Journal: bioRxiv

Article Title: Repetitive magnetic stimulation induces plasticity of excitatory synapses through cooperative pre- and postsynaptic activity

doi: 10.1101/2024.11.04.621890

Figure Lengend Snippet: Pre- and postsynaptic activation of excitatory synapses leads to Schaffer collateral LTP and potentiation of CA1 excitatory synapses. (A) Overview of a tissue culture transduced with AAV1-hSyn-CrimsonR-tdTomato in the CA1 and CA3 region. DG, Dentate gyrus; EC, entorhinal cortex; CA1 and CA3, Cornu Ammonis areas 1 and 3. Scale bar, 500 μm. (B) Sample trace of light induced responses of a patched CA1 neuron expressing the opsin CrimsonR. (C, D) Excitatory post synaptic field potential (fEPSP) slopes recorded in the CA1 stratum radiatum following a 10 Hz, 900 pulses optical stimulation of the transduced CA3 and CA1 pyramidal neurons (n = 4 tissue cultures) (E) Excitatory post synaptic field potential (fEPSP) slopes recorded in the CA1 stratum radiatum following a 10 Hz, 900 pulses optical stimulation of the transduced CA3 pyramidal neurons (n = 5 tissue cultures). (F) Excitatory post synaptic field potential (fEPSP) slopes recorded in the CA1 stratum radiatum following a 10 Hz, 900 pulses optical stimulation of the transduced CA1 pyramidal neurons (n = 3 tissue cultures). (G) Overview of a tissue culture transduced with AAV1-hSyn-hM4D(Gi)-tdTomato in the CA1 region. DG, Dentate gyrus; EC, entorhinal cortex; CA1 and CA3, Cornu Ammonis areas 1 and 3. Scale bar, 500 μm. (H) Group data of AMPA receptor-mediated mEPSCs recorded 2 – 4 h after stimulation from virus expressing, or control, CA1 pyramidal neurons in presence or absence of CNO (n sham+CNO = 15 neurons, n sham+virus+CNO = 20 neurons, n rms(2-4h)+virus = 16 neurons, n rms(2-4h)+virus+CNO = 17 neurons; Kruskal-Wallis test). Data are mean ± SEM. NS, Not significant. **p < 0.01

Article Snippet: For local viral transduction approximately 100 nL of AAV-hSyn-hM4D(Gi)-mCherry [gift from Bryan Roth (Addgene viral prep # 50475-AAV9 ; http://n2t.net/addgene:50475 ; RRID:Addgene_50475)] or AAV-Syn-ChrimsonR-tdT [gift from Edward Boyden (Addgene viral prep # 59171-AAV9; http://n2t.net/addgene:59171 ; RRID:Addgene_59171] were injected within the area of interest using a glass pipette with the help of a micromanipulator.

Techniques: Activation Assay, Transduction, Expressing, Virus, Control

Journal: bioRxiv

Article Title: Repetitive magnetic stimulation induces plasticity of excitatory synapses through cooperative pre- and postsynaptic activity

doi: 10.1101/2024.11.04.621890

Figure Lengend Snippet: Calcium imaging during rMS indicates simultaneous activation of CA1 and CA3. (A) Overview of an AAV1-hSyn-GCaMP6f transduced tissue culture. DG, Dentate gyrus; CA1 and CA3, Cornu Ammonis areas 1 and 3. (B) GCaMP6f fluorescence (ΔF/F 0 ) during baseline and rMS (10 Hz, 900 pulses) from 10 CA1 and 10 CA3 pyramidal neurons (n = 3 tissue cultures). (C) Overview of an AAV1-hSyn-GCaMP6f and AAV1-hSyn-hM4D(Gi)-tdTomato transduced tissue culture. DG, Dentate gyrus; CA1 and CA3, Cornu Ammonis areas 1 and 3 (n = 2 tissue cultures). (D) GCaMP6f fluorescence (ΔF/F 0 ) during baseline and rMS (10 Hz, 900 pulses) from 10 CA1 and 10 CA3 pyramidal neurons in the presence of CNO. (E, F) Computational modeling revealing LTP when both pre- and postsynaptic neurons are activated simultaneously while no change in excitatory neurotransmission is evident when only the presynaptic neurons is activated. (G) Computational modeling for the frequency dependent effects of rTMS in silico . (H, I) Sample traces and group data of AMPA receptor-mediated miniature excitatory post synaptic currents (mEPSCs) recorded from mouse CA1 pyramidal neurons in control (sham) and stimulated (rMS) cultures 2 – 4 h post stimulation at 1, 5 and 10 Hz (n sham = 14 neurons, n 1Hz = 17 neurons, n 5Hz = 15 neurons, n 10Hz = 14 neurons; Kruskal-Wallis test). Data are mean ± SEM. NS, Not significant. *p<0.05; **p < 0.01.

