Journal: The Journal of Neuroscience

Article Title: SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons

doi: 10.1523/JNEUROSCI.1367-20.2020

Figure Lengend Snippet: Development of isogenic SYNGAP1 KO hiPSCs. A, Diagram represents CRISPR targeting within exon 7 (complete coding sequence) of the SYNGAP1 gene. Blue sequence represents the sgRNA. Red sequence represents the PAM site. B, Sanger sequencing for two WT and two SYNGAP1 mutant clones derived from the CRISPR experiment. C, Individual WES paired-end reads from clone #4 near the CRISPR targeted region within SYNGAP1. D, Normalized mapped reads for the same samples around the Cas9 target sequence. E, Genomic PCR to amplify DNA sequence flanking the CRISPR target site. F, Normalized mapped reads from the entire coding sequence of the SYNGAP1 gene in the four clone hiPSCs. Red arrow indicates predicted Cas9 cut site. Numbers indicate clonal reads relative to Cas9 hiPSC reads. G, Indels from each clone identified from WES analysis. Indels were identified by clonal sequence differences from the original Cas9 hiPSCs (reference sequence). Indel threshold was determined by at least 50% of the reads differing from the reference sequence with a minimum of at least 10 reads. Indels with frequency >0.8 were used to determine frequency of homozygous variants. *(in clone #4) indicates the compound heterozygous deletion of neighboring base pairs on each copy of SYNGAP1 within exon 7 (B, C). *(in clone #38) indicates the 8 bp deletion present in one copy of SYNGAP1. The other copy contains a large deletion encompassing the entirety of exons 6 and 7. H, Whole-genome view of iNeurons from WT#6, WT#30, KO#4, and KO#38 clones represents a copy number value of 2 cross all chromosomes (except for the Y-chromosome, which is not detected), revealing normal (female) karyotype with no chromosomal aberrations. Pink, green, and yellow represent the raw signal for each individual chromosome probe. Blue signal represents the normalized probe signal that is used to identify copy number and aberrations (if any).

Article Snippet: Half of the medium was changed with fresh iNeuron Maintenance Media every 4–5 d. Dendritic tracing Each well of a 96-well imaging plate contained ∼50,000 cells per well, consisting of ∼32,000 iNeurons + ∼18,000 primary rat astrocytes along with 0.1% (∼50 per well) of eGFP-positive human-iNeurons derived from the same clone. eGFP-positive iNeurons were created through a separate induction as stated above, except that an additional lentivirus expressing eGFP under the control of a TET-responsive promoter was included (Addgene, catalog #30130). eGFP-positive neurons were mixed with eGFP-negative neurons in the 96-well plates. iNeurons derived from either of the WT or KO clones were compared by tracing primary (originating from the soma), secondary, and tertiary dendrites, as well as total dendrite length.

Techniques: CRISPR, Sequencing, Mutagenesis, Clone Assay, Derivative Assay

Journal: The Journal of Neuroscience

Article Title: SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons

doi: 10.1523/JNEUROSCI.1367-20.2020

Figure Lengend Snippet: Increased dendrite length in iNeurons derived from KO iPSC clones. A, Representative images of eGFP-expressing iNeurons from the four different clones at DIV45. Scale bars: Inset, 200 µm. B–E, Histograms represent average length per cell of total (B), primary (C), secondary (D), and tertiary dendrites (E) of the four clones. F–I, Graphs represent average number of dendrites per cell of total (F), primary (G), secondary (H), and tertiary (I) dendrites of the four clones. In box-and-whisker plots, the middle, boxes, and whiskers represent the median, interquartile range, and min to max, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Half of the medium was changed with fresh iNeuron Maintenance Media every 4–5 d. Dendritic tracing Each well of a 96-well imaging plate contained ∼50,000 cells per well, consisting of ∼32,000 iNeurons + ∼18,000 primary rat astrocytes along with 0.1% (∼50 per well) of eGFP-positive human-iNeurons derived from the same clone. eGFP-positive iNeurons were created through a separate induction as stated above, except that an additional lentivirus expressing eGFP under the control of a TET-responsive promoter was included (Addgene, catalog #30130). eGFP-positive neurons were mixed with eGFP-negative neurons in the 96-well plates. iNeurons derived from either of the WT or KO clones were compared by tracing primary (originating from the soma), secondary, and tertiary dendrites, as well as total dendrite length.

Techniques: Derivative Assay, Clone Assay, Expressing, Whisker Assay

Journal: The Journal of Neuroscience

Article Title: SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons

doi: 10.1523/JNEUROSCI.1367-20.2020

Figure Lengend Snippet: Increased dendritic area and more numerous postsynaptic structures in SYNGAP1 KO iNeurons. A, B, Representative images showing MAP2 labeling (top), PSD95 labeling (middle), and GluA1 labeling (middle bottom), and merge of MAP2, PSD95, and GLUA1 (bottom) of iNeurons from WT#30 (A) and KO#4 (B) at DIV45. Yellow arrows indicate cell bodies. Scale bars: yellow, 10 µm; white, 2 µm. C, Graphs represent MAP2 area and number of PSD95 and GluA1 objects (punctate labeling) per FOV in WT#30 and KO#4. D, Number of MAP2-postive somas detected per image FOV. E, MAP2 signal normalized to MAP2 = positive somas per image FOV. F, Graphs represent quantification of PSD95 and GluA1 expression in WT#30 and KO#4 normalized to MAP2 area. In box-and-whisker plots, the middle, boxes, and whiskers represent the median, interquartile range, and min to max, respectively. Bar graph represents mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Half of the medium was changed with fresh iNeuron Maintenance Media every 4–5 d. Dendritic tracing Each well of a 96-well imaging plate contained ∼50,000 cells per well, consisting of ∼32,000 iNeurons + ∼18,000 primary rat astrocytes along with 0.1% (∼50 per well) of eGFP-positive human-iNeurons derived from the same clone. eGFP-positive iNeurons were created through a separate induction as stated above, except that an additional lentivirus expressing eGFP under the control of a TET-responsive promoter was included (Addgene, catalog #30130). eGFP-positive neurons were mixed with eGFP-negative neurons in the 96-well plates. iNeurons derived from either of the WT or KO clones were compared by tracing primary (originating from the soma), secondary, and tertiary dendrites, as well as total dendrite length.

