Excitatory and inhibitory receptors utilize distinct post- and trans-synaptic mechanisms in vivo

  1. Taisuke Miyazaki
  2. Megumi Morimoto-Tomita
  3. Coralie Berthoux
  4. Kotaro Konno
  5. Yoav Noam
  6. Tokiwa Yamasaki
  7. Matthijs Verhage
  8. Pablo E Castillo
  9. Masahiko Watanabe
  10. Susumu Tomita  Is a corresponding author
  1. Department of Cellular and Molecular Physiology, Department of Neuroscience, Yale University School of Medicine, United States
  2. Department of Health Sciences, School of Medicine, Hokkaido University, Japan
  3. Department of Anatomy, Faculty of Medicine, Hokkaido University, Japan
  4. Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, United States
  5. Department of Clinical Genetics, Center for Neurogenomics and Cognitive Research (CNCR), VU University Amsterdam and VU Medical Center, Netherlands
7 figures, 2 videos, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Adult cerebellar organization after cell-autonomous elimination of neurons.

(A) Synaptic circuit organization in the adult cerebellum. Granule cells (GCs) send excitatory inputs to Purkinje cells (PCs) and molecular layer interneurons (MLIs). MLIs form inhibitory synapses …

Figure 1—source data 1

Adult cerebellar organization after cell-autonomous elimination of neurons.

https://cdn.elifesciences.org/articles/59613/elife-59613-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Normal cerebellar histology of mouse brains at P7−9.

Cerebellar granule cell (GC)-specific and Purkinje cell (PC)-specific conditional Stxbp1 homozygous mice (ΔGC mice and ΔPC mice, respectively) are generated, and cerebellar structures are examined …

Excitatory AMPARs remain at spiny synapses without presynaptic terminals.

(A) Vesicular glutamate transporter (VGluT1) signal from GC axons, that is, parallel fibers (PFs), is significantly decreased in the molecular layer (ML) of the ΔGC mice (n = 6 images from three …

Figure 2—source data 1

Excitatory AMPARs remain at spiny synapses without presynaptic terminals.

https://cdn.elifesciences.org/articles/59613/elife-59613-fig2-data1-v2.xlsx
Excitatory AMPARs remain at shaft synapses without presynaptic terminals.

(A) The number of postsynaptic density (PSD)-95 puncta is reduced (n = 6 images from three mice each) and individual puncta is enlarged in molecular layer (ML) of the ΔGC mice (n = 1421 puncta in …

Figure 3—source data 1

Excitatory AMPARs remain at shaft synapses without presynaptic terminals.

https://cdn.elifesciences.org/articles/59613/elife-59613-fig3-data1-v2.xlsx
Inhibitory GABAARs require presynaptic terminals in the deep cerebellar nuclei (DCN).

(A) Immunofluorescence showing marked reductions of calbindin (Calb)-labeled Purkinje cells (PCs) in the cerebellar cortex and of vesicular inhibitory amino acid transporter (VIAAT)-labeled …

Figure 4—source data 1

Inhibitory GABAARs require presynaptic terminals in the deep cerebellar nuclei (DCN).

https://cdn.elifesciences.org/articles/59613/elife-59613-fig4-data1-v2.xlsx
Inhibitory postsynaptic sites at Purkinje cell (PC) somas require presynaptic terminals.

(A) Wiring diagram between molecular layer interneurons (MLIs) and PCs. Lower MLIs, also known as basket cells, innervate PC somata and surround the axon initial segment of PCs with the pinceau …

Figure 5—source data 1

Inhibitory postsynaptic sites at Purkinje cell (PC) somas require presynaptic terminals.

https://cdn.elifesciences.org/articles/59613/elife-59613-fig5-data1-v2.xlsx
Ectopic Nrxn3 expression in excitatory climbing fibers (CFs) recruits inhibitory GABARs.

(A) Adenoassociated viruses (AAVs) carrying GFP or Nrxn3α fused with GFP (Nrxn3α GFP) are injected into the inferior olivary nucleus (IONs) that project excitatory CFs to Purkinje cells (PCs). (B) …

Figure 6—source data 1

Ectopic Nrxn3 expression in excitatory climbing fibers (CFs) recruits inhibitory GABARs.

https://cdn.elifesciences.org/articles/59613/elife-59613-fig6-data1-v2.xlsx
Author response image 1
Elimination of presynaptic neurons reduces synapse number without producing free spines in hippocampus.

