rabbit anti α2δ 1  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti α2δ 1
    <t>α2δ−1</t> 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
    Rabbit Anti α2δ 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti α2δ 1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti α2δ 1 - by Bioz Stars, 2022-01
    94/100 stars

    Images

    1) Product Images from "Increased α2δ−1–NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain"

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

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14627

    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
    Figure Legend 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

    Techniques Used: Injection

    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.
    Figure Legend 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.

    Techniques Used: Knock-Out, Mouse Assay

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Whisker Assay

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Whisker Assay

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Whisker Assay

    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
    Figure Legend 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

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

    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
    Figure Legend 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

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

    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
    Figure Legend 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

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

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Whisker Assay

    2) Product Images from "Increased α2δ−1–NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain"

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

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14627

    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
    Figure Legend 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

    Techniques Used: Injection

    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.
    Figure Legend 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.

    Techniques Used: Knock-Out, Mouse Assay

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Whisker Assay

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Whisker Assay

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Whisker Assay

    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
    Figure Legend 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

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

    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
    Figure Legend 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

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

    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
    Figure Legend 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

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

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Whisker Assay

    3) Product Images from "α2δ-1–Bound NMDA Receptors Mediate Morphine-induced Hyperalgesia and Analgesic Tolerance by Potentiating Glutamatergic Input in Rodents"

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

    Journal: Anesthesiology

    doi: 10.1097/ALN.0000000000002648

    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
    Figure Legend 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

    Techniques Used:

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Mouse Assay

    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
    Figure Legend 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

    Techniques Used: Activity Assay, Mouse Assay

    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
    Figure Legend 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

    Techniques Used: Activity Assay, Incubation

    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
    Figure Legend 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

    Techniques Used: Activity Assay

    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
    Figure Legend 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

    Techniques Used: Activation Assay

    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
    Figure Legend 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

    Techniques Used: Mouse Assay, Injection

    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
    Figure Legend 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

    Techniques Used: Activity Assay

    4) Product Images from "Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity"

    Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1532-17.2018

    Subretinal hUTC transplantation preserves α2δ-1-containing synapses in the IPL of RCS rats. A , Representative images of the IPL labeled for VGluT1 (green) and PSD95 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. B , Quantification of excitatory synapses in the IPL revealed that synapse numbers did not differ between RCS+BSS and RCS+hUTC P21 and P60. C , Representative images of the IPL labeled for Bassoon (green) and α2δ-1 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. D , The α2δ-1-containing synapses were specifically preserved with hUTC transplantation. E , Representative images of the IPL stained for Bassoon (green) and Gephyrin (blue) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. F , hUTC transplantation did not preserve inhibitory synapses. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. * p
    Figure Legend Snippet: Subretinal hUTC transplantation preserves α2δ-1-containing synapses in the IPL of RCS rats. A , Representative images of the IPL labeled for VGluT1 (green) and PSD95 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. B , Quantification of excitatory synapses in the IPL revealed that synapse numbers did not differ between RCS+BSS and RCS+hUTC P21 and P60. C , Representative images of the IPL labeled for Bassoon (green) and α2δ-1 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. D , The α2δ-1-containing synapses were specifically preserved with hUTC transplantation. E , Representative images of the IPL stained for Bassoon (green) and Gephyrin (blue) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. F , hUTC transplantation did not preserve inhibitory synapses. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. * p

