glua2  (Alomone Labs)

Journal: Journal of neurochemistry

Article Title: Increased α2δ−1–NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain

doi: 10.1111/jnc.14627

Figure Lengend Snippet: α2δ−1 at the spinal cord level is involved in paclitaxel-induced pain hypersensitivity. ( A–C ). Time course of the effect of intrathecal injection with vehicle, 5 μg pregabalin, 1 μg α2δ−1Tat peptide, or 1 μg control peptide on the paw withdrawal thresholds measured with von Frey filaments (A), a pressure stimulus (B), and a heat stimulus (C) in paclitaxel-treated rats (n = 8 rats per group). Data are expressed as means ± SEM. *P

Article Snippet: The membrane was treated with 5% nonfat dry milk in Tris-buffered saline (TBS) at 25°C for 1 h and then incubated in TBS supplemented with 0.1% Triton X-100, 1% bovine serum albumin, and rabbit anti-α2δ−1 (#ACC-015, 1:500, Alomone Labs, Jerusalem, Israel), rabbit anti-GluN1 (#G8913, 1:1,000, Sigma-Aldrich), rabbit anti-GAPDH (#14C10, 1:5,000, Cell Signaling Technology, Danvers, MA), or mouse anti-PSD95 (#75–348, 1:1,000, NeuroMab, Davis, CA) antibodies overnight at 4°C.

Techniques: Injection

Journal: Journal of neurochemistry

Article Title: Increased α2δ−1–NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain

doi: 10.1111/jnc.14627

Figure Lengend Snippet: Flowchart diagrams show the timeline of experimental procedures used in the study. Rats were treated with either paclitaxel or vehicle (top panels) and then used for behavioral, biochemical or electrophysiological experiments. Wild-type and α2δ−1 knockout mice were treated with paclitaxel or vehicle (lower panels), and nociceptive tests or electrophysiological recordings were performed at the time indicated. The number of animals used for each group was indicated in parenthesis.

Article Snippet: The membrane was treated with 5% nonfat dry milk in Tris-buffered saline (TBS) at 25°C for 1 h and then incubated in TBS supplemented with 0.1% Triton X-100, 1% bovine serum albumin, and rabbit anti-α2δ−1 (#ACC-015, 1:500, Alomone Labs, Jerusalem, Israel), rabbit anti-GluN1 (#G8913, 1:1,000, Sigma-Aldrich), rabbit anti-GAPDH (#14C10, 1:5,000, Cell Signaling Technology, Danvers, MA), or mouse anti-PSD95 (#75–348, 1:1,000, NeuroMab, Davis, CA) antibodies overnight at 4°C.

Techniques: Knock-Out, Mouse Assay

Journal: Journal of neurochemistry

Article Title: Increased α2δ−1–NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain

doi: 10.1111/jnc.14627

Figure Lengend Snippet: Inhibiting α2δ−1 with pregabalin normalizes paclitaxel-induced activation of presynaptic NMDARs at primary afferent terminals. ( A,B ). Original recording traces (A) and box-and-whisker plots (B) show the effect of bath application of 50 μM AP5 on evoked monosynaptic EPSCs of lamina II neurons from paclitaxel-treated rats. Neurons were treated with 20 μM pregabalin (+pregabalin, n = 8 neurons from 4 rats) or untreated (–pregabalin, n = 12 neurons from 4 rats). ( C,D ). Representative recording traces (C) and box-and-whisker plots (D) shows the effect of bath application of 50 μM AP5 on evoked monosynaptic EPSCs of lamina II neurons from vehicle-treated rats. Neurons treated with 20 μM pregabalin (+pregabalin, n = 9 neurons from 4 rats) or untreated (–pregabalin, n = 8 neurons from 4 rats). In B and D (right panels), values are normalized to their respective baselines. ( E,F ). Original recording traces (E) and box-and-whisker plots (F) shows the effect of bath application of AP5 on the paired-pulse ratio (PPR) of lamina II neurons treated with pregabalin (+pregabalin) and of untreated neurons (–pregabalin) from vehicle-treated (n = 9 neurons with pregabalin from 4 rats; n = 8 neurons without pregabalin from 4 rats) and paclitaxel-treated rats (n = 8 neurons with pregabalin from 4 rats; n = 9 neurons without pregabalin from 4 rats). *P

Article Snippet: The membrane was treated with 5% nonfat dry milk in Tris-buffered saline (TBS) at 25°C for 1 h and then incubated in TBS supplemented with 0.1% Triton X-100, 1% bovine serum albumin, and rabbit anti-α2δ−1 (#ACC-015, 1:500, Alomone Labs, Jerusalem, Israel), rabbit anti-GluN1 (#G8913, 1:1,000, Sigma-Aldrich), rabbit anti-GAPDH (#14C10, 1:5,000, Cell Signaling Technology, Danvers, MA), or mouse anti-PSD95 (#75–348, 1:1,000, NeuroMab, Davis, CA) antibodies overnight at 4°C.