(A–C) Electron micrographs showing CA1 region of wild-type (A) and Stxbp1fl/fl mice injected with AAV-synapsin promoter-driven (pSynapsin) Cre (B) and wild-type injected with AAV-pSynapsin Cre and …

Videos

Video 1
Sample video of two mice eliminating cerebellar granule cells (ΔGC) and two control mice.
Video 2
Sample video of two mice eliminating cerebellar Purkinje cells (ΔPC) and two control mice.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)Putative Nos1 promoterBACPAC Resources Center (BPRC)BAC: RP24-84E3
Strain, strain background (Escherichia coli)Putative Grin3a promoterBACPAC Resources Center (BPRC)BAC: RP23-104D24
Strain, strain background (Escherichia coli)Putative Kit promoterBACPAC Resources Center (BPRC)BAC: RP23-232H18
Strain, strain background (Mus musculus)Wild-type (C57BL/6 J)The Jackson LaboratoryStock# 00064
Strain, strain background (Mus musculus)Mouse: Stxbp1lox/loxVerhage et al., 2000N/A
Strain, strain background (Mus musculus)Mouse: Tg(Gabra6-Cre)MMRRCID# 015966-UCD
Strain, strain background (Mus musculus)Mouse:Tg(Pcp2-Cre)The Jackson LaboratoryStock# 004146
Cell line (Homo sapiens)293AAVCell BiolabCat#: AAV-100
Antibodyanti- GABAAR α1(Rabbit polyclonal)Frontier Inst.Cat# GABAARa1-Rb-Af660 RRID:AB_2571571IHC(1 μg/ml)
Antibodyanti-GluD2(Rabbit polyclonal)Frontier Inst.Cat# GluD2C-Rb-Af1200 RRID:AB_2571601IHC (1 μg/ml)
Antibodyanti-panAMPAR(Guinea pig polyclonal)Frontier Inst.Cat# panAMPAR-GP-Af580, RRID:AB_2571610IHC (1 μg/ml)IEM (5 μg/ml)
Antibodyanti-VGluT1(Guinea pig polyclonal)Frontier Inst.Cat# VGluT1-GP-Af570, RRID:AB_2571534IHC (1 μg/ml)IEM (10 μg/ml)
Antibodyanti-VGluT2(Guinea pig polyclonal)Frontier Inst.Cat# VGluT2-GP-Af810, RRID:AB_2341096IHC (1 μg/ml)
Antibodyanti-VIAAT(Guinea pig polyclonal)Frontier Inst.Cat# VGAT-GP-Af1000, RRID:AB_2571624IHC (1 μg/ml)
Antibodyanti-PSD95(Guinea pig polyclonal)Frontier Inst.Cat# PSD95-GP-Af660, RRID:AB_2571539IHC (1 μg/ml)
Antibodyanti-GFP(Goat polyclonal)Frontier Inst.Cat# GFP-Go-Af1480, RRID:AB_2571574IHC (1 μg/ml)
Antibodyanti-calbindin(Goat polyclonal)Frontier Inst.Cat# Calbindin-Go-Af1040, RRID:AB_2532104IHC (1 μg/ml)
Antibodyanti-MAP2(Goat polyclonal)Frontier Inst.Cat# MAP2-Go-Af860, RRID:AB_2571557IHC (1 μg/ml)
Antibodyanti-Car8(Goat/Rabbit polyclonal)Frontier Inst.Cat# Car8-Go-Af780, RRID:AB_2571668Cat# Car8-Rb-Af330, RRID:AB_2571667IHC (1 μg/ml)IEM (10 μg/ml)
Antibodyanti-Bassoon(Mouse monoclonal)EnzoCat# SAP7F407, RRID:AB_2313990IHC (1 μg/ml)IEM (20 μg/ml)
Antibodyanti-GABAAR γ2(Rabbit polyclonal)MilliporeCat#: AB5559, RRID: AB_11211236WB (1:2000)
