Journal: bioRxiv

Article Title: Smoothened receptor Signaling regulates the developmental shift of GABA polarity in rat somatosensory cortex

doi: 10.1101/799015

Figure Lengend Snippet: ( A ) Gramicidin perforated patch clamp recordings of I-V relationships for isoguvacine currents in mCherry only (control) or mCherry plus Smo-CA or Smo-DN in rat hippocampal primary neuronal cultures (9 DIV). Inserts depict the isoguvacine currents for control condition. Scale bar, 500 ms, 50 pA. ( B ) Box plots of E GABA in the indicated conditions. The number of cells recorded and cultures used are indicated in parenthesis. ( C ) Typical images of Cl-Sensor fluorescence excited at 500nm in slice from P30 rat electroporated with Cl-Sensor plus Smo-CA. Rats were electroporated in utero at embryonic day 15 with Cl-Sensor plus mCherry (control) or Cl-Sensor plus Smo-CA (Smo-CA) or Cl-Sensor plus Smo-DN (Smo-DN). The regions of interest were drawn around the soma of cells located in the focal plane. 5 to 10 neurons were analyzed per image. Data represent results obtained from 4-5 rats per experimental condition and 1-2 slices were recorded per animal. ( D and E ) Example of typical ratiometric fluorescence (R430/500) recordings at P10 ( D ) and P30 ( E ) from control, Smo-CA and Smo-DN rats. Horizontal bars indicate the times of application of ACSF containing isoguvacine. Arrows and arrowheads indicate different types of responses between control, Smo-CA and Smo-DN neurons at P10 and P30. Data represent results obtained from 4-5 rats per experimental condition and 1-2 slices were recorded per animal. ( D ) Box plots quantification of fluorescence change of ΔR/R, where R is the mean of 5 measurements before isoguvacine application and ΔR is the difference between absolute maximum of isoguvacine-induced response. Number of slices and rats are indicated in parenthesis. * p < 0.01, ** p < 0.01 when compared to control at the same age; Mann-Whitney U test.

Article Snippet: Thus, the overall effects of isoguvacine on the spiking activity in the electroporated cortical slices suggest that the constitutively active and the dominant negative forms of Smo accelerate and delay respectively the developmental hyperpolarizing GABA shift ( p < 0.0001, Chi-square test; ).

Techniques: Patch Clamp, Control, Fluorescence, In Utero, MANN-WHITNEY

Journal: bioRxiv

Article Title: Smoothened receptor Signaling regulates the developmental shift of GABA polarity in rat somatosensory cortex

doi: 10.1101/799015

Figure Lengend Snippet: ( A ) Gramicidin perforated patch clamp recordings I-V relationships for isoguvacine currents in KCC2-pH ext alone or co-transfected with Smo-CA (green) or Smo-DN (red) in hippocampal primary culture at 9 DIV. Inserts depict the isoguvacine currents for control condition. Scale bar, 500 ms, 50 pA. ( B ) Box plots of E GABA in the indicated conditions. Left panel show neurons transfected without KCC2-pH ext from data shown in Fig. 4A and B. Right panel show neurons co-transfected with KCC2-pH ext . The number of cells recorded, and cultures used are indicated in parenthesis. ( C ) Representative images illustrating total, membrane and internalized pools of KCC2 with external tag (KCC2-pHext) in vehicle (control), and Smo-related transfected constructs in hippocampal primary culture neurons. KCC2 construction stable to membrane (A/A KCC2) was used as positive control for KCC2 membrane trafficking. Neurons expressing a KCC2 mutant construction known to not be addressed to membrane (ΔNTD-KCC2) were proceeded in parallel experiments to ensure that immunocytochemistry on living neurons does not permeabilized the membrane. Scale bar 20μm. ( D ) Box plots of total protein (F t ), ( E ) normalized membrane (F m ), ( F ) internalized pool (F i ) fluorescence and ( G ) single membrane cluster size in cultured neurons expressing the indicated constructs. The number of cells and cultures used indicated in parenthesis are identical for all box plots. The sample size is the same for all box plots. * p <0.05, ** p < 0.01, *** p <0.005, **** p <0.001; Mann-Whitney U test.

Article Snippet: Thus, the overall effects of isoguvacine on the spiking activity in the electroporated cortical slices suggest that the constitutively active and the dominant negative forms of Smo accelerate and delay respectively the developmental hyperpolarizing GABA shift ( p < 0.0001, Chi-square test; ).

Techniques: Patch Clamp, Transfection, Control, Membrane, Construct, Positive Control, Expressing, Mutagenesis, Immunocytochemistry, Fluorescence, Cell Culture, MANN-WHITNEY