Presentation_1_GABAAR-mediated tonic inhibition differentially modulates intrinsic excitability of VIP- and SST- expressing interneurons in layers 2/3 of the somatosensory cortex.PPTX

Extrasynaptic GABAA receptors (GABAARs) mediating tonic inhibition are thought to play an important role in the regulation of neuronal excitability. However, little is known about a cell type-specific tonic inhibition in molecularly distinctive types of GABAergic interneurons in the mammalian neocortex. Here, we used whole-cell patch-clamp techniques in brain slices prepared from transgenic mice expressing red fluorescent protein (TdTomato) in vasoactive intestinal polypeptide- or somatostatin- positive interneurons (VIP-INs and SST-INs, respectively) to investigate tonic and phasic GABAAR-mediated inhibition as well as effects of GABAA inhibition on intrinsic excitability of these interneurons in layers 2/3 (L2/3) of the somatosensory (barrel) cortex. We found that tonic inhibition was stronger in VIP-INs compared to SST-INs. Contrary to the literature data, tonic inhibition in SST-INs was comparable to pyramidal (Pyr) neurons. Next, tonic inhibition in both interneuron types was dependent on the activity of delta subunit-containing GABAARs. Finally, the GABAAR activity decreased intrinsic excitability of VIP-INs but not SST-INs. Altogether, our data indicate that GABAAR-mediated inhibition modulates neocortical interneurons in a type-specific manner. In contrast to L2/3 VIP-INs, intrinsic excitability of L2/3 SST-INs is immune to the GABAAR-mediated inhibition.</p

Genetic ablation of β4 results in larger BK channel currents in CA3 pyramidal neurons.

<p>(A) Fluorescent image of a living slice from a β4−/− knock-out mouse showing GFP-signal in hippocampus. Scale bar: 500 µm. (B) Mean total potassium current from β4 heterozygote (β4+/−) and β4 knock-out (β4−/−) mice. (C) Example paxilline-sensitive BK channel current from representative CA3 neurons in β4+/− and β4−/− mice. (D) Mean amplitude of paxilline-sensitive BK channel current in β4+/− and β4−/− mice.</p

An ER-retention/retrieval sequence at the C-terminus of β4 inhibits the surface expression of BKα and β4.

<p>(A) Schematic of the mutant β4 constructs showing the C-terminal amino acids. (B) Fluorescence from binding of cell-impermeable dye to cell-surface BKα channels in cells transfected with FAP-BKα (red) and GFP (green). (C) Same as (B) but in cells transfected with FAP-BKα and wild-type β4. (D) Same as (B) but in cells transfected with FAP-BKα and β4-Ala. (E) Same as (B) but in cells transfected with FAP-BKα and β4-polySer. Scale bar = 20 µm (B–E). (F) Mean intensity of surface fluorescence for β4 constructs normalized to fluorescence from cells transfected with FAP-BKα alone. (G) Histogram showing distribution of fluorescence intensity in cells transfected with FAP-BKα alone (red), FAP-BKα+β4-Ala (green) and FAP-BKα+β4-polySer (blue). Scale bar: 20 µm (B–E).</p

Co-expression of the β4 subunit reduces cell-surface trafficking of BK channels.

<p>(A) Membrane topology of the FAP-BKα and β4 proteins. The FAP tag is at the extracellular, N-terminus. The C-terminus of the BKα subunit is indicated. (B) Schematic of binding of cell-impermeable dye (pink) to the FAP results in significant increase in fluorescence (red). (C) Application of cell-impermeable dye labels only surface BKα channels in live HEK-293 cells co-transfected with FAP-BKα (red) and GFP (green). (D) Same as (C) but in cells co-transfected with β4, FAP-BKα, and GFP showing reduced surface expressed of the FAP-BKα. (E) Application of cell-permeable dye labels intracellular stores of channel in cells transfected with FAP-BKα (red) and GFP (green). (F) Same as (E) but in cells transfected with β4, FAP-BKα, and GFP. Scale bar = 20 µm (C–F). (G) Distribution of surface fluorescence intensity values after application of cell-impermeable dye in transfected cells for FAP-BKα (red bars) or FAP-BKα+β4 (black bars). (H–I) Proposed model for surface distribution of BK channels in the absence (H) or presence (I) of the β4 subunit.</p

Immunofluorescence reveals that BKα and β4 are substantially localized to the ER and that β4 is difficult to detect on the cell surface.

