPFC, have axonal projections exclusively in the thalamus, including the MD, and that optogenetic suppression of these neurons leads to reduced behavioral flexibility.²⁷⁾ In this study, we found that clozapine-activated neurons in the mPFC had denser axonal projections to the MD than to other areas, although only 6.2% of MD-projecting neurons in the mPFC showed clozapine-induced c-Fos expression. Therefore, the slight but distinct activation of the corticothalamic pathway may be crucial for the pharmacological action of clozapine in optimal regulation of brain functions.

The difference between clozapine-responsive and non-responsive neurons in the MD-projecting PFC neurons may be attributed to the varied expression profiles of 5-HT2A receptor in the subpopulations of PFC neurons. Previous studies reported that 29% of layer 6 neurons and 23% of parvalbumin-expressing interneurons exhibit 5-HT2A receptor expression,^30,31) which is known to exert Gq signaling-mediated excitatory effects. Consequently, the impact of clozapine treatment could differ among neuronal subpopulations, depending on their 5-HT2A receptor expression levels. In addition, blockade of 5-HT2A receptor on projection neurons and interneurons is considered to result in direct inhibition and interneuron-mediated disinhibition of projection neurons, respectively. This modulation of neuronal subpopulations and their local networks may contribute to the activation of a subset of MD-projecting PFC neurons by clozapine. Given that other atypical antipsychotics also have an affinity for the 5-HT2A receptor, it is possible that MD-projecting PFC neurons could be universally activated by such drugs. Alternatively, our findings might be associated with distinct action of clozapine via dopamine D4 receptor because of its predominant expression in the deep layers of PFC.³²⁾ Considering that stimulation of the D4 receptor reduces the excitability of projection neurons,³³⁾ its blockade by clozapine could play a role in activating MD-projecting PFC neurons. Although the neuronal circuitry activated by other antipsychotics with lesser affinity for the D4 receptor was not examined in this study, future research, including comparisons with other antipsychotics, will be necessary for our understanding of the circuit-level mechanisms underlying the action of clozapine. Technical advancements for selective targeting and manipulation of this small population may enable further investigation of causal role of this subset of the corticothalamic pathway.

In summary, using brain-wide anatomical analysis and labeling of functionally identified neurons, this study bridges the gap between drug-induced neuronal activity and anatomical properties from the local to the whole-brain level. These findings may help in elucidating the pharmacological mechanisms of clozapine at the neuronal circuit level and in understanding neuronal network-based therapeutic interventions that extend beyond clozapine treatment.

MATERIALS AND METHODS

Animals

All animal care and handling procedures were approved by the Animal Care and Use Committee of Osaka University (Approval Number: R02-8-7). All efforts were made to minimize the number of animals used. Male TRAP2 mice (Fos 2A-iCreER knock-in; JAX Stock No. 030323, The Jackson Laboratory, Bar Harbor, ME, U.S.A.),¹⁷⁾ Df/+ mice (mouse models carrying a deletion of the chromosomal region corresponding to the human 3q29 region),²⁵⁾ and C57BL/6N (C57BL/6NCrSlc, SLC, Shizuoka, Japan), aged 7–14 weeks were used in this study. The mice were maintained in group housing (three–six mice per cage), except for the singly housed mice. These were kept on a 12-h light–dark cycle (lights on at 8:00 a.m.) with controlled room temperature and humidity. Water and food (CMF, Oriental Yeast, Osaka, Japan) were provided ad libitum.

Viral Vector Production and Purification

To produce the AAV, Lenti-X 293T cells (632180, Clontech, Mountain View, CA, U.S.A.) were used and cultured in Dulbecco’s modified Eagle’s medium with high glucose and GlutaMAX supplement (DMEM, high glucose, GlutaMAX™ Supplement; 10566024, Thermo Fisher Scientific, Waltham, MA, U.S.A.) containing 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, U.S.A.) at 37 °C with 5% CO₂. AAV vectors were produced using a helper-free triple-transfection procedure, as described previously³⁴⁾ with minor modifications. Briefly, an AAV transgene plasmid was transfected into Lenti-X 293T cells using polyethyleneimine (PEI MAX; 24765, Polyscience Inc., Warrington, PA, U.S.A.) with a pAAV2/9n plasmid (#112865, Addgene, Watertown, MA, U.S.A.) that supplied AAV2 replication and AAV9 capsid proteins and a pHelper plasmid that supplied the adenovirus gene products required for AAV production. The cells were harvested 72 h after transfection and suspended in a buffer containing 100 mM Tris (pH 7.6), 100 mM MgCl2, and 500 mM NaCl. The cell suspension was subjected to three freeze-thawed cycles and treated with ≥250 U/µL of Benzonase nuclease (Sigma-Aldrich) at 37 °C for at least 40 min. The suspension was centrifuged at 3000 × g for 15 min at 4 °C, and the supernatant was loaded onto an iodixanol step gradient (15, 25, 40, and 60%; Optiprep, Cosmo Bio, Tokyo, Japan) and centrifuged at 58400 rpm using Optima L-90K and Type 70 Ti rotor (Beckman Coulter, Pasadena, CA, U.S.A.) for 2 h 25 min at 18 °C. After centrifugation, the 40/60% interface and the 40% layer containing AAV vectors were collected and replaced with phosphate-buffered saline (PBS) containing 0.001% (v/v) Pluronic F-68 via ultrafiltration using an Amicon Ultra-15 centrifugal filter (100-kDa cutoff; UFC910024, Merck Millipore, Burlington, MA, U.S.A.) to concentrate the AAV vectors. The AAV titer was quantified using a quantitative real-time PCR with GoTaq qPCR Master Mix (Cat# A6001, Promega, Madison, WI, U.S.A.), with a linearized AAV genome plasmid serving as a standard.

