Journal: iScience

Article Title: Prkn knockout mice show autistic-like behaviors and aberrant synapse formation

doi: 10.1016/j.isci.2022.104573

Figure Lengend Snippet:

Article Snippet: Rabbit anti-GluA3 (1:2000) , Cell Signaling Technology , Cat. #5117S; RRID: AB_10544796.

Techniques: Software

Journal: Frontiers in Molecular Neuroscience

Article Title: Somatic Accumulation of GluA1-AMPA Receptors Leads to Selective Cognitive Impairments in Mice

doi: 10.3389/fnmol.2018.00199

Figure Lengend Snippet: Expression of the L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits and subunit assemblies in WT and Gria1 R/R mice. (A) Hippocampal expression of GluA1–3, GluN1, αCaMKII and ß-actin in WT and Gria1 R/R mice from P2 till P90. (B) Co-immunoprecipitations (IPs) using polyclonal anti-GluA1 and anti-GluA2/3 antibodies show the presence of GluA1–3 in AMPAR assemblies from hippocampal membrane preparations at P > 60 of WT , Gria1 R/R (R/R) and Gria1 −/− (−/−) mice. (C) Schematic representation of the Gria1 R “knock-in” ( Gria1 tm1Erk ) allele and the Gria1 + allele ( WT ). Below the gene segments, the putative AMPAR subtypes, that can operate at CA3-to-CA1 synapses in Gria1 R/R and WT mice, are schematically depicted (GluA1(R) = GluA1(Q600R)). Large AMPAR symbols for high abundance; small symbols for low abundance; transparent for AMPARs with low single channel conductance. The inset shows the position of the Q600R mutations (R) in two out of the four P-loop segments that form the ion pore of an AMPAR. Exons are in boxes, loxP sites in black triangles and the M1 and P-loop coding sequence in black squares. The position of the mutated codon Q600R codon and codon Q600 in Gria1 tm1Erk and Gria1 are indicated, respectively (see Sprengel et al., ). High resolution images of (A,B) are accessible at https://dx.doi.org/10.17617/3.1i .

Article Snippet: The blotted proteins were probed with polyclonal antibodies against GluA1 (Merck Millipore 0.1 μg/ml), anti-GluA2 (Merck Millipore 0.16 μg/ml), anti-GluN1 (Merck Millipore, 0.25 μg/ml, monoclonal anti-GluA3 (ThermoFisher, 1:250), anti-CaMKII (MAB 8699 Merck Millipore 0.1 μg/ml) and anti-ß-actin (AC-15 Ascites, Sigma, 1:25,000), followed by peroxidase-linked anti-rabbit or -mouse secondary antibodies (Jackson Immuno Res., 1:20,000).

Techniques: Expressing, Knock-In, Sequencing

Journal: Frontiers in Molecular Neuroscience

Article Title: Somatic Accumulation of GluA1-AMPA Receptors Leads to Selective Cognitive Impairments in Mice

doi: 10.3389/fnmol.2018.00199

Figure Lengend Snippet: Expression of the L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits and subunit assemblies in WT and Gria1 R/R mice. (A) Hippocampal expression of GluA1–3, GluN1, αCaMKII and ß-actin in WT and Gria1 R/R mice from P2 till P90. (B) Co-immunoprecipitations (IPs) using polyclonal anti-GluA1 and anti-GluA2/3 antibodies show the presence of GluA1–3 in AMPAR assemblies from hippocampal membrane preparations at P > 60 of WT , Gria1 R/R (R/R) and Gria1 −/− (−/−) mice. (C) Schematic representation of the Gria1 R “knock-in” ( Gria1 tm1Erk ) allele and the Gria1 + allele ( WT ). Below the gene segments, the putative AMPAR subtypes, that can operate at CA3-to-CA1 synapses in Gria1 R/R and WT mice, are schematically depicted (GluA1(R) = GluA1(Q600R)). Large AMPAR symbols for high abundance; small symbols for low abundance; transparent for AMPARs with low single channel conductance. The inset shows the position of the Q600R mutations (R) in two out of the four P-loop segments that form the ion pore of an AMPAR. Exons are in boxes, loxP sites in black triangles and the M1 and P-loop coding sequence in black squares. The position of the mutated codon Q600R codon and codon Q600 in Gria1 tm1Erk and Gria1 are indicated, respectively (see Sprengel et al., ). High resolution images of (A,B) are accessible at https://dx.doi.org/10.17617/3.1i .