<p>(A) Immunocytochemistry against fixed, permeabilized HEK-293 cells for the FAP-BKα subunit (anti-BKα: green) and the ER-marker mRFP-KDEL (intrinsic fluorescence; red) in cells transfected with BKα alone. Inset shows a cell with overlapping expression of BKα and mRFP-KDEL with arrows pointing to regions containing only BKα. (B) Quantitation of normalized intensity versus normalized distance from the cell nucleus for anti-BKα (green) and mRFP (red) in transfected cells. (C) As in (A) but for cells expressing BKα (green), β4 (unlabeled; not visualized) and mRFP-KDEL (red). (D) Same as (B) but in cells expressing BKα, β4 and mRFP-KDEL. (E) Localization of the β4 subunit (anti-β4: green) and mRFP- KDEL (intrinsic fluorescence; red) in cells expressing BKα (unlabeled; not visualized), β4 and mRFP-KDEL. Scale bar = 20 µm or 5 µm for inset. (F) As in (B), but for β4 and mRFP-KDEL. (G) Localization of the β4 subunit (anti-β4: green), mRFP-KDEL (intrinsic fluorescence; red) and BKα (anti-α: blue) in transfected, fixed HEK-293 cells expressing BKα, β4, and mRFP-KDEL. (H) Expression pattern of the β4 subunit (anti-β4: green) and mRFP- KDEL (intrinsic fluorescence; red) under non-permeabilized immunohistochemistry conditions. Note that immunohistochemical detection of cell-surface BKα is difficult because of an intracellular epitope for BKα. Scale bar = 10 µm (G,H).</p

β4-containing BK channels do not contribute to whole-cell BK channel currents.

<p>(A) In situ hybridization showing regions of β4 expression in a mouse brain. Arrow points to high levels of expression in the CA3. (B) Hippocampal area CA3 shows robust β4 expression. Scale bar = 200 µm. (C) Bright field image showing a whole cell electrode in the CA3. Scale bar = 500 µm. (D) Fluorescent image of a CA3 pyramidal cell filled with Alexa fluor 568 after whole cell patch clamp recording. Scale bar = 50 µm. (E) Overlaid traces from a representative cell showing total potassium current and the residual potassium current after application of a BK channel blocker. (F) Schematic overlap between paxilline- and iberiotoxin-sensitive currents when β4-containing BK channels contribute to whole-cell current. (G) Schematic overlap between paxilline- and iberiotoxin-sensitive currents when β4 containing BK channels do not contribute to whole-cell current. (H) Example paxilline-sensitive BK channel current from a representative cell. (I) Example iberiotoxin-sensitive BK channel current from a representative cell. (J) Quantitation of mean paxilline (pax)- and iberiotoxin (ibtx)-sensitive currents from CA3 pyramidal cells.</p

Mutation of the C-terminal ER-retention/retrieval sequence in β4 liberates β4 to the surface.

<p>(A) Expression pattern of the cotransfected FAP-BKα (unlabeled; not visualized), β4-Ala mutant (anti-β4 immunocytochemistry: green) and mRFP- KDEL (intrinsic fluorescence; red) in fixed, permeabilized HEK-293 cells. (B) Same as (A) but under non-permeabilized conditions. (C) Same as for (A) but for β4-polySer mutant under permeabilized conditions. (D) Same as (C) but under non-permeabilized conditions. Scale bar = 10 µm (A–D).</p