    Techniques Used: Transplantation Assay, Labeling, Staining

    KO of MERTK in MG results in impaired synapse development in vivo . A , Representative images of the OPL synapses labeled by Bassoon (green) and mGluR6 (red) from Control (CTR, right eyeballs) and MERTK KO (KO, left eyeballs) at P21 and P45. B , Quantification of synapses in the OPL revealed that synapse development is impaired in MG-specific MERTK-KO at both P21 and P45. C , IPL excitatory synapses are visualized by VGluT1 (green) and PSD95 (red). D , Quantification of synapse number demonstrates that early synaptic development at P21 is significantly affected by MERTK KO but not at P45. Representative images of the ( E ) OPL and ( G ) IPL synapses labeled for Bassoon (green) and α2δ-1 (red). Quantification of the number of α2δ-1-containing synapses in both ( F ) OPL and ( H ) IPL revealed that KO of MERTK in MG induces severely reduced number of α2δ-1-containing synapses at P21 and P45. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. * p
    Figure Legend Snippet: KO of MERTK in MG results in impaired synapse development in vivo . A , Representative images of the OPL synapses labeled by Bassoon (green) and mGluR6 (red) from Control (CTR, right eyeballs) and MERTK KO (KO, left eyeballs) at P21 and P45. B , Quantification of synapses in the OPL revealed that synapse development is impaired in MG-specific MERTK-KO at both P21 and P45. C , IPL excitatory synapses are visualized by VGluT1 (green) and PSD95 (red). D , Quantification of synapse number demonstrates that early synaptic development at P21 is significantly affected by MERTK KO but not at P45. Representative images of the ( E ) OPL and ( G ) IPL synapses labeled for Bassoon (green) and α2δ-1 (red). Quantification of the number of α2δ-1-containing synapses in both ( F ) OPL and ( H ) IPL revealed that KO of MERTK in MG induces severely reduced number of α2δ-1-containing synapses at P21 and P45. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. * p

    Techniques Used: In Vivo, Labeling

    TSP-receptor α2δ-1 is synaptically expressed in the retina, and its expression is reduced in RCS rats. A , Representative images of the retina stained for α2δ-1 from LE (healthy) and RCS (degenerative) retinas at P14 and ( B ) P30. C , Quantitative staining intensity analysis demonstrated that α2δ-1 expression was reduced in RCS rat as early as P14. D , α2δ-1 was enriched in both the OPL and IPL, and the expression gap became more distinct at P30. Representative images of the ( E ) OPL and ( F and G ) IPL with the synapses labeled for Bassoon ( F , green), VGluT1 ( G , green), α2δ-1 (red), and NR1 (blue) from LE retina on P21 demonstrated postsynaptic expression of α2δ-1. Representative images of the ( H ) OPL and ( J ) IPL synapses labeled for Bassoon (green) and α2δ-1 (red) from LE (healthy) and RCS (degenerative) retinas on P21. Quantification of α2δ-1-containing synapses formed in the ( I ) OPL and ( K ) IPL reveals that the number of α2δ-1 synapse is already reduced in RCS rat by P21. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *** p
    Figure Legend Snippet: TSP-receptor α2δ-1 is synaptically expressed in the retina, and its expression is reduced in RCS rats. A , Representative images of the retina stained for α2δ-1 from LE (healthy) and RCS (degenerative) retinas at P14 and ( B ) P30. C , Quantitative staining intensity analysis demonstrated that α2δ-1 expression was reduced in RCS rat as early as P14. D , α2δ-1 was enriched in both the OPL and IPL, and the expression gap became more distinct at P30. Representative images of the ( E ) OPL and ( F and G ) IPL with the synapses labeled for Bassoon ( F , green), VGluT1 ( G , green), α2δ-1 (red), and NR1 (blue) from LE retina on P21 demonstrated postsynaptic expression of α2δ-1. Representative images of the ( H ) OPL and ( J ) IPL synapses labeled for Bassoon (green) and α2δ-1 (red) from LE (healthy) and RCS (degenerative) retinas on P21. Quantification of α2δ-1-containing synapses formed in the ( I ) OPL and ( K ) IPL reveals that the number of α2δ-1 synapse is already reduced in RCS rat by P21. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *** p

    Techniques Used: Expressing, Staining, Labeling

    Subretinal hUTC transplantation preserves OPL synapses in RCS rats. Representative images of the OPL with the PR ribbon synapses labeled for ( A ) Bassoon (green) and mGluR6 (red) and ( B ) Bassoon (green) and α2δ-1 (red) from LE (control), RCS + BSS, and RCS + hUTC P21 and P60 retinas on P95. Quantification of the number of synapses in the OPL revealed that hUTC transplantation protected ( C ) ribbon synapses. D , Particularly, α2δ-1-containing synapses were specifically preserved following hUTC treatment. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *** p
    Figure Legend Snippet: Subretinal hUTC transplantation preserves OPL synapses in RCS rats. Representative images of the OPL with the PR ribbon synapses labeled for ( A ) Bassoon (green) and mGluR6 (red) and ( B ) Bassoon (green) and α2δ-1 (red) from LE (control), RCS + BSS, and RCS + hUTC P21 and P60 retinas on P95. Quantification of the number of synapses in the OPL revealed that hUTC transplantation protected ( C ) ribbon synapses. D , Particularly, α2δ-1-containing synapses were specifically preserved following hUTC treatment. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *** p