Article Snippet: The blotted proteins were probed with polyclonal antibodies against GluA1 (Merck Millipore 0.1 μg/ml), anti-GluA2 (Merck Millipore 0.16 μg/ml), anti-GluN1 (Merck Millipore, 0.25 μg/ml, monoclonal anti-GluA3 (ThermoFisher, 1:250), anti-CaMKII (MAB 8699 Merck Millipore 0.1 μg/ml) and anti-ß-actin (AC-15 Ascites, Sigma, 1:25,000), followed by peroxidase-linked anti-rabbit or -mouse secondary antibodies (Jackson Immuno Res., 1:20,000).

Techniques: Expressing, Knock-In, Sequencing

Journal: Frontiers in Molecular Neuroscience

Article Title: Somatic Accumulation of GluA1-AMPA Receptors Leads to Selective Cognitive Impairments in Mice

doi: 10.3389/fnmol.2018.00199

Figure Lengend Snippet: Subcellular AMPAR subunit distribution in the hippocampus. (A) Distribution of GluA1–3 subunits in hippocampi of WT controls and Gria1 R/R mice at P14 and P42. The GluA1–3 subunit expression levels were detected by GluA1, GluA2 and GluA2/A3 subunit specific antibodies. The ratios of the average signal intensity in the str. pyramidale (py) vs. the signal intensity in the str. oriens (or) and radiatum (ra) are indicated (Somatic Accumulation index; SAi). Scale bar = 0.9 mm. Insets show higher magnifications of the respective str. pyramidale; scale bar = 50 μm. (B) Anti-GluA1 immunogold labeling of GluA1 and GluA1(Q600R) at an excitatory CA1 synapse taken from an adult WT and a Gria1 R/R mouse, respectively. Scale bar = 0.1 μm. (C) Quantification of the immunogold signal at CA3-to-CA1 synapses containing GluA1, GluA1(Q600R) and GluA2 subunits. Intensity of GluA1- and GluA2-immunoreactivity expressed as number of gold particles per synapse. Gria1 R/R mice showed significant fewer anti-GluA1 gold particles per synapse in comparison to WT . Moreover, the number of anti-GluA1 labeled synapses was significantly lower in Gria1 R/R mice. Similar concentrations of anti-GluA2 gold particles were found in Gria1 R/R and WT control mice; n = 6 Gria1 R/R and 6 WT control mice. Error bars indicate SEM. Unpaired two-tailed student’s t -test was used (* P ≤ 0.05). For original data used for the quantification see https://dx.doi.org/10.17617/3.1i .

Article Snippet: The blotted proteins were probed with polyclonal antibodies against GluA1 (Merck Millipore 0.1 μg/ml), anti-GluA2 (Merck Millipore 0.16 μg/ml), anti-GluN1 (Merck Millipore, 0.25 μg/ml, monoclonal anti-GluA3 (ThermoFisher, 1:250), anti-CaMKII (MAB 8699 Merck Millipore 0.1 μg/ml) and anti-ß-actin (AC-15 Ascites, Sigma, 1:25,000), followed by peroxidase-linked anti-rabbit or -mouse secondary antibodies (Jackson Immuno Res., 1:20,000).