Stereotaxic Surgery

Mice were deeply anesthetized by intraperitoneal injection of an anesthetic cocktail containing 0.75 mg/kg medetomidine (Nippon Zenyaku Kogyo, Fukushima, Japan), 4 mg/kg midazolam (Sandoz Pharma, Basel, Switzerland), and 5 mg/kg butorphanol (Meiji Seika Pharma, Tokyo, Japan) and placed in a stereotaxic instrument. The AAV vectors were unilaterally injected into mPFC (anteroposterior (AP) +1.9 mm, mediolateral (ML) +0.4 mm, and dorsoventral (DV) −2.3 mm from the bregma) using a 10-µL-Gastight Syringe (Cat# 1701RN, Hamilton, Reno, NV, U.S.A.) with a 30-gauge needle and an UltraMicroPumpIII with a Micro4 controller (World Precision Instruments, Sarasota, FL, U.S.A.) at a rate of 50 nL/min. For retrograde tracing, 200 nL of RetroBeads IX Red (Cat# 1RX, Lumafluor, Naples, FL, U.S.A.) was unilaterally injected into MD (AP −1.2 mm, ML +0.3 mm, and DV −3.2 mm from the bregma) or VM (AP −1.5 mm, ML +0.9 mm, and DV −4.4 mm from the bregma) at a rate of 100 nL/min. The needle was maintained in place for 5 min before its slow removal to avoid backflow. Following stereotaxic surgery, the mice immediately received an intraperitoneal injection of atipamezole (7.5 mg/kg; Nippon Zenyaku Kogyo) and gentamicin (10 mg/kg; Sigma-Aldrich).

Drug Administration

For its administration, clozapine (Nacalai Tesque, Kyoto, Japan) was first dissolved in saline (0.9% NaCl solution) (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 20% (v/v) acetic acid as a stock solution (50 mg/mL clozapine). This stock solution was subsequently diluted 1 : 100 with saline to obtain a working solution of clozapine for intraperitoneal injection at a dosage of 5 mg/kg. Saline containing 0.2% (v/v) acetic acid was used as vehicle.

For 4-hydroxytamoxifen (4-OHT) administration, 4-OHT (H6278, Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) and subsequently diluted in PBS containing 1% Tween 80 (final concentration, 2.5 mg/mL 4-OHT, 5% DMSO and 1% Tween 80 dissolved in PBS). TRAP2 mice were administrated intraperitoneal injection of 4-OHT at a dosage of 25 mg/kg.

Tissue Preparation

The mice were deeply anesthetized as described above, and transcardially perfused with saline followed by 4% paraformaldehyde (Nacalai Tesque) in PBS. Brain tissues were excised and immersed in a 4% paraformaldehyde solution until use. For immunohistochemical analysis, fixed brains were cryoprotected in 20% (w/v) sucrose dissolved in PBS for two days, embedded in OCT compound (Sakura Finetek, Osaka, Japan) and quickly frozen on liquid nitrogen. For whole-brain imaging, fixed brains were embedded in a 4% (w/v) agarose gel (A6013, Sigma-Aldrich).

Anterograde Tracing from the mPFC Using AAV Vectors

To label axonal projections of the neurons in the mPFC in wild-type mice, C57BL/6N mice received a unilateral injection of 200 nL of a viral cocktail including AAV9-CamKII-Cre (5.0 × 10¹¹ viral genomes (vg)/mL), AAV9-ihSyn-DIO-tTA (1.0 × 10¹¹ vg/mL; Plasmid #99121, Addgene), and AAV9-TRE-pal-mScarlet (5.0 × 10¹² vg/mL). Two weeks after surgery, the mice were transcardially perfursed and fixed for whole-brain imaging.