    Techniques Used: Transplantation Assay, Labeling

    5) Product Images from "Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity"

    Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1532-17.2018

    Subretinal hUTC transplantation preserves α2δ-1-containing synapses in the IPL of RCS rats. A , Representative images of the IPL labeled for VGluT1 (green) and PSD95 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. B , Quantification of excitatory synapses in the IPL revealed that synapse numbers did not differ between RCS+BSS and RCS+hUTC P21 and P60. C , Representative images of the IPL labeled for Bassoon (green) and α2δ-1 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. D , The α2δ-1-containing synapses were specifically preserved with hUTC transplantation. E , Representative images of the IPL stained for Bassoon (green) and Gephyrin (blue) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. F , hUTC transplantation did not preserve inhibitory synapses. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. * p
    Figure Legend Snippet: Subretinal hUTC transplantation preserves α2δ-1-containing synapses in the IPL of RCS rats. A , Representative images of the IPL labeled for VGluT1 (green) and PSD95 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. B , Quantification of excitatory synapses in the IPL revealed that synapse numbers did not differ between RCS+BSS and RCS+hUTC P21 and P60. C , Representative images of the IPL labeled for Bassoon (green) and α2δ-1 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. D , The α2δ-1-containing synapses were specifically preserved with hUTC transplantation. E , Representative images of the IPL stained for Bassoon (green) and Gephyrin (blue) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. F , hUTC transplantation did not preserve inhibitory synapses. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. * p

    Techniques Used: Transplantation Assay, Labeling, Staining

    KO of MERTK in MG results in impaired synapse development in vivo . A , Representative images of the OPL synapses labeled by Bassoon (green) and mGluR6 (red) from Control (CTR, right eyeballs) and MERTK KO (KO, left eyeballs) at P21 and P45. B , Quantification of synapses in the OPL revealed that synapse development is impaired in MG-specific MERTK-KO at both P21 and P45. C , IPL excitatory synapses are visualized by VGluT1 (green) and PSD95 (red). D , Quantification of synapse number demonstrates that early synaptic development at P21 is significantly affected by MERTK KO but not at P45. Representative images of the ( E ) OPL and ( G ) IPL synapses labeled for Bassoon (green) and α2δ-1 (red). Quantification of the number of α2δ-1-containing synapses in both ( F ) OPL and ( H ) IPL revealed that KO of MERTK in MG induces severely reduced number of α2δ-1-containing synapses at P21 and P45. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. * p
    Figure Legend Snippet: KO of MERTK in MG results in impaired synapse development in vivo . A , Representative images of the OPL synapses labeled by Bassoon (green) and mGluR6 (red) from Control (CTR, right eyeballs) and MERTK KO (KO, left eyeballs) at P21 and P45. B , Quantification of synapses in the OPL revealed that synapse development is impaired in MG-specific MERTK-KO at both P21 and P45. C , IPL excitatory synapses are visualized by VGluT1 (green) and PSD95 (red). D , Quantification of synapse number demonstrates that early synaptic development at P21 is significantly affected by MERTK KO but not at P45. Representative images of the ( E ) OPL and ( G ) IPL synapses labeled for Bassoon (green) and α2δ-1 (red). Quantification of the number of α2δ-1-containing synapses in both ( F ) OPL and ( H ) IPL revealed that KO of MERTK in MG induces severely reduced number of α2δ-1-containing synapses at P21 and P45. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. * p