Techniques: Expressing, Labeling, Two Tailed Test

Journal: Frontiers in Molecular Neuroscience

Article Title: Somatic Accumulation of GluA1-AMPA Receptors Leads to Selective Cognitive Impairments in Mice

doi: 10.3389/fnmol.2018.00199

Figure Lengend Snippet: Expression of the L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits and subunit assemblies in WT and Gria1 R/R mice. (A) Hippocampal expression of GluA1–3, GluN1, αCaMKII and ß-actin in WT and Gria1 R/R mice from P2 till P90. (B) Co-immunoprecipitations (IPs) using polyclonal anti-GluA1 and anti-GluA2/3 antibodies show the presence of GluA1–3 in AMPAR assemblies from hippocampal membrane preparations at P > 60 of WT , Gria1 R/R (R/R) and Gria1 −/− (−/−) mice. (C) Schematic representation of the Gria1 R “knock-in” ( Gria1 tm1Erk ) allele and the Gria1 + allele ( WT ). Below the gene segments, the putative AMPAR subtypes, that can operate at CA3-to-CA1 synapses in Gria1 R/R and WT mice, are schematically depicted (GluA1(R) = GluA1(Q600R)). Large AMPAR symbols for high abundance; small symbols for low abundance; transparent for AMPARs with low single channel conductance. The inset shows the position of the Q600R mutations (R) in two out of the four P-loop segments that form the ion pore of an AMPAR. Exons are in boxes, loxP sites in black triangles and the M1 and P-loop coding sequence in black squares. The position of the mutated codon Q600R codon and codon Q600 in Gria1 tm1Erk and Gria1 are indicated, respectively (see Sprengel et al., ). High resolution images of (A,B) are accessible at https://dx.doi.org/10.17617/3.1i .

Article Snippet: The blotted proteins were probed with polyclonal antibodies against GluA1 (Merck Millipore 0.1 μg/ml), anti-GluA2 (Merck Millipore 0.16 μg/ml), anti-GluN1 (Merck Millipore, 0.25 μg/ml, monoclonal anti-GluA3 (ThermoFisher, 1:250), anti-CaMKII (MAB 8699 Merck Millipore 0.1 μg/ml) and anti-ß-actin (AC-15 Ascites, Sigma, 1:25,000), followed by peroxidase-linked anti-rabbit or -mouse secondary antibodies (Jackson Immuno Res., 1:20,000).

Techniques: Expressing, Membrane, Knock-In, Sequencing

Journal: Frontiers in Molecular Neuroscience

Article Title: Somatic Accumulation of GluA1-AMPA Receptors Leads to Selective Cognitive Impairments in Mice

doi: 10.3389/fnmol.2018.00199

Figure Lengend Snippet: Subcellular AMPAR subunit distribution in the hippocampus. (A) Distribution of GluA1–3 subunits in hippocampi of WT controls and Gria1 R/R mice at P14 and P42. The GluA1–3 subunit expression levels were detected by GluA1, GluA2 and GluA2/A3 subunit specific antibodies. The ratios of the average signal intensity in the str. pyramidale (py) vs. the signal intensity in the str. oriens (or) and radiatum (ra) are indicated (Somatic Accumulation index; SAi). Scale bar = 0.9 mm. Insets show higher magnifications of the respective str. pyramidale; scale bar = 50 μm. (B) Anti-GluA1 immunogold labeling of GluA1 and GluA1(Q600R) at an excitatory CA1 synapse taken from an adult WT and a Gria1 R/R mouse, respectively. Scale bar = 0.1 μm. (C) Quantification of the immunogold signal at CA3-to-CA1 synapses containing GluA1, GluA1(Q600R) and GluA2 subunits. Intensity of GluA1- and GluA2-immunoreactivity expressed as number of gold particles per synapse. Gria1 R/R mice showed significant fewer anti-GluA1 gold particles per synapse in comparison to WT . Moreover, the number of anti-GluA1 labeled synapses was significantly lower in Gria1 R/R mice. Similar concentrations of anti-GluA2 gold particles were found in Gria1 R/R and WT control mice; n = 6 Gria1 R/R and 6 WT control mice. Error bars indicate SEM. Unpaired two-tailed student’s t -test was used (* P ≤ 0.05). For original data used for the quantification see https://dx.doi.org/10.17617/3.1i .