Techniques: Activation Assay, Whisker Assay

Journal: Journal of neurochemistry

Article Title: Increased α2δ−1–NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain

doi: 10.1111/jnc.14627

Figure Lengend Snippet: Disrupting the α2δ−1–NMDAR interaction using α2δ−1Tat peptide reverses paclitaxel-induced tonic activation of presynaptic NMDARs in the spinal cord. ( A,B ). Representative recording traces and cumulative plots (A) and box-and-whisker plots (B) show the effect of bath application of 50 μM AP5 on the frequency and amplitude of mEPSCs of lamina II neurons from paclitaxel-treated rats, Neurons were treated with 1 μM α2δ−1Tat peptide (n = 10 neurons from 5 rats) or 1 μM control peptide (n = 11 neurons from 5 rats). ( C,D ) Original recording traces and cumulative plots (C) and box-and-whisker plots (D) show the effect of bath application of AP5 on the frequency and amplitude of mEPSCs of lamina II neurons from vehicle-treated rats. Neurons were treated with 1 μM α2δ−1Tat peptide (n = 11 neurons from 5 rats) or 1 μM control peptide (n = 12 neurons from 5 rats). *P

Article Snippet: The membrane was treated with 5% nonfat dry milk in Tris-buffered saline (TBS) at 25°C for 1 h and then incubated in TBS supplemented with 0.1% Triton X-100, 1% bovine serum albumin, and rabbit anti-α2δ−1 (#ACC-015, 1:500, Alomone Labs, Jerusalem, Israel), rabbit anti-GluN1 (#G8913, 1:1,000, Sigma-Aldrich), rabbit anti-GAPDH (#14C10, 1:5,000, Cell Signaling Technology, Danvers, MA), or mouse anti-PSD95 (#75–348, 1:1,000, NeuroMab, Davis, CA) antibodies overnight at 4°C.

Techniques: Activation Assay, Whisker Assay

Journal: Journal of neurochemistry

Article Title: Increased α2δ−1–NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain

doi: 10.1111/jnc.14627

Figure Lengend Snippet: Inhibiting α2δ−1 with pregabalin reverses paclitaxel-induced tonic activation of presynaptic NMDARs in the spinal cord. ( A,B ) Representative recording traces and cumulative plots (A) and box-and-whisker plots (B) show the effect of bath application of 50 μM AP5 on the frequency and amplitude of mEPSCs of lamina II neurons from paclitaxel-treated rats. Neurons were treated with 20 μM pregabalin (+pregabalin, n = 10 neurons from 4 rats) or untreated (–pregabalin, n = 12 neurons from 4 rats). ( C,D ) Original recording traces and cumulative plots (C) and box-and-whisker plots (D) show the effect of bath application of AP5 on the frequency and amplitude of mEPSCs of lamina II neurons from vehicle-treated rats. Neurons were treated with 20 μM pregabalin (+pregabalin, n = 10 neurons from 4 rats) or untreated (–pregabalin, n = 12 neurons from 4 rats). *P

Article Snippet: The membrane was treated with 5% nonfat dry milk in Tris-buffered saline (TBS) at 25°C for 1 h and then incubated in TBS supplemented with 0.1% Triton X-100, 1% bovine serum albumin, and rabbit anti-α2δ−1 (#ACC-015, 1:500, Alomone Labs, Jerusalem, Israel), rabbit anti-GluN1 (#G8913, 1:1,000, Sigma-Aldrich), rabbit anti-GAPDH (#14C10, 1:5,000, Cell Signaling Technology, Danvers, MA), or mouse anti-PSD95 (#75–348, 1:1,000, NeuroMab, Davis, CA) antibodies overnight at 4°C.

Techniques: Activation Assay, Whisker Assay

Journal: Journal of neurochemistry

Article Title: Increased α2δ−1–NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain

doi: 10.1111/jnc.14627

Figure Lengend Snippet: Paclitaxel increases α2δ−1 expression levels and synaptic trafficking of α2δ−1–bound NMDARs in the spinal cord. ( A–C ) Original gel images (A and B, 2 pairs of samples) and quantification (C) of the α2δ−1 protein level in the DRG and dorsal spinal cord tissues of paclitaxel-treated (P) and vehicle-treated (V) rats (n = 9 rats per group). The amount of α2δ−1 proteins was normalized to that of GAPDH on the same blot. ( D–F ) Quantification of the mRNA level of α2δ−1, α2δ−2, α2δ−3, GluN1 (GluN1), GluN2A, and GluN2B in the DRG and dorsal spinal cord tissues of paclitaxel-treated (P) and vehicle-treated (V) rats (n = 6 rats per group). ( G,H ) co-IP analysis showing the interaction between α2δ−1 and GluN1 in the membrane extracts of dorsal spinal cord tissues from rats treated with paclitaxel (P) or vehicle (V) (n = 6 rats per group). Proteins were immunoprecipitated initially with a mouse anti-GluN1 or anti-IgG antibody. Immunoblotting was performed by using rabbit anti-α2δ−1 and anti-GluN1 antibodies. The amount of α2δ−1 proteins was normalized to that of GluN1 on the same blot. ( I,J ) Representative gel images (I, 3 pairs of samples) and quantification (J) of the protein levels of α2δ−1, GluN1, and PSD-95 (a synaptic marker) in synaptosomes isolated from dorsal spinal cord tissues of paclitaxel-treated (P) and vehicle-treated (V) rats (n = 8 rats per group). The amount of α2δ−1 and GluN1 proteins was normalized to that of PSD-95 on the same blot. Values in C-F, H, and J are normalized to expression levels in vehicle-treated rats. *P

Article Snippet: The membrane was treated with 5% nonfat dry milk in Tris-buffered saline (TBS) at 25°C for 1 h and then incubated in TBS supplemented with 0.1% Triton X-100, 1% bovine serum albumin, and rabbit anti-α2δ−1 (#ACC-015, 1:500, Alomone Labs, Jerusalem, Israel), rabbit anti-GluN1 (#G8913, 1:1,000, Sigma-Aldrich), rabbit anti-GAPDH (#14C10, 1:5,000, Cell Signaling Technology, Danvers, MA), or mouse anti-PSD95 (#75–348, 1:1,000, NeuroMab, Davis, CA) antibodies overnight at 4°C.

Techniques: Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, Marker, Isolation

Journal: Journal of neurochemistry

Article Title: Increased α2δ−1–NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain

doi: 10.1111/jnc.14627

Figure Lengend Snippet: Ablation of α2δ−1 prevents paclitaxel-induced tonic activation of presynaptic NMDARs in the spinal cord in mice. ( A–C ). Representative recording traces (A), cumulative plots (B), and box-and-whisker plots (C) show the effect of bath application of 50 μM AP5 on the frequency and amplitude of mEPSCs of lamina II neurons from wild-type (WT, n = 11 neurons from 5 mice) and α2δ−1 knockout (KO, n = 16 neurons from 5 mice) mice treated with paclitaxel. *P

Article Snippet: The membrane was treated with 5% nonfat dry milk in Tris-buffered saline (TBS) at 25°C for 1 h and then incubated in TBS supplemented with 0.1% Triton X-100, 1% bovine serum albumin, and rabbit anti-α2δ−1 (#ACC-015, 1:500, Alomone Labs, Jerusalem, Israel), rabbit anti-GluN1 (#G8913, 1:1,000, Sigma-Aldrich), rabbit anti-GAPDH (#14C10, 1:5,000, Cell Signaling Technology, Danvers, MA), or mouse anti-PSD95 (#75–348, 1:1,000, NeuroMab, Davis, CA) antibodies overnight at 4°C.

Techniques: Activation Assay, Mouse Assay, Whisker Assay, Knock-Out

Journal: Journal of neurochemistry

Article Title: Increased α2δ−1–NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain

doi: 10.1111/jnc.14627

Figure Lengend Snippet: Ablation of α2δ−1 abolishes paclitaxel-induced activation of presynaptic NMDARs at primary afferent terminals in mice. ( A,B ). Original recording traces (A) and box-and-whisker plots (B) shows the effect of bath application of 50 μM AP5 on evoked monosynaptic EPSCs of lamina II neurons from wild-type (WT, n = 13 neurons from 5 mice) and α2δ−1 knockout (KO, n = 11 neurons from 5 mice) mice treated with paclitaxel. In B (right panel), values are normalized to the respective baselines. ( C,D ). Original recording traces (C) and box-and-whisker plots (D) shows the effect of bath application of AP5 on the paired-pulse ratio (PPR) of lamina II neurons from WT (n = 13 neurons from 5 mice) and α2δ−1 KO (n = 10 neurons from 5 mice) mice treated with paclitaxel. *P

Article Snippet: The membrane was treated with 5% nonfat dry milk in Tris-buffered saline (TBS) at 25°C for 1 h and then incubated in TBS supplemented with 0.1% Triton X-100, 1% bovine serum albumin, and rabbit anti-α2δ−1 (#ACC-015, 1:500, Alomone Labs, Jerusalem, Israel), rabbit anti-GluN1 (#G8913, 1:1,000, Sigma-Aldrich), rabbit anti-GAPDH (#14C10, 1:5,000, Cell Signaling Technology, Danvers, MA), or mouse anti-PSD95 (#75–348, 1:1,000, NeuroMab, Davis, CA) antibodies overnight at 4°C.