Techniques: Labeling, Expressing, Whisker Assay

Journal: The Journal of Neuroscience

Article Title: SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons

doi: 10.1523/JNEUROSCI.1367-20.2020

Figure Lengend Snippet: SYNGAP1 expression in human iNeurons regulates excitatory synapse function. A, Flow diagram of iNeuron generation from WT and SYNGAP1 KO iPSCs for whole-cell electrophysiological experiments (recording days within red boxes). B, Representative DIC image of patched iNeurons cells from WT#6. C–E, Bar graphs represent intrinsic membrane properties measured at DIV20-DIV30 as resting membrane potential (C), capacitance (D), and input resistance (E) from the four clones. F, Representative traces of mEPSCs of iNeurons from WT and KO clones at DIV20-DIV30. Calibration: 2 s, 20 pA. G, Percentage of successful observations of mEPSCs in iNeurons from the four clones at DIV20-DIV30. H, I, Cumulative plots of mEPSC interevent interval and frequency (inset) of the different clones individually (H) and grouped by genotype (I) at DIV20-DIV30. J, K, Cumulative probability plots of mEPSC amplitude of the different clones individually (J) and grouped by genotype (K) at DIV20-DIV30. L, Representative traces of mEPSCs of iNeurons from WT and KO clones at DIV40-DIV50. Calibration: 2 s, 20 pA. M, Percentage of successful observations of mEPSCs in iNeurons from the four clones at DIV40-DIV50. N, O, Cumulative probability plots of mEPSC interevent interval (IEI) and frequency (inset) of the different clones individually (N) and grouped by genotype (O) at DIV40-DIV50. P, Q, Cumulative probability plots of mEPSC amplitude of the different clones individually (P) and grouped by genotype (Q) at DIV40-DIV50. In box-and-whisker plots, the middle, boxes, and whiskers represent the median, interquartile range, and min to max, respectively. Bar graphs represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Half of the medium was changed with fresh iNeuron Maintenance Media every 4–5 d. Dendritic tracing Each well of a 96-well imaging plate contained ∼50,000 cells per well, consisting of ∼32,000 iNeurons + ∼18,000 primary rat astrocytes along with 0.1% (∼50 per well) of eGFP-positive human-iNeurons derived from the same clone. eGFP-positive iNeurons were created through a separate induction as stated above, except that an additional lentivirus expressing eGFP under the control of a TET-responsive promoter was included (Addgene, catalog #30130). eGFP-positive neurons were mixed with eGFP-negative neurons in the 96-well plates. iNeurons derived from either of the WT or KO clones were compared by tracing primary (originating from the soma), secondary, and tertiary dendrites, as well as total dendrite length.

Techniques: Expressing, Clone Assay, Whisker Assay

Journal: The Journal of Neuroscience

Article Title: SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons

doi: 10.1523/JNEUROSCI.1367-20.2020

Figure Lengend Snippet: Earlier onset and elevated levels of network activity in SYNGAP1 KO iNeurons. A, Representative bright-field image of 1-week-old iNeurons differentiated from iPSC-derived NPCs plated on a 16-electrode array of an MEA well. Spontaneous action potentials were recorded from the homozygous SYNGAP1 null (KO#4 and #38) and control (WT#6 and #30) neurons. B, Representative waveforms of spiking behavior from a single electrode for each Homo and WT neuronal culture. C, Representative temporal raster plots of KO iNeurons (KO#4 and #38) and WT isogenic control iNeurons (WT#6 and #30) over 5 min of continuous recording during culture week 3. D, E, Cumulative plots of mean firing rates for all four clones individually (D) and grouped together by genotype (E), along a developmental timeline. F, G, Cumulative plots of average number of bursts for individual clones (F) and grouped together by genotype (G). H, I, Cumulative plots of average number of network bursts for all clone individually (H) and grouped together by genotype (I). KO neurons display synaptic connections as early as week 3 of maturation compared with the WT controls. For each clone, four replicates of iNeurons were plated and differentiated concurrently. Graphs represent mean ± SEM.

Article Snippet: Half of the medium was changed with fresh iNeuron Maintenance Media every 4–5 d. Dendritic tracing Each well of a 96-well imaging plate contained ∼50,000 cells per well, consisting of ∼32,000 iNeurons + ∼18,000 primary rat astrocytes along with 0.1% (∼50 per well) of eGFP-positive human-iNeurons derived from the same clone. eGFP-positive iNeurons were created through a separate induction as stated above, except that an additional lentivirus expressing eGFP under the control of a TET-responsive promoter was included (Addgene, catalog #30130). eGFP-positive neurons were mixed with eGFP-negative neurons in the 96-well plates. iNeurons derived from either of the WT or KO clones were compared by tracing primary (originating from the soma), secondary, and tertiary dendrites, as well as total dendrite length.

Techniques: Activity Assay, Derivative Assay, Clone Assay