Article Snippet: The blotted proteins were probed with polyclonal antibodies against GluA1 (Merck Millipore 0.1 μg/ml), anti-GluA2 (Merck Millipore 0.16 μg/ml), anti-GluN1 (Merck Millipore, 0.25 μg/ml, monoclonal anti-GluA3 (ThermoFisher, 1:250), anti-CaMKII (MAB 8699 Merck Millipore 0.1 μg/ml) and anti-ß-actin (AC-15 Ascites, Sigma, 1:25,000), followed by peroxidase-linked anti-rabbit or -mouse secondary antibodies (Jackson Immuno Res., 1:20,000).

Techniques: Expressing, Labeling, Comparison, Control, Two Tailed Test

Journal: Frontiers in Molecular Neuroscience

Article Title: Somatic Accumulation of GluA1-AMPA Receptors Leads to Selective Cognitive Impairments in Mice

doi: 10.3389/fnmol.2018.00199

Figure Lengend Snippet: Whole-soma currents, excitatory synaptic transmission, synaptic excitability and paired-pulse facilitation in the CA1 hippocampal region. (A) Recordings of glutamate activated AMPAR and N -methyl-D-aspartate receptor (NMDAR) currents from nucleated patches of CA1 pyramidal cells obtained from P42 WT (left) and Gria1 R/R (right) mice. I AMPAR/NMDAR ratio is significantly different at P42; I AMPAR/NMDAR ratio 1.87 ± 0.68 in Gria1 R/R , n = 7 from 3 mice; 5.43 ± 0.97 in WT , n = 9 from 4 mice; p < 0.01. (B) Left: stimulation strengths (in nC) necessary to elicit a prevolley of a given amplitude (0.5 mV, 1.0 mV and 1.5 mV) in slices from WT (open columns) and Gria1 R/R (filled columns) mice. Middle left: fEPSP amplitudes in the two genotypes as a function of the three prevolley amplitudes. Middle right: (1) the fEPSP amplitudes in slices from the two genotypes necessary to elicit a just detectable population spike; and (2) a population spike of 2 mV amplitude. Right: paired-pulse facilitation (PPF) ratio in the two genotypes at an inter-stimulus interval of 50 ms. Lower panels: each trace is the mean of five consecutive synaptic responses in str. radiatum elicited by different stimulation strengths in slices from WT (left) and Gria1 R/R (right) mice. The prevolleys preceding the fEPSPs are indicated by circles. The lower recordings show traces from str. pyramidale elicited by paired-pulse stimulation (50 ms interstimulus interval). Arrowheads indicate the population spike threshold. The number of experiments ranged from 30 to 82. Data are shown as mean + SEM.

Article Snippet: The blotted proteins were probed with polyclonal antibodies against GluA1 (Merck Millipore 0.1 μg/ml), anti-GluA2 (Merck Millipore 0.16 μg/ml), anti-GluN1 (Merck Millipore, 0.25 μg/ml, monoclonal anti-GluA3 (ThermoFisher, 1:250), anti-CaMKII (MAB 8699 Merck Millipore 0.1 μg/ml) and anti-ß-actin (AC-15 Ascites, Sigma, 1:25,000), followed by peroxidase-linked anti-rabbit or -mouse secondary antibodies (Jackson Immuno Res., 1:20,000).

Techniques: Transmission Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Somatic Accumulation of GluA1-AMPA Receptors Leads to Selective Cognitive Impairments in Mice

doi: 10.3389/fnmol.2018.00199

Figure Lengend Snippet: Diminished long-term potentiation (LTP) in Gria1 R/R mice. (A) Normalized and pooled fEPSP slopes evoked in str. radiatum at CA3-to-CA1 synapses in slices from Gria1 R/R and WT mice. LTP was induced by a single tetanization. For the sake of clarity, only the non-tetanized control pathway in control WT mice is shown. Arrow at the abscissa indicates the time of tetanic stimulation. Vertical bars indicate SEM. (B) As in (A) , but four tetanizations were used for LTP induction. Insets show means of six consecutive synaptic responses in the tetanized pathway before (open arrowhead) and 45 min after (filled arrowhead) tetanization in an experiment from a Gria1 R/R (left sweeps) and a WT (right sweeps) control. (C) As in (A) , but LTP was induced by a theta-burst paradigm where synaptically and antidromically evoked responses were paired. For comparison, experiments performed in slices from the brains of Gria1 −/− mice are also shown.