Techniques: Activation Assay, Mouse Assay, Whisker Assay, Knock-Out

Journal: Journal of neurochemistry

Article Title: Increased α2δ−1–NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain

doi: 10.1111/jnc.14627

Figure Lengend Snippet: Disrupting the α2δ−1–NMDAR interaction using α2δ−1Tat peptide abrogates paclitaxel-induced activation of presynaptic NMDARs at primary afferent terminals. ( A,B ). Original recording traces (A) and box-and-whisker plots (B) show the effect of bath application of 50 μM AP5 on evoked monosynaptic EPSCs of lamina II neurons from paclitaxel-treated rats. Neurons were treated with 1 μM α2δ−1Tat peptide (n = 9 neurons from 4 rats) or 1 μM control peptide (n = 12 neurons from 4 rats). ( C,D ). Representative recording traces (C) and box-and-whisker plots (D) show the effect of bath application of 50 μM AP5 on evoked monosynaptic EPSCs of lamina II neurons from vehicle-treated rats. Neurons were treated with 1 μM α2δ−1Tat peptide (n = 10 neurons from 4 rats) or 1 μM control peptide (n = 9 neurons from 4 rats). In B and D (right panels), values are normalized to their respective baselines. ( E,F ). Original recording traces (E) and box-and-whisker plots (F) shows the effect of bath application of 50 μM AP5 on the paired-pulse ratio (PPR) of lamina II neurons treated with α2δ−1Tat peptide or control peptide from vehicle-treated (n = 10 neurons with α2δ−1Tat peptide from 4 rats; n = 9 neurons with control peptide from 4 rats) and paclitaxel-treated rats (n = 9 neurons with α2δ−1Tat peptide from 4 rats; n = 9 neurons with control peptide from 4 rats). *P

Article Snippet: The membrane was treated with 5% nonfat dry milk in Tris-buffered saline (TBS) at 25°C for 1 h and then incubated in TBS supplemented with 0.1% Triton X-100, 1% bovine serum albumin, and rabbit anti-α2δ−1 (#ACC-015, 1:500, Alomone Labs, Jerusalem, Israel), rabbit anti-GluN1 (#G8913, 1:1,000, Sigma-Aldrich), rabbit anti-GAPDH (#14C10, 1:5,000, Cell Signaling Technology, Danvers, MA), or mouse anti-PSD95 (#75–348, 1:1,000, NeuroMab, Davis, CA) antibodies overnight at 4°C.

Techniques: Activation Assay, Whisker Assay

Journal: Cell reports

Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

doi: 10.1016/j.celrep.2021.109396

Figure Lengend Snippet: α2δ-1 reduces surface and synaptic expression of GluA2 and heteromeric GluA1/GluA2 receptors (A) Original blots and quantification show the protein levels of GluA1 and GluA2 in HEK293 cells cotransfected with GluA1/GluA2 and control vector (pcDNA3, P3), α2δ-1, α2δ-2, or α2δ-3 (n = 4 per group). ***p

Article Snippet: Samples were immunoblotted after being washed three times with IP buffer. α2δ-1 was detected using rabbit anti-α2δ-1 antibody (#C5105, 1:1,000; Sigma-Aldrich), GluA1 was detected using mouse anti-GluA1 antibody (#75-327, 1:1,000; NeuroMab) or rabbit anti-GluA1 antibody (#ACC-015, 1:1,000; Alomone Labs), GluA2 was detected by using mouse anti-GluA2 antibody (#75-002, 1:1,000; NeuroMab) or rabbit anti-GluA2 antibody (#AGC-005, 1:1,000; Alomone Labs), and Flag Tag was detected using mouse anti-Flag antibody (#F1804, 1:1,000; Sigma-Aldrich) or rabbit anti-Flag antibody (#F7425, 1:1,000; Sigma-Aldrich).

Techniques: Expressing, Plasmid Preparation

Journal: Cell reports

Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

doi: 10.1016/j.celrep.2021.109396

Figure Lengend Snippet: α2δ-1 disrupts the heteromeric assembly of GluA1/GluA2 and increases GluA2 retention in the endoplasmic reticulum (A and B) Original blots (A) and quantification (B) show that α2δ-1 coexpression diminishes heteromeric GluA1/GluA2 receptors in the ER of HEK293 cells. n = 5 per group. *p

Article Snippet: Samples were immunoblotted after being washed three times with IP buffer. α2δ-1 was detected using rabbit anti-α2δ-1 antibody (#C5105, 1:1,000; Sigma-Aldrich), GluA1 was detected using mouse anti-GluA1 antibody (#75-327, 1:1,000; NeuroMab) or rabbit anti-GluA1 antibody (#ACC-015, 1:1,000; Alomone Labs), GluA2 was detected by using mouse anti-GluA2 antibody (#75-002, 1:1,000; NeuroMab) or rabbit anti-GluA2 antibody (#AGC-005, 1:1,000; Alomone Labs), and Flag Tag was detected using mouse anti-Flag antibody (#F1804, 1:1,000; Sigma-Aldrich) or rabbit anti-Flag antibody (#F7425, 1:1,000; Sigma-Aldrich).