    Techniques Used: In Vivo, Labeling

    TSP-receptor α2δ-1 is synaptically expressed in the retina, and its expression is reduced in RCS rats. A , Representative images of the retina stained for α2δ-1 from LE (healthy) and RCS (degenerative) retinas at P14 and ( B ) P30. C , Quantitative staining intensity analysis demonstrated that α2δ-1 expression was reduced in RCS rat as early as P14. D , α2δ-1 was enriched in both the OPL and IPL, and the expression gap became more distinct at P30. Representative images of the ( E ) OPL and ( F and G ) IPL with the synapses labeled for Bassoon ( F , green), VGluT1 ( G , green), α2δ-1 (red), and NR1 (blue) from LE retina on P21 demonstrated postsynaptic expression of α2δ-1. Representative images of the ( H ) OPL and ( J ) IPL synapses labeled for Bassoon (green) and α2δ-1 (red) from LE (healthy) and RCS (degenerative) retinas on P21. Quantification of α2δ-1-containing synapses formed in the ( I ) OPL and ( K ) IPL reveals that the number of α2δ-1 synapse is already reduced in RCS rat by P21. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *** p
    Figure Legend Snippet: TSP-receptor α2δ-1 is synaptically expressed in the retina, and its expression is reduced in RCS rats. A , Representative images of the retina stained for α2δ-1 from LE (healthy) and RCS (degenerative) retinas at P14 and ( B ) P30. C , Quantitative staining intensity analysis demonstrated that α2δ-1 expression was reduced in RCS rat as early as P14. D , α2δ-1 was enriched in both the OPL and IPL, and the expression gap became more distinct at P30. Representative images of the ( E ) OPL and ( F and G ) IPL with the synapses labeled for Bassoon ( F , green), VGluT1 ( G , green), α2δ-1 (red), and NR1 (blue) from LE retina on P21 demonstrated postsynaptic expression of α2δ-1. Representative images of the ( H ) OPL and ( J ) IPL synapses labeled for Bassoon (green) and α2δ-1 (red) from LE (healthy) and RCS (degenerative) retinas on P21. Quantification of α2δ-1-containing synapses formed in the ( I ) OPL and ( K ) IPL reveals that the number of α2δ-1 synapse is already reduced in RCS rat by P21. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *** p

    Techniques Used: Expressing, Staining, Labeling

    Subretinal hUTC transplantation preserves OPL synapses in RCS rats. Representative images of the OPL with the PR ribbon synapses labeled for ( A ) Bassoon (green) and mGluR6 (red) and ( B ) Bassoon (green) and α2δ-1 (red) from LE (control), RCS + BSS, and RCS + hUTC P21 and P60 retinas on P95. Quantification of the number of synapses in the OPL revealed that hUTC transplantation protected ( C ) ribbon synapses. D , Particularly, α2δ-1-containing synapses were specifically preserved following hUTC treatment. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *** p
    Figure Legend Snippet: Subretinal hUTC transplantation preserves OPL synapses in RCS rats. Representative images of the OPL with the PR ribbon synapses labeled for ( A ) Bassoon (green) and mGluR6 (red) and ( B ) Bassoon (green) and α2δ-1 (red) from LE (control), RCS + BSS, and RCS + hUTC P21 and P60 retinas on P95. Quantification of the number of synapses in the OPL revealed that hUTC transplantation protected ( C ) ribbon synapses. D , Particularly, α2δ-1-containing synapses were specifically preserved following hUTC treatment. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *** p

    Techniques Used: Transplantation Assay, Labeling

    6) Product Images from "α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels"

    Article Title: α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0511-18.2018