Article Snippet: The blotted proteins were probed with polyclonal antibodies against GluA1 (Merck Millipore 0.1 μg/ml), anti-GluA2 (Merck Millipore 0.16 μg/ml), anti-GluN1 (Merck Millipore, 0.25 μg/ml, monoclonal anti-GluA3 (ThermoFisher, 1:250), anti-CaMKII (MAB 8699 Merck Millipore 0.1 μg/ml) and anti-ß-actin (AC-15 Ascites, Sigma, 1:25,000), followed by peroxidase-linked anti-rabbit or -mouse secondary antibodies (Jackson Immuno Res., 1:20,000).

Techniques: Control, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Somatic Accumulation of GluA1-AMPA Receptors Leads to Selective Cognitive Impairments in Mice

doi: 10.3389/fnmol.2018.00199

Figure Lengend Snippet: Effects of GluA1(Q600R) on locomotor activity and spatial working memory during the spontaneous alternation T-maze test.

Article Snippet: The blotted proteins were probed with polyclonal antibodies against GluA1 (Merck Millipore 0.1 μg/ml), anti-GluA2 (Merck Millipore 0.16 μg/ml), anti-GluN1 (Merck Millipore, 0.25 μg/ml, monoclonal anti-GluA3 (ThermoFisher, 1:250), anti-CaMKII (MAB 8699 Merck Millipore 0.1 μg/ml) and anti-ß-actin (AC-15 Ascites, Sigma, 1:25,000), followed by peroxidase-linked anti-rabbit or -mouse secondary antibodies (Jackson Immuno Res., 1:20,000).

Techniques: Activity Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Somatic Accumulation of GluA1-AMPA Receptors Leads to Selective Cognitive Impairments in Mice

doi: 10.3389/fnmol.2018.00199

Figure Lengend Snippet: Spatial memory in Gria1 R/R mice. (A) Left: Gria1 R/R mice are impaired on a spatial working memory task on the elevated T-maze. Mean percentage correct responses (± SEM) for WT littermates (white circles; n = 14) and Gria1 R/R mice (black squares; n = 15) during spatial non-matching to place testing on the elevated T-maze. Right: Gria1 R/R mice show normal spatial reference memory acquisition on the elevated Y-maze. Mean percentage of correct responses (± SEM) during acquisition of an appetitive spatial reference memory task for male WT littermates (white circles; n = 8) and Gria1 R/R mice (black squares; n = 7). (B) Left: Gria1 R/R mice acquired a standard spatial reference memory version of the Morris water-maze task. Mean escape latency (± SEM) for each day of testing during acquisition for WT littermates (white circles; n = 14) and Gria1 R/R mice (black squares, n = 15). Right, top: mean percentage of time (± SEM) spent in the four quadrants of the pool during the 90 s probe test, conducted at the end of spatial training (after 36 trials) for WT littermates (left; n = 14) and Gria1 R/R mice (right; n = 15). The platform had previously been located in the training quadrant (G) during acquisition. Right, bottom: mean percentage of time (± SEM) spent in the training quadrant during the three 30 s time blocks of the 90 s probe trial by WT littermates (white circles; n = 14) and Gria1 R/R mice (black squares, n = 15).

Article Snippet: The blotted proteins were probed with polyclonal antibodies against GluA1 (Merck Millipore 0.1 μg/ml), anti-GluA2 (Merck Millipore 0.16 μg/ml), anti-GluN1 (Merck Millipore, 0.25 μg/ml, monoclonal anti-GluA3 (ThermoFisher, 1:250), anti-CaMKII (MAB 8699 Merck Millipore 0.1 μg/ml) and anti-ß-actin (AC-15 Ascites, Sigma, 1:25,000), followed by peroxidase-linked anti-rabbit or -mouse secondary antibodies (Jackson Immuno Res., 1:20,000).

Techniques: