Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Synaptic Gα12/13 signaling establishes hippocampal PV inhibitory circuits
doi: 10.1073/pnas.2407828121
Figure Lengend Snippet: The Gα12/13 pathway is essential for inhibitory synaptic function in hippocampal neurons. ( A ) representative primary hippocampal neuron immunolabeled for Gα13 together with HA-tagged endogenous LPHN3 and the somatodendritic marker MAP2. ( B-D ), colocalization of Gα13 with LPHN1-3 in primary hippocampal neurons. Primary neurons from mouse lines with endogenously tagged LPHN1-3 ( , , ) were colabeled with Gα13, MAP2, and Myc-LPHN1 ( B ), LPHN2-mVenus ( C ), or HA-LPHN3 ( D ). ( E – H ) primary neurons colabeled for Gα13 and Syn1/2 ( E ), vGAT ( F ), SHANK2 ( G ), or Homer1 ( H ). MAP2 was used as a somatodendritic marker. ( I ) validation of Gα12/13 KD and rescue system. Primary hippocampal neurons were infected with lentiviruses encoding indicated conditions together with mClover3 as a reporter and immunolabeled for Gα13 and MAP2. ( J – L ) mIPSC recordings from primary hippocampal neurons infected with lentiviruses expressing either Gα12/13 shRNA scramble (Ctl) or Gα12/13 shRNA (KD), together with mClover3. ( J ) representative mIPSC traces. ( K ) cumulative probability plot of interevent intervals and summary graph of the mean mIPSC frequency [ Inset ]. ( L ) cumulative probability plot and summary graph [ Inset ] of mIPSC amplitude measurements. ( M – O ), similar to ( J – L ), except for mEPSC measurements. Numerical data are cumulative histograms or means ± SEM. Statistical significance was determined via Kolmogorov–Smirnov test for cumulative histograms or two-tailed t test for summary graphs using the number of neurons as “n” values (*** P < 0.001; * P < 0.05). SI Appendix , Fig. S4 for colocalization measurements, additional validation of shRNA constructs, and additional electrophysiological parameters.
Article Snippet: The following antibodies and reagents were used: HA rabbit (Cell Signaling Technologies, cat# 3724), GFP rabbit (Life Technologies, cat# A11122), Myc rabbit (Sigma, cat# C3956), Homer1 rabbit (Synaptic Systems, cat# 160 003), MAP2 mouse (Sigma, cat# M1406), MAP2 chicken (EnCor Biotechnology, cat# CPCA-MAP2), SHANK2 guinea pig (Synaptic Systems, cat# 162204), Syn1/2 rabbit (Synaptic Systems, cat# 106 002), VGAT guinea pig (Synaptic Systems, cat# 131004), vGLUT1 guinea pig (Millipore, cat# AB5905), Gephyrin mouse (Synaptic Systems cat# 147111), GNA13 mouse (Life Technologies, cat# 67188), β-actin mouse (Sigma, A1978), Parvalbumin rabbit (SWANT, cat # PV27a), Alexa Fluor 647 Phalloidin (Invitrogen cat# A22287), and corresponding fluorescently-conjugated goat secondary antibodies from Life Technologies.
Techniques: Immunolabeling, Marker, Biomarker Discovery, Infection, Expressing, shRNA, Two Tailed Test, Construct
Synaptic Systems manufactures this product 
![The Gα12/13 pathway is essential for inhibitory synaptic function in hippocampal neurons. ( A ) representative primary hippocampal neuron immunolabeled for Gα13 together with HA-tagged endogenous LPHN3 and the somatodendritic marker MAP2. ( B-D ), colocalization of Gα13 with LPHN1-3 in primary hippocampal neurons. Primary neurons from mouse lines with endogenously tagged LPHN1-3 ( , , ) were colabeled with Gα13, MAP2, and Myc-LPHN1 ( B ), LPHN2-mVenus ( C ), or HA-LPHN3 ( D ). ( E – H ) primary neurons colabeled for Gα13 and Syn1/2 ( E ), vGAT ( F ), <t>SHANK2</t> ( G ), or Homer1 ( H ). MAP2 was used as a somatodendritic marker. ( I ) validation of Gα12/13 KD and rescue system. Primary hippocampal neurons were infected with lentiviruses encoding indicated conditions together with mClover3 as a reporter and immunolabeled for Gα13 and MAP2. ( J – L ) mIPSC recordings from primary hippocampal neurons infected with lentiviruses expressing either Gα12/13 shRNA scramble (Ctl) or Gα12/13 shRNA (KD), together with mClover3. ( J ) representative mIPSC traces. ( K ) cumulative probability plot of interevent intervals and summary graph of the mean mIPSC frequency [ Inset ]. ( L ) cumulative probability plot and summary graph [ Inset ] of mIPSC amplitude measurements. ( M – O ), similar to ( J – L ), except for mEPSC measurements. Numerical data are cumulative histograms or means ± SEM. Statistical significance was determined via Kolmogorov–Smirnov test for cumulative histograms or two-tailed t test for summary graphs using the number of neurons as “n” values (*** P < 0.001; * P < 0.05). SI Appendix , Fig. S4 for colocalization measurements, additional validation of shRNA constructs, and additional electrophysiological parameters.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0215/pmc11670215/pmc11670215__pnas.2407828121fig02.jpg)
