cnqx  (Tocris)

Journal: bioRxiv

Article Title: Inhibitory and disinhibitory VIP IN-mediated circuits in neocortex

doi: 10.1101/2025.02.26.640383

Figure Lengend Snippet: A) Schematic representation of the experimental procedure to study vM1 inputs onto L2-4 VIP INs in wS1, showing the injection of AAV-ChR2-EYFP into vM1, leading to the expression of ChR2 in vM1 axons projecting to Layer 1 and Layer 6 of wS1. B) Differential interference contrast (DIC) (top panels) and epifluorescence images (bottom panels) of the vM1 injection site (left) and the projection/recording site in wS1 (right), illustrating the ChR2-EYFP injection site and the vM1 axons in wS1. Scale bar = 200 μm. C) Schematic of the electrophysiological experiment in FLTG mice with the exception of VIP-Cre;SNCG-Flp;Ai65, where different VIP INs populations in wS1 were patched to measure input from L1-projecting vM1 axons upon light stimulation. D) Confocal image showing two patched VIP neurons, with a VIP/CR neuron (left, white) and a VIP/CCK neuron (right, white), both receiving vM1 input from L1-projecting axons (green top). Scale bar = 200 μm. E) Representative voltage-clamp (left, V-clamp, V_hold = -90 mV) and current-clamp (right, I-clamp) recordings from the same cells of vM1 evoked synaptic input to VIP/CCK (blue), VIP/nCCK nCR (green), and VIP/CR (red) neurons. F) Quantification of vM1 evoked excitatory postsynaptic currents (eEPSCs) in different VIP IN populations (SNCG+CCK H N=15, CCK V N=10, nonCCK nonCR N=11, CR N=15). Box-and-whisker plots the box represents the interquartile range (IQR: 25th to 75th percentile), the horizontal line within the box indicates the median, and whiskers extend to 1.5x the IQR unless outliers are present. Outliers are plotted individually as separate points. The stronger vM1 response was evoked in nCCK nCR VIP INs and was significantly stronger than VIP/CCK V and VIP/CR (Kruskal-Wallis p = 0.042, Dunn-Sidak nCCK nCR vs CCK V p = 0.0166 and nCCK nCR vs CR p = 0.0191). The VIP/SNCG group received the second strongest input and was not significantly different from the rest of the groups (SNCG vs CCK V p = 0.2037, SNCG vs nCCK nCR p = 0.8228, SNCG vs CR p = 0.2682) and the least responsive were VIP/CCK V and VIP/CR (CCK V vs CR p = 0.9997). G) Stacked bar plots of normalized mRNA levels (CPM weighted mean, the mean of all CPM values from all transcriptomic bins for the indicated IN subtype weighted by the number of cells in each transcriptomic bin; Allen Institute scRNAseq data;) for nicotinic cholinergic receptor subunits (Chrna3, Chrna4, Chrna5, Chrna7) across four VIP IN populations (VIP/CCK SNCG, VIP/CCK nSNCG, VIP/nCCK nCR, and VIP/CR) showing that although all VIP populations express α nicotinic receptor subunits the VIP/nCCK nCR expresses the least. H) Schematic of the cholinergic input experiment, where L2-4 VIP INs were patched in a Cholinergic/VIP mouse model (ChAT-ires-Flpo; VIP-Cre; Ai80F + Cre-dependent tdTomato virus to label all VIP INs) in Layer 2/3, measuring cholinergic light-evoked currents in the presence of synaptic blockers (Gabazine and CNQX 10 µM; D-AP5 25 µM). Soma location, morphology, electrophysiological properties, and CR immunohistochemistry were used for VIP IN classification. I) Post hoc immunohistochemistry in brain slices showing biocytin-filled patched neurons. The images show CR+ (top) and CR-(bottom) neurons, co-labeled with VIP (red) and Biocytin (blue). Scale bar = 10 μm. J) Representative voltage-clamp (V-clamp) recordings of cholinergic light-evoked currents in VIP/CCK (blue), VIP/nCCK nCR (green), and VIP/CR (red) neurons. K) Quantification of cholinergic input (eEPSCs) to VIP neurons (CCK, N=5, nCCK nCR N=6, CR N=11, Kruskal-Wallis test p=0.0127, Dunn-Sidak CCK vs nCCK nCR p=0.3129, CCK vs CR p>0.9999, nCCK nCR vs CR p=0.0147). L) Stacked bar plots of normalized mRNA levels (CPM weighted mean as in G ; Allen Institute scRNAseq data;) for α1-adrenergic receptor subunits (Adra1a, Adra1b) across the four VIP interneuron subpopulations (VIP/CCK SNCG, VIP/CCK nSNCG, VIP/nCCK nCR, VIP/ CR). The height of each colored segment represents the relative contribution of each subunit to the total expression. M) Representative traces of noradrenaline (NA, 10 μM) application in the presence of glutamate blockers, showing differential effects in a VIP/CR vs. a VIP/nonCR neuron The VIP/CR neuron exhibited a modest membrane potential depolarization, whereas the VIP/nonCR neuron showed a strong depolarization with spiking. N) Quantification of membrane potential change (ΔV, if cell began spiking than spiking threshold was used as the final Vm reached) after NA application demonstrating a significantly greater NA-induced depolarization in VIP/CCK and VIP/nCCK nCR neurons, with many reaching spiking threshold, compared to VIP/CR neurons (CCK, N=9, nCCK nCR N=7, CR N=5, Kruskal-Wallis test p=0.0091, Dunn-Sidak CCK vs nCCK nCR p>0.9999, CCK vs CR p=0.0336, nCCK nCR vs CR p=0.0225).

Article Snippet: In select experiments, the bath solution was supplemented with the NMDA receptor antagonist D-AP5 (25 µM; Abcam) and/or the AMPA receptor antagonist CNQX (10 µM; Abcam).

Techniques: Injection, Expressing, Whisker Assay, Virus, Immunohistochemistry, Labeling, Membrane