Presynaptic transmitter release is regulated by the expression levels of DISC1.

<p>A) Representative EPSCs recorded from an untransfected neuron and evoked from presynaptic neurons expressing ChR2 and either dsRed (control) or wtDISC1. B) Summary graph showing the mean±SEM PPR recorded from control and wtDISC1 conditions. C) Summary plot comparing the CV values vs PPR for each condition. D) Representative EPSCs evoked from neurons expressing DISC1 RNAi (D1 RNAi) and D1 RNAi+DISC1-GFP (rescue). E) Summary graph showing the mean±SEM PPR recorded from D1 RNAi and rescue conditions. F) Summary plot comparing CV values vs PPR for each condition. All EPSC traces are normalized to the first EPSC peak amplitude.</p

Postnatal expression of DISC1ΔC enhances the frequency of mEPSCs.

<p>A) Expression of DISC1ΔC is induced by postnatal administration of 4-OHT as seen by expression of GFP fused to DISC1ΔC in this P28 brain slice. No GFP expression is observed in vehicle treated animals (- 4-OHT). Scale bar equals 100 µm. B) DISC1ΔC-GFP is localized to the soma, dendrite (arrow), and axons (arrowhead) of layer 2/3 pyramidal cells. Expression is also observed in axon terminal field of layer 5 and axon tracts of the corpus callosum. Scale bar equals 20 µm. C) Example mEPSC sweeps from transfected layer 2/3 neurons expressing either GFP (control), DISC1ΔC, wtDISC1 or D1 RNAi. Traces shown above are the average of all the captured mEPSC from every recording for each condition. D) Summary graph showing the frequency of mEPSCs are nearly doubled by DISC1ΔC expression. This increase in synaptic activity was present to the same extent in both transfected cells expressing DISC1ΔC and neighboring non-transfected cells. All recordings performed in the presence of gabazine (5 µM) and TTX (1 µM).</p

Presynaptic expression of DISC1ΔC alters the kinetics of evoked glutamate release and inhibits synchronous glutamate release.

<p>A) Normalized EPSCs recorded from an untransfected neuron and evoked from presynaptic terminals expressing ChR2 and either dsRed (control), DISC1ΔC, wtDISC1 or D1 RNAi. B) Summary graphs showing the mean ± SEM of four different kinetic measures of EPSCs evoked from presynaptic terminals expressing ChR2 and either dsRed (control), DISC1ΔC, wtDISC1 or D1 RNAi. C) Two representative recordings from a neuron in control or DISC1ΔC conditions showing 10 consecutive traces that are normalized and overlaid (top traces). The average trial-to-trial variance for the traces above is displayed over the duration of the waveform (bottom trace). D) A histogram depicting the average trial-to-trial variance for all neurons in each condition (bin = 0.001 A²). E) The ratio of the average trial-to-trial variance for each condition (black trace) is displayed over the duration of the EPSC waveform. The dotted blue line indicates the threshold for statistical significance (p<0.05; F-test). A representative DISC1ΔC EPSC (red trace) is overlaid to emphasize the aspects of the EPSC waveform that produce the most variance.</p

Recombinant and endogenous ZNF804a is localized to the nucleus of neural progenitor cells.

<p><b>A</b>) pCAG-ZNF804A was transfected into rat neural progenitor cells for twenty-four hours and processed for immunocytochemistry with antibodies against an myc-tag fused to ZNF804a. Expression of ZNF804a co-localizes with the nuclear counter stain DAPI (Scale bar = 10 µm). <b>B</b>) Immunohistochemistry using anti-ZNF804a antibodies showing endogenous ZNF804a protein (left panel) co-localizes (right panel) with the nuclear counter stain TOPRO (middle panel) in E11 neural progenitor cells within the ventricle zone. <b>C</b>) Endogenous ZNF804a protein is devoid of the cytoplasmic fraction and observed in the nuclear fraction. As a control to demonstrate proper cellular fractionation, Nestin, a cytoplasmic marker of neural progenitor cells, is observed in the cytoplasmic fraction. Likewise, Histone H3, a chromatin marker, is observed in the nuclear fraction.</p

ZNF804a regulates the transcription of several Schizophrenia- associated genes.

<p><b>A</b>) Quantitative RT-PCR was performed on 37 SZ associated gene transcripts following ZNF804A transfection. PRSS16 and COMT (red) showed robust upregulation of transcription twenty-four hours after transfection (Bonferroni corrected p<0.05; n = 5). Conversely, PDE4B and DRD2 (green) transcript levels were downregulated following ZNF804a transfection (Bonferroni corrected p<0.05; n = 5).</p

Recombinant ZNF804a binds to the DNA regions directly upstream of the transcription start site of PRSS16 and COMT.

<p><b>A</b>) Chromatin was immunoprecipitated with antibody directed against the myc-tag or IgG as control and then probed with anti-ZNF804a antibody. Recombinant ZNF804a was correctly identified by anti-ZNF804a antibody. <b>B–C</b>) Tiling qRT-PCR of the promoter sequences of PRSS16 and COMT following ChIP against the myc-tag reveals enrichment for ZNF804A. Enrichment of ZNF804A on the PRSS16 promoter appears 1.5 Kb 5′ upstream of the transcription start sites (TSS) while enrichment on the COMT promoter appears 1 Kb 5′ upstream of the TSS <b>D</b>) Tiling qRT-PCR of the promoter sequences of DRD2 and PDE4B following ChiP did not result in enrichment of ZNF804a (all figures *p<0.05, <i>Students</i> t-test, Error bars indicate ± SEM).</p

Endogenous ZNF804a binds to the DNA regions directly upstream of the transcription start site of PRSS16 and COMT.

<p><b>A</b>) Tiling qRT-PCR of the promoter sequences of PRSS16 and COMT following ChIP using anti-ZNF804a reveals enrichment for ZNF804A. Enrichment of ZNF804A on the PRSS16 promoter appears 1.5 Kb 5′ upstream of the transcription start sites (TSS) while enrichment on the COMT promoter appears 1 Kb 5′ upstream of the TSS.</p

Membrane potential responses to sine-wave modulation of light intensity express stronger attenuation of high frequencies than responses to intracellular current injection.

<p><b>A:</b> Membrane potential responses (blue traces) of a layer 2/3 pyramidal neuron expressing ChR2 to a 488 nm diode light modulated by sine-wave signals at 10, 50 and 100 Hz (light blue traces). The amplitude of the sine-wave modulation of light intensity was kept constant. <b>B:</b> Membrane potential responses (red traces) of a neuron to injection of sine-wave current at 10, 50 and 100 Hz (green traces). Two-electrode experiment. One electrode was used for current injection, and another electrode for recording of membrane potential responses. The amplitude of the sine-wave current was kept constant. <b>C:</b> Attenuation of the amplitude of membrane potential modulation in responses to sine-wave signals of different frequencies injected through the intracellular electrode or induced by photostimulation.</p

Malyshev et al PONE

<p>Data for the paper:</p> <p>Advantages and limitations of the use of optogenetic approach in studying fast-scale spike encoding</p> <p>Aleksey Malyshev, Roman Goz, Joseph J. LoTurco, Maxim Volgushev</p> <p>PLos ONE 2015</p

Frequency response of the membrane potential to fluctuating current injected through intracellular electrode or photoinduced in somatic region and in distal dendrites.

<p><b>A:</b> Membrane potential responses of layer 2/3 pyramidal neuron to injection of subthreshold fluctuating current through intracellular electrode. Two-electrode experiment. <b>B:</b> Membrane potential responses of the same neuron to illumination of somatic region or distal dendrites by the light from 488 nm diode with intensity modulated by the same fluctuating current as used for intracellular injection. The illumination field for dendritic stimulation was shifted ~150 μm from the soma. For dendritic stimulation light intensity was increased 2.5 times in order to induce membrane potential fluctuations of the the same amplitude range. <b>C:</b> Normalized power spectra of the current used for injection or modulation of light intensity, and of membrane potential responses to intracellular current injection and to photostimulation of the somatic region and of distal dendrites.</p