To label neurons and their axons activated by clozapine, TRAP2 mice received a unilateral injection of 200 nL of a viral cocktail including AAV9-ihSyn-DIO-tTA (1.0 × 10¹¹ vg/mL; Plasmid #99121, Addgene) and AAV9-TRE-pal-mScarlet (5.0 × 10¹² vg/mL). One week after surgery, mice received an intraperitoneal injection of clozapine (5 mg/kg) or vehicle, followed by an injection of 4-OHT (25 mg/kg) one hour after the clozapine or vehicle administration. After two weeks, the mice were transcardially perfused and fixed for whole-brain imaging as previously described.^18,19)

Whole-Brain Imaging and Data Processing

Whole-brain imaging was performed using our recently developed high-speed serial-section imaging system, FAST.^18,19) Briefly, brain tissue blocks embedded in 4% agarose gel (A6013, Sigma-Aldrich) were placed in the sample chamber of the FAST system. Brain section images were acquired as a mosaic of fields of view with 20% overlap in the x–y plane and 25-µm overlap in the z-direction, with a resolution of 1.0 × 1.0 × 5.0 µm. Red fluorescence was excited by a 561 nm laser. The x–y plane section images were reconstructed by overlapping the alignment of consecutive sections using the stitching program, FASTicher,¹⁹⁾ with minor modifications in Python 3.8. Reconstructed serial section images were downsized to 5.0 × 5.0 × 50 µm and exported to Imaris software (Bitplane, Zurich, Switzerland) for three-dimensional (3D) volume rendering and preparation of Supplementary movies of the 3D whole brains. To evaluate the axonal innervation from the mPFC, the target brain regions were manually defined with a square box (250 × 250 pixels), and the number of fluorescence-positive pixels in each brain region was quantified using ImageJ (NIH, Bethesda, MD, U.S.A.).

Immunohistochemistry and Histological Analysis

Brain tissues sectioned at a 25 µm thickness using a cryostat (Leica, Wetzlar, Germany, CM1860) were incubated with 1% bovine serum albumin (Nacalai Tesque) in PBS containing 0.3% (v/v) Triton X-100 (Nacalai Tesque) for 1 h at room temperature for blocking and permeabilization. For the primary antibody reaction, the sections were incubated with rabbit anti-c-Fos monoclonal antibody (1 : 5000 dilution; catalog number #2250, Cell Signaling Technology, MA, U.S.A.) overnight at 4 °C. For the secondary antibody reaction and nuclear staining, the sections were incubated with Alexa Fluor^® 488 Goat anti-rabbit immunoglobulin G (IgG) (1 : 1000 dilution; catalog number A-11008, Invitrogen, CA, U.S.A.) and 10 µg/mL Hoechst 33258 (Catalog Number: H3569, Invitrogen) for 1 h at room temperature. Images were acquired under a BZ-X810 microscope (Keyence, Osaka, Japan). The number of labeled cells in the mPFC was manually counted using the ImageJ software (NIH) and averaged across six sections per mouse.

Statistical Analysis

All data are presented as the mean ± standard error of the mean (S.E.M.). Statistical analyses were performed using the R software (version 4.2.2; https://www.r-project.org/) and the GraphPad Prism 9.5.0 (GraphPad Software, CA, U.S.A.). To compare differences in axonal density, one-way or two-way repeated measures ANOVA followed by Tukey’s or Bonferroni post hoc tests were used, where applicable. For two-group comparisons in the immunohistochemical analysis, the unpaired Student’s t-test or Welch’s t-test was used, where applicable, depending on the equality of variances assessed by the F-test. Statistical information, including the sample size, is indicated in the figure legends. Statistical significance was set at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Acknowledgments

This work was supported in part by JSPS KAKENHI Grant Numbers, JP23H00395 (H.H.), JP20H00492 (H.H.), JP20H03556 (K.S.), JP22J13200 (Y.H.), JP22KJ2157 (Y.H.), and JP23KJ1519 (T.T.); MEXT KAKENHI Grant Number, JP18H05416 (H.H.); AMED Grant Numbers, JP21dm0207117 (H.H.), JP23ama121052 (H.H.), JP23ama121054 (H.H.), JP21wm0525005 (K.S.), JP19gm1310003 (T.N.), and JP21wn0425012 (T.N.); Research Grant from the Takeda Science Foundation (T.N. and H.H., respectively); and Research Grant from the Asahi Glass Foundation (T.N.).

We are grateful to James M. Wilson for giving pAAV2/9n plasmid (Addgene plasmid # 112865; http://n2t.net/addgene:112865; RRID:Addgene_112865) and Viviana Gradinaru for giving pAAV-ihSyn1-DIO-tTA plasmid (Addgene plasmid # 99121; http://n2t.net/addgene:99121; RRID:Addgene_99121).

Author Contributions

K.S. and H.H. conceived and designed the study; Y.H. performed stereotaxic surgery, immunohistochemical analysis, and image analysis of whole-brain data. L.K. and S.Y. performed whole-brain imaging and image data processing for analysis. K.S., L.K., and H.R. performed AAV production. T.T. and T.N. provided resources for the mouse model of human 3q29 deletion syndrome (Df/+ mice) and advice on the experimental design of Df/+ mice. T.N. and A.K. provided advice on the experimental design and data interpretation. Y.H., K.S., and H.H. wrote the manuscript with input from all coauthors.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

All the data used in this study are available upon reasonable request from the corresponding authors.

Supplementary Materials

This article contains supplementary materials.

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