Journal: Cell Reports

Article Title: The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity

doi: 10.1016/j.celrep.2023.112456

Figure Lengend Snippet: RACK1 KO in astrocytes leads to higher levels of Kir4.1 in astrocyte somata and PAPs (A) Generation of a mouse line with RACK1 KO in astrocytes (RACK1 cKO). Shown is a schematic of the RACK1 fl/fl and Aldh1l1-Cre/ERT2 alleles. Deletion of exon 2 in Gnb2l1 (the gene coding for RACK1) is induced in astrocytes by tamoxifen injection; this results in a frameshift and premature termination of Gnb2l1 translation. Primers are indicated by red arrows. (B) PCR assays for Gnb2l1 KO in brain DNA from RACK1 fl/fl or Aldh1l1-CreERT2: RACK1 fl/fl tamoxifen-injected mice (RACK1 cKO). The 898-bp band corresponds to the floxed allele ( Table S4 ). The 672-bp band corresponds to the exon 2-deleted allele. (C) Confocal images of RACK1 immunofluorescence (red) in the hippocampus in RACK1 fl/fl and RACK1 cKO mice. Astrocytes are co-immunolabeled for GFAP (green). Some astrocytes are indicated by white arrowheads. Nuclei are stained with DAPI. The bottom panel gives a higher-magnification view of the boxed areas in the RACK1 fl/fl and RACK1 cKO images, which shows that RACK1 is specifically depleted in astrocytes ( ∗ ) and is still expressed by neurons (°). Scale bars, 20 μm. (D), Western blot detection and analysis of Kir4.1 and GLT-1 in protein extracts from whole brain, hippocampus, whole-brain synaptogliosomes, hippocampal synaptogliosomes, or whole-brain microvessels purified from RACK1 fl/fl or RACK1 cKO mice. The position of molecular weight markers is indicated on the right. Signals were normalized against that of stain-free membranes except for the experiment on purified microvessels, where histone 3 was used. The data are quoted as the mean ± SD (N = 5 samples per genotype, 1 mouse per sample); two-tailed unpaired t test or Mann-Whitney test. ns, p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. The raw data are presented in Table S3 and Figure S2 .

Article Snippet: Rabbit polyclonal anti-KIR4.1 , Alomone labs , Cat#APC-035; RRID: AB_2040120.

Techniques: Injection, Immunofluorescence, Immunolabeling, Staining, Western Blot, Purification, Molecular Weight, Two Tailed Test, MANN-WHITNEY

Journal: Cell Reports

Article Title: The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity

doi: 10.1016/j.celrep.2023.112456

Figure Lengend Snippet: Absence of RACK1 in astrocytes leads to higher Kir4.1-mediated astrocytic inward K + currents and cell volumes (A) Schematic of electrode positions used to record astrocyte whole-cell currents evoked by Schaffer collateral (SC) stimulation in the CA1 region of hippocampal slices. (B) Left: representative traces of astrocytic whole-cell currents induced by 150-ms voltage steps (from −200 mV to +100 mV, 10-mV steps; black traces at the bottom) in RACK1 fl/fl and RACK1 cKO mice before (black and pink) and after (blue) application of a KIR 4.1 antagonist (VU). Scale bars, 50 ms, 5 nA. Right: current-voltage (I-V) plot in RACK1 fl/fl (white filled dots) and in RACK1 cKO (pink-filled dots) mice before (black) and after (blue) application of VU (N = 7 and 10 astrocytes for RACK1 fl/fl and RACK1 cKO mice, respectively; repeated-measures two-way ANOVA with Sidak’s correction for multiple comparisons) The data are quoted as the mean ± SD. (C) Left: representative astrocytic Kir4.1 (VU-sensitive) currents induced by SC stimulation (10 Hz, 1 s) in RACK1 fl/fl (black) and RACK1 cKO (pink) mice. Scale bars, 200 ms, 50 pA. Right: quantification of astrocytic Kir4.1 current peak amplitude after each stimulus during SC stimulation (10 Hz, 1 s) in RACK1 fl/fl (white filled dots) and RACK1 cKO (pink filled dots) mice (N = 6 and 5 astrocytes for RACK1 fl/fl and RACK1 cKO mice, respectively; repeated-measures two-way ANOVA with Sidak’s correction for multiple comparisons). The data are quoted as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (D) Illustration of the imaging method with a representative raw confocal image of an isolated RACK1 fl/fl CA1 astrocyte expressing tdTomato. (E–G) Imaris analysis: filament tracing (E), convex hull volume (F), and a 3D Sholl analysis (G). (H) Mean territory volume and filament length of RACK1 fl/fl and RACK1 cKO astrocytes. Shown is a histogram of the data, presented as the mean ± SD (N = 4 mice per genotype, 45 astrocytes); two-tailed t test. The data are quoted as the mean ± SD. (I) A Sholl analysis of the ramification complexity of RACK1 fl/fl and RACK1 cKO astrocytes. Two-way analysis of variance. ∗ p < 0.05, ∗∗ p < 0.01. The data are quoted as the mean ± SD. The raw data are presented in Table S3 .

Article Snippet: Rabbit polyclonal anti-KIR4.1 , Alomone labs , Cat#APC-035; RRID: AB_2040120.

Techniques: Imaging, Isolation, Expressing, Two Tailed Test

Journal: Cell Reports

Article Title: The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity

doi: 10.1016/j.celrep.2023.112456

Figure Lengend Snippet: Absence of RACK1 in astrocytes alters network population activity and neuronal responses to intense stimulation (A) Schematic of electrode positions used to record field excitatory postsynaptic potentials (fEPSPs) evoked by SC stimulation in the CA1 region of hippocampal slices. (B) Input-output curves for basal synaptic transmission. Left: representative recordings in RACK1 fl/fl mice (black) and RACK1 cKO mice before (pink) and after (blue) application of a Kir 4.1 antagonist (VU). Scale bars, 10 ms, 0.5 mV. Right: quantification of the fEPSP slope for different fiber volley amplitudes after SC stimulation. The data are quoted as the mean ± SD. RACK1 fl/fl: n = 5 slices from 4 mice; p = 0.0087; RACK1 cKO: n = 5 slices from 5 mice; repeated-measures two-way ANOVA with Sidak’s correction for multiple comparisons. (C) Top: a representative recording of fEPSPs evoked by repetitive stimulation (10 Hz, 30 s) of CA1 SCs in RACK1 fl/fl mice under control conditions. Scale bars, 5 s, 0.2 mV. Bottom: enlarged view of fEPSPs evoked by the first 10 stimuli. Scale bars, 200 ms, 0.2 mV. (D) Changes in the fEPSP slope induced by 10-Hz stimulation relative to responses measured before the onset of stimulation (baseline responses) in RACK1 fl/fl mice (white filled dots) and in RACK1 cKO mice (pink-filled dots) before (black) and after (blue) application of VU. The data are quoted as the mean ± SD. RACK1 fl/fl: n = 5 from 5 mice; RACK1 cKO: n = 6 slices from 4 mice; repeated-measures two-way ANOVA with Sidak’s correction for multiple comparisons. (E) Schematic (left) and picture (right) of a hippocampal slice placed on a multielectrode array (MEA). Scale bar, 200 μm. (F) Representative MEA recordings of burst activity induced in hippocampal slices of RACK1 fl/fl (black) and RACK1 cKO (pink) mice by incubation in Mg 2+ -free ACSF containing 6 mM KCl. The expanded recordings of the bursts (surrounded by gray rectangles) are shown on the right. Scale bars, 10 s (left)/200 ms (right), 50 μV. (G) Quantification of burst frequency (top) and burst duration (bottom) in RACK1 fl/fl (white) and RACK1 cKO (pink) hippocampal slices. The data are quoted as the mean ± SD. n = 15 slices from 5 mice for RACK1 fl/fl and n = 18 slices from 6 mice for RACK1 cKO; unpaired t test. (H) Representative MEA recordings of hippocampal bursting activity in RACK1 fl/fl (top) and RACK1 cKO (bottom) slices in control (Ct) and during 25-min treatment with the Kir4.1 blocker VU0134992 (VU). The corresponding time-frequency plots are shown under the traces. Scale bar, 20 s/2 min, 50 μV. (I) Quantification of VU’s effect on burst frequency (top) and duration (bottom) in RACK1 fl/fl (white) and RACK1 cKO (pink) hippocampal slices (RACK1 fl/fl: n = 15 slices from 5 mice for burst frequency and duration, respectively; RACK1 cKO: n = 18 slices from 6 mice for burst frequency and duration; paired t test. The data are quoted as the mean ± SD. ns, p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. The raw data are presented in Table S3 .

Article Snippet: Rabbit polyclonal anti-KIR4.1 , Alomone labs , Cat#APC-035; RRID: AB_2040120.

Techniques: Activity Assay, Transmission Assay, Incubation

Journal: Cell Reports

Article Title: The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity

doi: 10.1016/j.celrep.2023.112456

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-KIR4.1 , Alomone labs , Cat#APC-035; RRID: AB_2040120.

Techniques: Transduction, Recombinant, Multiplex Assay, Mass Spectrometry, Software