Techniques:

Journal: Cell reports

Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

doi: 10.1016/j.celrep.2021.109396

Figure Lengend Snippet: α2δ-1 physically interacts with GluA1 and GluA2 in vitro and in vivo (A) CoIP shows the interaction between α2δ-1 and GluA1 and GluA2 in HEK293 cells. Cells cotransfected with GFP-tagged α2δ-1 and GluA1, GluA2, GluA1/GluA2, or FLAG-stargazin (STG). (B) CoIP shows the interaction of α2δ-1 with homomeric GluA1 or GluA2 in HEK293 cells. P3, control vector. (C) CoIP shows the interaction of α2δ-1 with heteromeric GluA1/GluA2 in HEK293 cells. (D) CoIP shows the interaction of α2δ-1 with GluA1 and GluA2 in the dorsal spinal cord of rats subjected to a sham procedure (S) or spinal nerve ligation (L). (E) CoIP shows the interaction of α2δ-1 with GluA1 and GluA2 subunits in the normal spinal cord tissue of two human donors (S1 and S2). (F) α2δ-1 interacts with GluA1 and GluA2 subunits via its C terminus. HEK293 cells were cotransfected with GluA1/GluA2 and various PC-tagged α2δ-1 constructs. δ-1ΔCT, δ-1 without the C terminus; CT, the C terminus of δ-1; VWA, von Willebrand factor type A domain. (G) CoIP shows that α2δ-1CT-Tat peptide disrupts the α2δ-1 interaction with GluA1 and GluA2 in HEK293 cells. Cell were cotransfected with GluA1/GluA2 and α2δ-1 or FLAG-α2δ-1 and were treated with 1 μM α2δ-1CT-Tat peptide (Pept) or 1 μM Tat-fused Cont peptide for 30 min. ***p

Article Snippet: Samples were immunoblotted after being washed three times with IP buffer. α2δ-1 was detected using rabbit anti-α2δ-1 antibody (#C5105, 1:1,000; Sigma-Aldrich), GluA1 was detected using mouse anti-GluA1 antibody (#75-327, 1:1,000; NeuroMab) or rabbit anti-GluA1 antibody (#ACC-015, 1:1,000; Alomone Labs), GluA2 was detected by using mouse anti-GluA2 antibody (#75-002, 1:1,000; NeuroMab) or rabbit anti-GluA2 antibody (#AGC-005, 1:1,000; Alomone Labs), and Flag Tag was detected using mouse anti-Flag antibody (#F1804, 1:1,000; Sigma-Aldrich) or rabbit anti-Flag antibody (#F7425, 1:1,000; Sigma-Aldrich).

Techniques: In Vitro, In Vivo, Co-Immunoprecipitation Assay, Plasmid Preparation, Ligation, Construct

Journal: Cell reports

Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

doi: 10.1016/j.celrep.2021.109396

Figure Lengend Snippet: Gabapentin and the α2δ-1CT-Tat peptide normalize synaptic expression of GluA2-containing AMPARs in the spinal cord diminished in neuropathic pain (A and B) Original blots and quantification show the effect of gabapentin and α2δ-1CT-Tat peptide on the protein levels of GluA1 and GluA2 in the spinal cord synaptosome (A) and the ER (B) of sham and SNL rats (n = 6 rats per group). Spinal cord slices were treated with vehicle (Cont), 100 μM GBP, 1 μM control peptide (P(−)), or 1 μM α2δ-1CT-Tat peptide (P(+)). **p

Article Snippet: Samples were immunoblotted after being washed three times with IP buffer. α2δ-1 was detected using rabbit anti-α2δ-1 antibody (#C5105, 1:1,000; Sigma-Aldrich), GluA1 was detected using mouse anti-GluA1 antibody (#75-327, 1:1,000; NeuroMab) or rabbit anti-GluA1 antibody (#ACC-015, 1:1,000; Alomone Labs), GluA2 was detected by using mouse anti-GluA2 antibody (#75-002, 1:1,000; NeuroMab) or rabbit anti-GluA2 antibody (#AGC-005, 1:1,000; Alomone Labs), and Flag Tag was detected using mouse anti-Flag antibody (#F1804, 1:1,000; Sigma-Aldrich) or rabbit anti-Flag antibody (#F7425, 1:1,000; Sigma-Aldrich).

