Journal: Nature Neuroscience

Article Title: Synaptic-like transmission between neural axons and arteriolar smooth muscle cells drives cerebral neurovascular coupling

doi: 10.1038/s41593-023-01515-0

Figure Lengend Snippet: a , Schematic showing the spatial arrangement of two electrodes, with the left one filled with glutamate (1 mM) for activating aSMCs through puff application, while the right one records membrane currents. b , A representative trace of glutamate-induced depolarizing currents at a holding potential of –40 mV in the absence (top) and presence (bottom) of D-AP5 and NBQX. c , The statistical analysis for b ( N = 5 cells). d , e , Still frame images of Ca 2+ spark-like ( d ) and Ca 2+ wave ( e ) in aSMCs upon 500 μM ( d ) or 1 mM ( e ) glutamate stimulation (red, high; blue, low). f , Representative trace of Ca 2+ spark-like kinetics and Ca 2+ wave of d and e . g , h , Quantification of the frequencies of Ca 2+ spark-like events and Ca 2+ waves in aSMCs treated with vehicle ( n = 4 cells), glutamate alone ( g , 500 μM; h , 1 mM; n = 5 cells for each dose), or together with D-AP5 ( n = 5 cells). These cells were collected from three independent experiments. The baseline F0 was determined by averaging 10 images without Ca2+ sparks. A Ca2+ spark was identified as a localized increase in ΔF/F0 greater than 0.2. Ca2+ waves were defined by an ΔF/F0 elevation >0.2 that propagated for more than 20 μm. i , Schematic illustration depicts the spatial configuration of electrodes, with the left one containing NMDA (100 μM) for puffing. j , Exemplary traces of NMDA-evoked currents at a holding potential of –40 mV under Mg 2+ -free artificial cerebrospinal fluid (aCSF) conditions. The first (left) and second (right) NMDA-induced currents were sequentially recorded on the same aSMC with intermediate aCSF washing. k , Quantification for j ( N = 12 cells). l , Model diagram shows neural optogenetics (OG) and responding GCamp6 dynamic signals in co-culture. m , Representative live image of a ChR2 and GCamp6 double-positive neuron connecting with a GCamp6 and tdTomato double-positive aSMCs; n = 6 co-cultures. n , Sequential Ca 2+ images of aSMCs upon OG. o , Quantitative analysis of the effect of D-AP5 on Ca 2+ dynamics in aSMCs during OG, as shown in n ( N = 8 cells without OG; 5 cells with OG; 6 cells with OG plus D-AP5; cells were collected from three independent experiments). Data are the mean ± s.e.m; nested unpaired two-tailed t -test ( c and o ), one-way ANOVA with Bonferroni multiple-comparison post hoc tests ( g and h ).

Article Snippet: For in vivo experiments, D-AP5 (500 μM), COX2 antagonist (100 μM, NS-398, MedChemExpress) or PAX (100 μM) was administered by intracisternal injection 15 min before whisker stimulation or 2P optogenetic stimulation.

Techniques: Membrane, Optogenetics, Co-Culture Assay, Two Tailed Test, Comparison

Journal: Nature Neuroscience

Article Title: Synaptic-like transmission between neural axons and arteriolar smooth muscle cells drives cerebral neurovascular coupling

doi: 10.1038/s41593-023-01515-0

Figure Lengend Snippet: (a) Schematic graph of the experimental workflow of broad 2 P optogenetics (b) Two-photon image of a p-arteriole (labeled with Hydrazide Alex 633, white arrows) at a cortical depth of 128 μm and surrounding ChR2-mCherry positive glutamatergic axons (green arrows). (c) Still frame images of p-arterioles before and during photostimulation in the littermate control ( Grin1 f/f ) and aSMC-cKO Grin1 mice ( SMACreER:Grin1 f/f ). The GCamp6s intensity further enhanced in the same axons when illumination power switched from 25 mW (white arrow) to 80 mW laser (green arrow). The white dashed lines outline the basal arteriole calibers. (d) Time course of changes in p-arteriolar diameter in response to 1100-nm laser at 25 mW and the following 960-nm laser with increasing power in c. (e) Maximum dilation amplitudes in c (magenta, n = 8 p-arterioles from 6 mice) or cistern magna injection of D-AP5 (100 μM, green, n = 4 p-arterioles from 3 mice) in control mice or aSMC-cKO Grin1 mice (orange, n = 6 p-arterioles from 5 mice). Data are shown as the mean ± s.e.m.; nested, one-way ANOVA after a post hoc Bonferroni multiple comparison adjustments (e) .

Article Snippet: For in vivo experiments, D-AP5 (500 μM), COX2 antagonist (100 μM, NS-398, MedChemExpress) or PAX (100 μM) was administered by intracisternal injection 15 min before whisker stimulation or 2P optogenetic stimulation.

Techniques: Optogenetics, Labeling, Control, Injection, Comparison

Journal: Neuron

Article Title: GluN3A excitatory glycine receptors control adult cortical and amygdalar circuits

doi: 10.1016/j.neuron.2022.05.016

Figure Lengend Snippet: eGlyRs control the resting membrane potential of neocortical SST-INs (A) Schematic representation of the canonical cellular organization of layer 2/3 in S1 depicting a whole-cell patch-clamped SST-IN. (B) Example traces, time course, and summary plot of the variations in the holding currents in SST-INs upon DCKA (500 μM) application in WT and GluN3A KO mice (WT [n = 18] versus GluN3A KO [n = 11], p = 0.0478, Kruskal-Wallis followed by Dunn’s multiple comparisons test). (C) Example traces and summary plot of the measured EPSPs obtained in response to the puff application of glycine (1 mM) in different cell populations of layer 2/3 of S1. Note significantly larger membrane depolarizations in SST-INs and absence in GluN3A KO mice (SST-INs [n = 11] versus PNs [n = 8], p = 0.0203; versus PV-INs [n = 8], p < 0.0001; versus VIP-INs [n = 8], p = 0.0016; SST-INs GluN3A KO [n = 8], p = 0.0012, Kruskal-Wallis followed by Dunn’s multiple comparisons test). (D) Left: representative current-clamp traces of membrane potential responses to injections of current steps applied to SST-INs (green) and PV-INs (fuchsia) in S1. Right: summary plot of the resting membrane potential values for SST-INs and PV-INs obtained in control conditions and after the addition of DCKA (500 μM) or D-AP5 (50 μM; SST versus SST + DCKA [n = 20], p = 0.0256; PV versus PV + DCKA [n = 15], p > 0.9999; control [n = 20] versus D-AP5 [n = 13], p > 0.99; Kruskal-Wallis followed by Dunn’s multiple comparisons test). (E) Same as (D) but for current-clamp recordings performed in the slices from WT and GluN3A KO mice. Left: electrophysiological traces represent the membrane potential responses to injections of current steps of increasing amplitude (−60, 0, 60, and 90 pA) to SST-INs. Right: SST-INs Vm rest values are more hyperpolarized in GluN3A-lacking slices (WT [n = 14] versus GluN3A KO [n = 12], p = 0.0356, Mann-Whitney U test). (F) Spiking frequency as a function of injected current for the SST-INs recorded in the slices from WT and GluN3A KO mice. Solid line represents the fit of Equation 1 (see ) that is statistically different between WT [n = 14] and GluN3A KO [n = 12] mice; p = 0.001, extra-sum-of-squares F test. (G) Same as (E) but for the current-clamp recordings performed in the slices treated with the enzyme glycine oxidase (GO). Reducing extracellular glycine levels with glycine oxidase depolarizes the Vm rest values of SST-INs in the slices from WT but not GluN3A KO mice (WT [n = 20] versus WT + GO [n = 16], p = 0.0012; GluN3A KO [n = 21] versus GluN3A KO + GO [n = 16], p = 0.6652, Kruskal-Wallis followed by Dunn’s multiple comparisons test). (H) Same as (F) but for the brain slices incubated with glycine oxidase (WT [n = 18] versus GO [n = 11], p = 0.001, extra-sum-of-squares F test). (B–G) Bars indicate mean ± SEM.

Article Snippet: With the exception of glycine and NMDA, all the drugs were bath applied: D-AP5 (HelloBio), NBQX (HelloBio or Tocris), Bicuculline (HelloBio or Tocris), Gabazine (Tocris), Picrotoxin (abcam), Strychnine (Sigma), TTX (HelloBio or Tocris), CGP-78608 (Tocris), 5,7-Dichlorokynurenic acid (DCKA; Tocris), Sarcosine (Sigma), D-serine (Sigma), CNQX (Tocris), mecamylamine (Sigma).

Techniques: MANN-WHITNEY, Injection, Incubation

Journal: Neuron

Article Title: GluN3A excitatory glycine receptors control adult cortical and amygdalar circuits

doi: 10.1016/j.neuron.2022.05.016

Figure Lengend Snippet:

Article Snippet: With the exception of glycine and NMDA, all the drugs were bath applied: D-AP5 (HelloBio), NBQX (HelloBio or Tocris), Bicuculline (HelloBio or Tocris), Gabazine (Tocris), Picrotoxin (abcam), Strychnine (Sigma), TTX (HelloBio or Tocris), CGP-78608 (Tocris), 5,7-Dichlorokynurenic acid (DCKA; Tocris), Sarcosine (Sigma), D-serine (Sigma), CNQX (Tocris), mecamylamine (Sigma).

Techniques: Recombinant, shRNA, Sequencing, Software