Neuronal DSCAM regulates the peri-synaptic localization of GLAST in Bergmann glia for functional synapse formation

In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

Fluorescence imaging was performed using a Zeiss LSM 780 confocal microscope system (Carl Zeiss, Oberkochen, Germany) and ZEN 2009 software (Carl Zeiss) or or spinning disk confocal super-resolution microscope (SpinSR10, Olympus Corporation, Tokyo, Japan) and cellSens Dimension (Ver 3.1.1,Olympus Corporation).To To detect DSCAM-ALFA at at both low and high magnifications, a Leica Stellaris 5 laser scanning confocal microscope (Leica, Wetzlar, Germany) was used and images were obtained with the LASX software (Leica).For high magnification, the Lightning 3D 3D deconvolution method was used for image acquisition.Nissle staining data were corrected by by all in in one fluorescent microscopy BZ700 (Keyence).Quantification of of the fluorescence intensity of of immunolabeled cells was performed using the "Measure" and "Plot Profile" functions of of ImageJ v1.46r.The number of of cells, dendritic height, and the height and number of of vGluT2 puncta were measured using Fiji/ImageJ 2.3.0/1.53.Electron microscopy data corrected by by TEM, JEM-3200FS.WB WB data collected by by ImageQuant LAS-4000 mini (Fujifilm).RNA-seq libraries were sequenced using Illumina NovaSeq platforms.Adapter and low-quality sequences were trimmed using Trimmomatic (version 0.36), and the trimmed reads were aligned to to the reference mouse genome (GRCm38/mm10) using HISAT2 (version 2.2.0).Genome-wide expression levels were measured as as a unit of of transcripts per kilobase million (TPM) using StringTie (version 1.3.6)and the number of of reads was counted per gene per sample using htseq-count within HTSeq (version 0.9.1).Differentially expressed genes were identified using DESeq2 (version 1.8.2).For gene ontology analysis, DAVID Bioinformatics Resources (version 6.8) was used (National Institute of of Allergy and Infectious Diseases, National Institutes of of Health; https://david.ncifcrf.gov).Current responses in in electrophysiological analyses were recorded using an an Axopatch 200 B amplifier, and the pCLAMP system (v9.2) was used for data acquisition and analysis.Statistical analyses were performed using Prism v7.0 (GraphPad Software, La La Jolla, CA, USA) or or R Studio v3.0 (R-Tools Technology, Boston, MA, USA).

Reporting on sex and gender
Reporting on race, ethnicity, or other socially relevant groupings

Ethics oversight
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research.If you are not sure, read the appropriate sections before making your selection.

Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.No other data were excluded from the analyses.
For each experimental condition, at least 3 sections were analyzed per animal and at least 3 animals were used per conditions.All attempts at replication were successful expect for the failures due to sickness or death of animals during the experimental period.
We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.In most of our experiments, especially in the electro physiological and mouse behavioral experiments, it was difficult to perform them in a blinded manner, because a single researcher with advanced skills had to concentrate on a single experiment.For general immunostaining and western blotting, a single researcher was in charge throughout a single experiment but the data from the second and third experiments were analyzed by a different researcher to confirm the reproducibility of the results.Furthermore, in most cases, we measured the data automatically by using some detectors or microscopy.A table summarizing whether the experiment was performed in a blind, automatic measurement, or non-blind manner is provided as Supplementary Table2in the supplementary information.
AuthenticationPlantsNot applicable to to this study Not applicable to to this study Not applicable to to this study