Techniques: Expressing

Journal: Cell reports

Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

doi: 10.1016/j.celrep.2021.109396

Figure Lengend Snippet: Gabapentin and the α2δ-1CT-Tat C terminus peptide restore heteromeric GluA1/GluA2 receptors diminished by α2δ-1 coexpression (A and B) Original GCaMP images and signals show intracellular Ca 2+ changes in response to 5 mM glutamate (Glut) in HEK293 cells transfected with GluA1/GluA2 (A) or GluA1/GluA2/α2δ-1 (B). (C) Mean data show effects of treatment with vehicle (n = 54 cells), gabapentin (100 μM, n = 28 cells), α2δ-1CT-Tat peptide (1 μM, n = 26 cells), or control peptide (1 μM, n = 21 cells) on the ratio (ΔF/F0) of GCaMP signals elicited by glutamate. ***p

Article Snippet: Samples were immunoblotted after being washed three times with IP buffer. α2δ-1 was detected using rabbit anti-α2δ-1 antibody (#C5105, 1:1,000; Sigma-Aldrich), GluA1 was detected using mouse anti-GluA1 antibody (#75-327, 1:1,000; NeuroMab) or rabbit anti-GluA1 antibody (#ACC-015, 1:1,000; Alomone Labs), GluA2 was detected by using mouse anti-GluA2 antibody (#75-002, 1:1,000; NeuroMab) or rabbit anti-GluA2 antibody (#AGC-005, 1:1,000; Alomone Labs), and Flag Tag was detected using mouse anti-Flag antibody (#F1804, 1:1,000; Sigma-Aldrich) or rabbit anti-Flag antibody (#F7425, 1:1,000; Sigma-Aldrich).

Techniques: Transfection

Journal: Anesthesiology

Article Title: α2δ-1–Bound NMDA Receptors Mediate Morphine-induced Hyperalgesia and Analgesic Tolerance by Potentiating Glutamatergic Input in Rodents

doi: 10.1097/ALN.0000000000002648

Figure Lengend Snippet: Chronic morphine treatment increases α2δ-1 association with NMDARs at spinal cord synapses. A and B, Representative blots and quantification of α2δ-1 protein levels in the DRG (A) and dorsal spinal cord (B) from vehicle-treated (V) and morphine-treated (M) rats (n = 6 rats in each group). C, Coimmunoprecipitation analysis shows that GluN1 coprecipitated with α2δ-1 in the membrane extracts of dorsal spinal cord tissues of rats treated with vehicle or morphine for 8 days (n = 6 rats in each group). The amount of α2δ-1 proteins was normalized to that of GluN1 in the same sample, and the mean α2δ-1 level in vehicle-treated rats was considered to be 1. D, Representative gel images and quantification of GluN1 and α2δ-1 protein amounts in dorsal spinal cord synaptosomes from vehicle- and morphine-treated rats (n = 6 rats in each group). E, Coimmunoprecipitation analysis shows the effect of treatment with 1 μM α2δ-1Tat peptide and scrambled control peptide on the α2δ-1-GluN1 complex level in spinal cord slices from morphine-treated rats (n = 6 rats in each group). Data are shown as means ± SD. *P

Article Snippet: The blots were probed with a rabbit anti-α2δ-1 antibody (1:500; #ACC-015, Alomone Labs, Jerusalem, Israel) or rabbit anti-GAPDH antibody (1:5000; #14C10, Cell Signaling Technology, Danvers, MA).

Techniques:

Journal: Anesthesiology

Article Title: α2δ-1–Bound NMDA Receptors Mediate Morphine-induced Hyperalgesia and Analgesic Tolerance by Potentiating Glutamatergic Input in Rodents

doi: 10.1097/ALN.0000000000002648

Figure Lengend Snippet: α2δ-1 is essential for the chronic morphine exposure-induced activation of presynaptic NMDARs in the spinal dorsal horn. A, Representative recording trace and cumulative plots show the effect of bath application of 50 μM AP5 on the frequency and amplitude of mEPSCs of a lamina II neuron from a morphine-treated WT mouse. B, Summary data show the effect of 50 μM AP5 on the mean frequency and amplitude of mEPSCs (n = 11 neurons) in spinal cord slices from morphine-treated WT mice. C, Representative recording traces and cumulative plots show no effect from AP5 on the frequency or amplitude of mEPSCs of a lamina II neuron from a morphine-treated α2δ-1 KO mouse. D, Summary data show no effect of AP5 on the mean frequency or amplitude of mEPSCs (n = 10 neurons) in spinal cord slices from morphine-treated α2δ-1 KO mice. Data are shown as means ± SD. ***P

Article Snippet: The blots were probed with a rabbit anti-α2δ-1 antibody (1:500; #ACC-015, Alomone Labs, Jerusalem, Israel) or rabbit anti-GAPDH antibody (1:5000; #14C10, Cell Signaling Technology, Danvers, MA).