Antibodyanti-GARLH4(Rabbit polyclonal)Yamasaki et al., 2017N/AWB (0.1 μg/ml)
Antibodyanti-GluA1(Rabbit polyclonal)MilliporeCat#: AB1504, RRID: AB_2113602WB (1:2000)
Recombinant DNA reagentpAAV-DJ(plasmid)Cell BiolabsVPK-410-DJ
Recombinant DNA reagentpHelper(plasmid)Cell BiolabsVPK-410-DJ
Recombinant DNA reagentpAAV-MCS(plasmid)Cell BiolabsVPK-410
Recombinant DNA reagentpAAV-Promoter less(plasmid)This paperN/ApAAV-MCS (Cell Biolabs)
Recombinant DNA reagentNrxn3alpha pXY-Asc(plasmid)HorizonCat# MMM1013-202798372
Sequence-based reagentB1.Nrxn3a.ForThis paperPCR primersGGGGACAAG
TTTGTACAAAA
AAGCAGGCTC
CACCATGAG
CTTTACCCTCCACTCAG
TTTTCTTC
Sequence-based reagentNrxn3a(-).B5.RevThis paperPCR primersGGGGACAACT
TTTGTATACAA
AGTTGTCACAT
AATACTCCTTG
TCCTTGTTTTT
CTGTTTC
Sequence-based reagentB5.(-M)AcGFP.FoThis paperPCR primersGGGGACAACTTTGTATACAAAAGTTGTGAGCAAGGGCGCC
GAGCTGTTC
Sequence-based reagentAcGFP*.B2.RevThis paperPCR primersGGGGACCAC
TTTGTACAAGA
AAGCTGGGT
TCACTTGTAC
AGCTCATCCATGCC
Sequence-based reagentAsCI.DEST.forThis paperPCR primersTACATGGCGC
GCCACAAGTT
TGTACAAAAAAGC
Sequence-based reagentDEST.BsiWI.revThis paperPCR primersATGTACGTAC
GACCACTTTG
TACAAGAAAGC
Sequence-based reagentB5.cre.ForThis paperPCR primersGGGGACAAC
TTTGTATACAA
AAGTTGCCACC
ATGTCCAATTT
ACTGACCG
TACACC
Sequence-based reagentcre*.B2.RevThis paperPCR primersGGGGACCACT
TTGTACAAGAA
AGCTGGGTTCA
ATCGCCATCTT
CCAGCAGGCG
Sequence-based reagentB1.pNOS.ForThis paperPCR primersGGGGACAAGT
TTGTACAAAAA
AGCAGGCTC
CCCTCACCCAT
CCCCACCCAC
CTCCATCCATAC
Sequence-based reagentpNOS.B5.RevThis paperPCR primersGGGGACAACTTTTGTATACAAAGTTGTT
GCCGTTCGGC
CTTGGGTGG
CATGATTTC
Sequence-based reagentB1.pGRIN3A.ForThis paperPCR primersGGGGACAAGTTTGTACA
AAAAAGCAG
GCTCCCTGC
CGTGCAAGGA
CCACACATT
CTACACTATAC
Sequence-based reagentpGRIN3A.B5.RevThis paperPCR primersGGGGACAACTT
TTGTATACAAA
GTTGTCGGCCA
CCTTACCGCGG
GCTCCCCCA
GCGCCTGG
Sequence-based reagentB1.pC-KIT.ForThis paperPCR primersGGGGACAAGTTTGTACAAA
AAAGCAGGCTG
TCCACCCCCG
GATAGCCACAG
TGACTGTGAAATG
Sequence-based reagentpC-KIT.B5.RevThis paperPCR primersGGGGACAACTTTTGTATACAAAGTTGT
GTGCACCGAG
CGCGGCAAA
GCCGAGC
Commercial assay or kitEndofree QIAGEN Maxi kitQIAGENQIAGEN Cat#12,362
Chemical compound, drugL-GlutamineGIBCO25030–081
Chemical compound, drugPenicillin– streptomycinGIBCO15140–122
Chemical compound, drugDMEM mediaSIGMA11965092
Chemical compound, drugIscove’s modified DM (IMDM)Thermo12440053
Chemical compound, drugBenzonase nucleaseSigmaE1014-25kU
Chemical compound, drugPfu Turbo DNA polymeraseAgilent600,250
Software, algorithmMetaMorphMolecular DevicesRRID:SCR_002368

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