Article Snippet: For local viral transduction approximately 100 nL of AAV-hSyn-hM4D(Gi)-mCherry [gift from Bryan Roth (Addgene viral prep # 50475-AAV9 ; http://n2t.net/addgene:50475 ; RRID:Addgene_50475)] or AAV-Syn-ChrimsonR-tdT [gift from Edward Boyden (Addgene viral prep # 59171-AAV9; http://n2t.net/addgene:59171 ; RRID:Addgene_59171] were injected within the area of interest using a glass pipette with the help of a micromanipulator.

Techniques: Imaging, Activation Assay, Fluorescence, In Silico, Control

Journal: eLife

Article Title: The anterior cingulate cortex and its role in controlling contextual fear memory to predatory threats

doi: 10.7554/eLife.67007

Figure Lengend Snippet:

Article Snippet: For the pharmacogenetic inhibition, ACA was injected bilaterally with 150 nl of AAV5-hSyn-HA-hM4D(Gi)-IRES-mCitrine (Dr. Bryan Roth; Addgene plasmid #50464) or AAV5-hSyn-eGFP (titer≥7×1012 vg/ml; Addgene viral prep #50465-AAV5) as control.

Techniques: Transfection, Construct, Virus, Plasmid Preparation, Software

Journal: Endocrine Oncology

Article Title: Characterisation of a GNAS variant linked to cortisol-producing adrenocortical adenoma

doi: 10.1530/EO-25-0009

Figure Lengend Snippet: The Gα s -Lys58Gln variant enhances constitutive activity of the ACTH receptor. (A) ACTH-mediated cAMP responses measured by GloSensor in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1. (B) Area under the curve was used to generate cAMP concentration–response curves. (C) Potency of ACTH in cells expressing either WT or Lys58Gln Gα s transfected with the ACTH receptor. n = 5 biological replicates. (D) ACTH-mediated phospho-CREB (pCREB) responses measured by AlphaLISA in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1, with (E) pEC 50 . n = 4 biological replicates. (F) ACTH-mediated CRE luciferase activity in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1, with (G) pEC 50 . n = 4 biological replicates. Statistical analyses were performed by two-way ANOVA with Bonferroni’s multiple-comparisons test in B and D, two-way ANOVA with Sidak’s multiple-comparisons test in F and unpaired t -test in C, E, G. **** P < 0.0001, *** P < 0.001, ns = not significant.

Article Snippet: MC2R-Tango (a gift from Bryan Roth; Addgene plasmid #66428 ( Kroeze et al. 2015 )) and MRAP1_pcDNA6.2/EmGFP-Bsd (a gift from Roger Reeves; Addgene plasmid #176937 ( Moyer et al. 2023 )) plasmids were purchased from Addgene and used as templates to generate pEGFP-C1-MC2R and pmCherry-C1-MRAP1 expression constructs by standard cloning procedures and oligonucleotides obtained from Merck (sequences listed in Supplementary Table 1).

Techniques: Variant Assay, Activity Assay, Expressing, Concentration Assay, Transfection, Luciferase

Journal: Endocrine Oncology

Article Title: Characterisation of a GNAS variant linked to cortisol-producing adrenocortical adenoma

doi: 10.1530/EO-25-0009

Figure Lengend Snippet: The Lys58Gln Gα s variant enhances cell growth and apoptosis. (A) Cell viability in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1 measured over 96 h. Data were expressed corrected to the total protein, then normalized to hour 0. (B) Apoptosis measured over 96 h was expressed corrected to the total protein, then normalised to hour 0. Statistical analyses were performed by unpaired t -test in n = 5 replicates. ** P < 0.01, * P < 0.05.

Article Snippet: MC2R-Tango (a gift from Bryan Roth; Addgene plasmid #66428 ( Kroeze et al. 2015 )) and MRAP1_pcDNA6.2/EmGFP-Bsd (a gift from Roger Reeves; Addgene plasmid #176937 ( Moyer et al. 2023 )) plasmids were purchased from Addgene and used as templates to generate pEGFP-C1-MC2R and pmCherry-C1-MRAP1 expression constructs by standard cloning procedures and oligonucleotides obtained from Merck (sequences listed in Supplementary Table 1).

Techniques: Variant Assay, Expressing