Techniques: Activation Assay, Mouse Assay

Journal: Anesthesiology

Article Title: α2δ-1–Bound NMDA Receptors Mediate Morphine-induced Hyperalgesia and Analgesic Tolerance by Potentiating Glutamatergic Input in Rodents

doi: 10.1097/ALN.0000000000002648

Figure Lengend Snippet: α2δ-1 is required for the chronic morphine exposure-induced increase in NMDAR activity at primary afferent terminals. A and B, Representative current traces show the effect of bath application of 50 μM AP5 on the amplitude of monosynaptic EPSCs (A) and the PPR (B) of a lamina II neuron from a morphine-treated WT mouse. C, Summary data show the effect of 50 μM AP5 on the mean amplitude ( n = 11 neurons) and PPR ( n = 11 neurons) of monosynaptic EPSCs of lamina II neurons from spinal cord slices of morphine-treated WT mice. C and D, Representative current traces show no effect of AP5 on the mean amplitude of evoked monosynaptic EPSCs (C) or PPR (D) of a lamina II neuron of a morphine-treated α2δ-1 KO mouse. E, Group data show the lack of effect of 50 μM AP5 on the amplitude (n = 11 neurons) and the PPR (n = 11 neurons) of monosynaptic EPSCs of lamina II neurons from spinal cord slices of morphine-treated α2δ-1 KO mice. Data are shown as means ± SD. **P

Article Snippet: The blots were probed with a rabbit anti-α2δ-1 antibody (1:500; #ACC-015, Alomone Labs, Jerusalem, Israel) or rabbit anti-GAPDH antibody (1:5000; #14C10, Cell Signaling Technology, Danvers, MA).

Techniques: Activity Assay, Mouse Assay

Journal: Anesthesiology

Article Title: α2δ-1–Bound NMDA Receptors Mediate Morphine-induced Hyperalgesia and Analgesic Tolerance by Potentiating Glutamatergic Input in Rodents

doi: 10.1097/ALN.0000000000002648

Figure Lengend Snippet: α2δ-1 is involved in chronic morphine exposure-induced potentiation of NMDAR activity at primary afferent terminals in the spinal dorsal horn. A and B, Representative recording traces show the effect of bath application of 50 μM AP5 on evoked monosynaptic EPSCs (A) and the paired-pulse ratio (PPR, B) of a vehicle-incubated lamina II neuron from a morphine-treated rat. C, Summary data show the effect of 50 μM AP5 on the amplitude (n = 10 neurons) and PPR (n = 10 neurons) of evoked monosynaptic EPSCs of vehicle-incubated lamina II neurons in morphine-treated rats. D and E, Representative recording traces show no effect from bath application of 50 μM AP5 on the amplitude of monosynaptically evoked EPSCs (D) or the PPR (E) of a lamina II neuron in spinal cord slices pretreated with 100 μM gabapentin in a morphine-treated rat. F, Summary data show no effect from 50 μM AP5 on the mean amplitude (n = 11 neurons) or PPR (n = 11 neurons) of monosynaptic EPSCs of lamina II neurons pretreated with 100 μM gabapentin from morphine-treated rats. Data are shown as means ± SD. *P

Article Snippet: The blots were probed with a rabbit anti-α2δ-1 antibody (1:500; #ACC-015, Alomone Labs, Jerusalem, Israel) or rabbit anti-GAPDH antibody (1:5000; #14C10, Cell Signaling Technology, Danvers, MA).

Techniques: Activity Assay, Incubation

Journal: Anesthesiology

Article Title: α2δ-1–Bound NMDA Receptors Mediate Morphine-induced Hyperalgesia and Analgesic Tolerance by Potentiating Glutamatergic Input in Rodents

doi: 10.1097/ALN.0000000000002648

Figure Lengend Snippet: α2δ-1 mediates chronic morphine exposure-induced potentiation of presynaptic NMDAR activity in the spinal dorsal horn. A, Representative recording traces and cumulative plots show the effect of bath application of 50 μM AP5 on the frequency and amplitude of mEPSCs of a lamina II neuron from a morphine-treated rat. B, Summary data show the effect of 50 μM AP5 on the mean frequency and amplitude of mEPSCs of lamina II neurons (n = 11 neurons) from morphine-treated rats. C, Representative recording traces and cumulative plots show that bath application of 50 μM AP5 had no effect on the frequency or amplitude of mEPSCs of a lamina II neuron pretreated with 100 μM gabapentin from a morphine-treated rat. D, Summary data show no effect from 50 μM AP5 on the mean frequency or amplitude of mEPSCs of lamina II neurons (n = 10 neurons) pretreated with 100 μM gabapentin from morphine-treated rats. Data are shown as means ± SD. **P

Article Snippet: The blots were probed with a rabbit anti-α2δ-1 antibody (1:500; #ACC-015, Alomone Labs, Jerusalem, Israel) or rabbit anti-GAPDH antibody (1:5000; #14C10, Cell Signaling Technology, Danvers, MA).

Techniques: Activity Assay

Journal: Anesthesiology

Article Title: α2δ-1–Bound NMDA Receptors Mediate Morphine-induced Hyperalgesia and Analgesic Tolerance by Potentiating Glutamatergic Input in Rodents

doi: 10.1097/ALN.0000000000002648

Figure Lengend Snippet: α2δ-1–bound NMDARs are critically involved in chronic morphine exposure-induced activation of NMDARs at primary afferent terminals. A and B, Representative current traces show the effect of bath application of 50 μM AP5 on the amplitude of monosynaptic EPSCs (A) and the PPR (B) of a lamina II neuron from a spinal cord slice pretreated with control peptide (1 μM) from a morphine-treated rat. C, Summary data show the effect of 50 μM AP5 on the mean amplitude ( n = 11 neurons) and PPR ( n = 11 neurons) of monosynaptic EPSCs of lamina II neurons from spinal cord slices pretreated with control peptide from morphine-treated rats. C and D, Representative current traces show no effect of AP5 on the amplitude of evoked monosynaptic EPSCs (C) or PPR (D) of a lamina II neuron from a spinal cord slice pretreated with α2δ-1Tat peptide (1 μM) from a morphine-treated rat. E, Summary data show no effect of AP5 on the mean amplitude (n = 11 neurons) or PPR (n = 11 neurons) of monosynaptic EPSCs of lamina II neurons from spinal cord slices pretreated with α2δ-1Tat peptide from morphine-treated rats. Data are shown as means ± SD. **P

Article Snippet: The blots were probed with a rabbit anti-α2δ-1 antibody (1:500; #ACC-015, Alomone Labs, Jerusalem, Israel) or rabbit anti-GAPDH antibody (1:5000; #14C10, Cell Signaling Technology, Danvers, MA).

Techniques: Activation Assay

Journal: Anesthesiology

Article Title: α2δ-1–Bound NMDA Receptors Mediate Morphine-induced Hyperalgesia and Analgesic Tolerance by Potentiating Glutamatergic Input in Rodents

doi: 10.1097/ALN.0000000000002648

Figure Lengend Snippet: α2δ-1 at the spinal cord level mediates chronic morphine exposure-induced hyperalgesia and analgesic tolerance. A and B, Time course of changes in the baseline mechanical (A) and thermal (B) withdrawal thresholds and the analgesic effect of morphine in rats treated with systemic morphine plus vehicle (n = 8 rats) or gabapentin (100 mg/kg, n = 8 rats). C and D, Time course of changes in the baseline mechanical (C) and thermal (D) withdrawal thresholds and the analgesic effect of morphine in rats treated with systemic morphine plus control peptide (1 μg) or α2δ-1Tat peptide (1 μg) (n = 10 rats in each group). E and F, Time course of changes in the baseline mechanical (E) and thermal (F) withdrawal thresholds and the analgesic effect of morphine in WT and α2δ-1 KO mice (n = 8 mice per group). The baseline withdrawal threshold was measured before each morphine injection, and the analgesic effect of morphine was tested 30 min after morphine injection. *P

Article Snippet: The blots were probed with a rabbit anti-α2δ-1 antibody (1:500; #ACC-015, Alomone Labs, Jerusalem, Israel) or rabbit anti-GAPDH antibody (1:5000; #14C10, Cell Signaling Technology, Danvers, MA).

Techniques: Mouse Assay, Injection

Journal: Anesthesiology

Article Title: α2δ-1–Bound NMDA Receptors Mediate Morphine-induced Hyperalgesia and Analgesic Tolerance by Potentiating Glutamatergic Input in Rodents

doi: 10.1097/ALN.0000000000002648

Figure Lengend Snippet: α2δ-1–bound NMDARs mediate the chronic morphine exposure-induced increase in presynaptic NMDAR activity in the spinal cord. A, Representative recording traces and cumulative plots show the effect of bath application of 50 μM AP5 on the frequency and amplitude of mEPSCs of a lamina II neuron pretreated with control peptide (1 μM) from a spinal cord slice of a morphine-treated rat. B, Summary data show the effect of 50 μM AP5 on the mean frequency and amplitude of mEPSCs (n = 10 neurons) in spinal cord slices pretreated with control peptide from morphine-treated rats. C, Representative recording traces and cumulative plots show no effect of AP5 on the frequency or amplitude of mEPSCs of a lamina II neuron pretreated with α2δ-1Tat peptide (1 μM) from a spinal cord slice of a morphine-treated rat. D, Summary data show no effect of AP5 on the mean frequency or amplitude of mEPSCs (n = 11 neurons) in spinal cord slices pretreated with α2δ-1Tat peptide from morphine-treated rats. Data are shown as means ± SD. ***P

Article Snippet: The blots were probed with a rabbit anti-α2δ-1 antibody (1:500; #ACC-015, Alomone Labs, Jerusalem, Israel) or rabbit anti-GAPDH antibody (1:5000; #14C10, Cell Signaling Technology, Danvers, MA).

Techniques: Activity Assay