BAD and KATP channels regulate neuron excitability and epileptiform activity
- Juan Ramón Martínez-François
- María Carmen Fernández-Agüera
- Nidhi Nathwani
- Carolina Lahmann
- Veronica L Burnham
- Nika N Danial
- Gary Yellen
- Harvard Medical School, United States
- Dana-Farber Cancer Institute, United States
Figures
Figure 1 with 2 supplements
Effect of BAD on KATP channels is cell-autonomous.
(A) Left: schematic representation of intracranial injection of AAV.Bad.2A.mCherry in the hippocampus of Bad−/− mice to reconstitute BAD expression in mCherry labeled cells. Right: epifluorescence …
Figure 1—figure supplement 1
‘Washdown’ of KATP channel conductance in Bad−/− or BAD-reconstituted DGNs.
Representative whole-cell current traces with high ATP (4 mM) in the patch electrode in a Bad−/− (A) or a BAD-reconstituted DGN (B). As the high ATP concentration washed into the cell, the …
Figure 1—figure supplement 2
Total activatable whole-cell KATP conductance is mostly unaffected by overexpressing BAD in Bad−/− DGNs.
(A,B) Representative whole-cell current traces with low ATP (0.3 mM) in the patch electrode (p. (e).) in a Bad−/− (A) or a BAD-reconstituted (B) DGN at the moment of break-in (black traces) or after …
Figure 2
Dentate granule neurons lacking BAD are less excitable.
(A) Bad−/− DGNs fire fewer action potentials that wild-type (WT), and 200 nM glibenclamide reverses Bad knockout effect on DGN excitability. Representative perforated-patch voltage recordings in …
Figure 3 with 1 supplement
Deletion of BAD reduces picrotoxin-elicited epileptiform activity.
(A) Top: Schematic representation of the analyzed regions of interest in EC-HC acute slices; dentate gyrus/hilus (DGH), CA3, CA1, medial entorhinal cortex (MEC) and lateral entorhinal cortex (LEC). …
Figure 3—figure supplement 1
Picrotoxin-triggered changes in GCaMP6f fluorescence intensity correspond to stereotypical epileptiform field potentials.
(A) Schematic representation of extracellular field potential electrode placement in CA1 or lateral entorhinal cortex (LEC). (B) Changes in GCaMP6f fluorescence faithfully represent …
Figure 4 with 1 supplement
Deletion of BAD decreases picrotoxin-triggered seizure-like activity in adult EC-HC slices.
Genetic deletion of BAD reduced the time hippocampal regions spent in SLEs in adult slices. Left column: fraction of time spent in SLEs (mean ± SEM) plotted versus time. The arrows indicate …
Figure 4—figure supplement 1
Picrotoxin-elicited epileptiform activity in adult EC-HP slices.
Picrotoxin application triggers SLEs in adult EC-HP slices. Representative ΔF/F traces (normalized from 0 to 1) in adult wild-type (A) or adult Bad−/− (B) slices for the indicated regions of …
Figure 5 with 2 supplements
Targeted knockout of Bad expression in the dentate gyrus but not in the rest of the hippocampus.
(A) Schematic illustration of the targeting construct and strategy to generate the conditional Badflox allele. Abbreviations: En2 SA: En2 splice acceptor; IRES: Internal ribosomal entry site; pA: …
Figure 5—figure supplement 1
Targeted knockout of Bad expression in the dentate gyrus: coronal sections.
Immunostaining for BAD in brain coronal sections from mice of the specified genotypes. BAD expression is only absent in DGNs from Badflox/flox; Dock10-Cre brain. Notice decreased BAD staining in …
Figure 5—figure supplement 2
Targeted knockout of Bad expression in the dentate gyrus: horizontal sections.
Immunostaining for BAD in brain horizontal sections from mice of the specified genotypes. BAD expression is only absent in DGNs from Badflox/flox; Dock10-Cre brain. Notice appreciable BAD expression …
Figure 6
DGN-specific BAD ablation is sufficient to reduce seizure-like activity.
Genetic deletion of Bad in DGNs alone (Badflox/flox; Dock10-Cre, orange) reduced the time hippocampal regions spent in SLEs in adult slices. Left column: fraction of time spent in SLEs (mean ± SEM) …
Videos
Video 1
Seizure-like events in the presence of 2.5 μM picrotoxin.
Fluorescence intensity as imaged in Figure 3 showing epileptiform activity in an acute EC-HP slice in the presence of 2.5 μM picrotoxin. Video was created using ImageJ 1.51 s (Fiji).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Bad | this paper | HEPD0750_4_D04 | for generation of Badflox/flox mice |
| Strain, strain background (Mus musculus) | male or female C57BL/6 mice | Charles River Laboratories | ||
| Genetic reagent (Mus musculus) | male or female homozygous Bad−/− mice | doi:10.1038/nm1717 | ||
| Genetic reagent (Mus musculus) | male or female homozygous Bad−/−; Kcnj11−/− mice | doi:10.1016/j.neuron. 2012.03.032 | ||
| Genetic reagent (Mus musculus) | female heterozygous Dock10-Cre+/− mice | doi:10.1038/nn.3614 | ||
| Genetic reagent (Mus musculus) | male or female homozygous Badflox/flox mice | this paper | ||
| Genetic reagent (Mus musculus) | male or female heterozygous Badflox/flox; Dock10-Cre+/− mice | this paper | ||
| Strain, strain background (Adeno-associated virus) | AAV2/8.CAG.BAD.2A.mCherry | BAD-2A-mCherry plasmid packed into AAV8 at Viral Core Boston Children’s Hospital | ||
| Strain, strain background (Adeno-associated virus) | AAV2/9.CAG.GCaMP6f | Penn Vector Core | Cat. no. AV-9-PV3081 | |
| Antibody | anti-Bad (rabbit monoclonal) | Abcam | ab32445; RRID:AB_725614 | (1:100) |
| Antibody | anti-Rabbit IgG (goat polyclonal), CF 594 | Sigma | SAB4600107 | (1:1000) |
| Other | VECTASHIELD mounting medium with DAPI | Vector Laboratories | H-1200; RRID:AB_2336790 | |
| Software | MATLAB 2016b | Mathworks | RRID:SCR_001622 | |
| Software | Fiji/ImageJ 1.51 s | NIH | RRID:SCR_002285 | |
| Software | Origin 9.1 | OriginLab | ||
| Software | Patchmaster v2x43 | HEKA | ||
| Software | TILLvisION 4.0.7.2 | Thermo Fisher Scientific |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.32721.015
