Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) An example for the immunostaining of the inhibitory neurotransmitter GABA (in green) and general neuronal marker MAP2 (in red) in differentiated inhibitory neurons (top) and excitatory neurons (bottom). The cell line in the images is hESC-H9. Scale bars, 50 microns. ( B ) Representative images showing a Calretinin + GAD67 + inhibitory neuron and a Somatostatin + GABA + inhibitory neuron. The cell line in the images is hESC-H9. ( C ) Percentage of Calretinin + or Somatostatin + inhibitory neurons from differentiation of hESC-H9 and control iPSC line 8858-C. ( D ) In inhibitory neuron cultures the expression of lentiviral reporter Dlxi1/2b-GFP overlapped with GABA immunostaining; on the contrary, in excitatory neuron cultures, CaMKII-GFP labeling and GABA immunostaining were mutually exclusive. Scale bars, 20 microns. ( E ) Around 65% of Dlxi1/2b-GFP + neurons were stained positive for GABA. Data represent two control iPSC lines 8858-C and 6593–7. ( F ) By single-cell RT-PCR paired with patch clamp recording, around 70% of Dlxi1/2b-GFP neurons express either GAD65 or GAD67, markers of inhibitory neurons. Data represent all Control (upper pie chart) and Dravet Syndrome (lower pie chart) pluripotent stem cell lines examined in this study. ( G ) Representative traces showing time-dependent maturation of the action potential firing pattern in inhibitory neurons. The cell line in this example is control iPSC 8858–3. ( H ) An example for the isolation of Gabazine-sensitive, spontaneous inhibitory synaptic currents (sIPSCs, insets in blue) and NBQX-sensitive, spontaneous excitatory synaptic currents (sEPSCs, insets in red) in cultures of differentiated inhibitory neurons. The detection of sEPSCs reflects the presence of a small fraction of excitatory neurons which likely arise from the low-percentage of PAX6 + progenitors in the ventral telencephalon patterning protocol . The cell line in this example is Dravet iPSC 6358–3. ( I ) Visualization of a hESC-H9-derived inhibitory neuron engrafted to the CA3 region of a rat hippocampal slice and co-cultured with the slice for 2 months. The human neuron was labeled with the lentiviral vector pLenti-Syn1-ChR2(H134R)-YFP. Rat neurons were visible by Ankyrin-G immunostaining (in red). Scale bar, 25 microns. ( J ) Under 470-nm light pulses, Gabazine-sensitive inhibitory postsynaptic currents (IPSCs, top trace) were detected in a rat neuron on a hippocampal slice engrafted with ChR2-YFP-expressing, hESC-H9-derived inhibitory neurons. Each trace represents the average of 20 consecutive trials (light pulses). DOI: http://dx.doi.org/10.7554/eLife.13073.015

Article Snippet: The GABA A receptor competitive antagonist Gabazine (SR 95,531 hydrobromide, Tocris) was dissolved in DMSO and used at a final concentration of 10 μM.

Techniques: Immunostaining, Marker, Control, Expressing, Labeling, Staining, Reverse Transcription Polymerase Chain Reaction, Patch Clamp, Isolation, Derivative Assay, Cell Culture, Plasmid Preparation

Journal: eLife

Article Title: A deleterious Na v 1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

doi: 10.7554/eLife.13073

Figure Lengend Snippet: ( A ) A low-magnification view of a hippocampal slice stained for CTIP2 (red, labeling excitatory pyramidal cells in the dentate-gyrus and CA1) and EYFP (green, labeling H9-derived and ChR2-EYFP-labeled inhibitory neurons, highlighted by unfilled arrows). Scale bar, 200 microns. ( B ) Bouton-like terminals of ChR2-EYFP-labeled human inhibitory neurons distributed over a cluster of rat CA1 pyramidal cells. Ankyrin-G (ANK-G, red) staining indicated the axon initial segments of the pyramidal cells. Scale bar, 25 microns. ( C ) Evoking action potentials by blue light pulses in an engrafted human inhibitory neuron. ( D ) Patch clamp recording of blue-light evoked inhibitory postsynaptic current (IPSCs) in a rat neuron in the vicinity of ChR2-EYFP-positive processes from an engrafted human inhibitory neuron. The arrow points to the patch clamp pipette. ( E ) IPSCs in response to repeated blue-light stimulation, without or with the GABA A receptor antagonist Gabazine (10 µM). DOI: http://dx.doi.org/10.7554/eLife.13073.019

Article Snippet: The GABA A receptor competitive antagonist Gabazine (SR 95,531 hydrobromide, Tocris) was dissolved in DMSO and used at a final concentration of 10 μM.

Techniques: Staining, Labeling, Derivative Assay, Patch Clamp, Transferring