    Nrxn1α does not engage in stable complexes with α2δ subunits. A , IP of cotransfected α2δ subunits and Nrxn1α or control membrane proteins from HEK293 cell lysates (top). IPs of HA-tagged α2δ-1 and α2δ-3 enrich Nrxn1α::GFP (lanes 4, 5) similar to the controls neuroligin-1 (Nlgn1), E-cadherin (E-Cad) and VE-cadherin (VE-Cad; lanes 6–8). Single transfections served as control for antibody specificity (lanes 1–3). Endogenous HSP70 indicates equal amounts of lysates used (botto). B , Co-secretion of extracellular domains of α2δ-3 (α2δ-3ECD::HA) and Nrxn1α (Nrxn1α::Fc) into HEK293 cell medium with subsequent binding of the Fc moiety to protein A beads. Lysates of cells show α2δ-3ECD::HA (lanes 1–2). Whereas the positive control, Nxph1-HA, is hardly detectable in cell lysates (lane 3, bottom), it is enriched with Nrxn1α::Fc (lane 6, bottom). α2δ-3ECD::HA is enriched similarly with Nrxn1α::Fc (lane 5, top) but also with the Fc-tag alone. C , Diagram of the cleavage experiment using HRV 3C protease to release the Nrxn1αECD from Fc-beads (immunoblot data in D ). Left, Nxph1 (magenta) is bound to Nrxn1αECD (green) as expected. Right, α2δ-3ECD (cyan) remains on Fc-coupled beads (orange) but does not interact with Nrxn1αECD. D , Immunoblot of the cleavage experiment ( C ) that starts from the precipitated samples in B . After addition of protease, α2δ-3ECD remains on Fc-tag bound to beads (lanes 7, 8) but is not found on Nrxn1αECD in the supernatant (lane 11). The positive control, Nxph1, is bound to the released Nrxn1αECD (lane 12). α2δ-3 and Nph1 are shown by immunoblot, Nrxn1α and Fc proteins are visualized by UV light.
    Figure Legend Snippet: Nrxn1α does not engage in stable complexes with α2δ subunits. A , IP of cotransfected α2δ subunits and Nrxn1α or control membrane proteins from HEK293 cell lysates (top). IPs of HA-tagged α2δ-1 and α2δ-3 enrich Nrxn1α::GFP (lanes 4, 5) similar to the controls neuroligin-1 (Nlgn1), E-cadherin (E-Cad) and VE-cadherin (VE-Cad; lanes 6–8). Single transfections served as control for antibody specificity (lanes 1–3). Endogenous HSP70 indicates equal amounts of lysates used (botto). B , Co-secretion of extracellular domains of α2δ-3 (α2δ-3ECD::HA) and Nrxn1α (Nrxn1α::Fc) into HEK293 cell medium with subsequent binding of the Fc moiety to protein A beads. Lysates of cells show α2δ-3ECD::HA (lanes 1–2). Whereas the positive control, Nxph1-HA, is hardly detectable in cell lysates (lane 3, bottom), it is enriched with Nrxn1α::Fc (lane 6, bottom). α2δ-3ECD::HA is enriched similarly with Nrxn1α::Fc (lane 5, top) but also with the Fc-tag alone. C , Diagram of the cleavage experiment using HRV 3C protease to release the Nrxn1αECD from Fc-beads (immunoblot data in D ). Left, Nxph1 (magenta) is bound to Nrxn1αECD (green) as expected. Right, α2δ-3ECD (cyan) remains on Fc-coupled beads (orange) but does not interact with Nrxn1αECD. D , Immunoblot of the cleavage experiment ( C ) that starts from the precipitated samples in B . After addition of protease, α2δ-3ECD remains on Fc-tag bound to beads (lanes 7, 8) but is not found on Nrxn1αECD in the supernatant (lane 11). The positive control, Nxph1, is bound to the released Nrxn1αECD (lane 12). α2δ-3 and Nph1 are shown by immunoblot, Nrxn1α and Fc proteins are visualized by UV light.

    Techniques Used: Transfection, Binding Assay, Positive Control

    Nrxn1α in combination with α2δ-1 facilitates Ca 2+ currents through recombinant Ca V 2.1 channels. A , Representative Ca V 2.1-mediated Ca 2+ current traces recorded from heterologous tsA201 cells expressing α1 A , β3 and α2δ-1 subunits alone (black) or together with Nrxn1α (red). Step potentials as shown (right) were used to elicit Ca 2+ currents. B , I–V relationships of Ca V 2.1/β3/α2δ-1 alone (black) or in combination with Nrxn1α (red). C , Similar analysis as in B but using α2δ-3; trace in combination with Nrxn1α in blue. D , Summary of maximum current densities for cells expressing Ca V 2.1(α1 A /β3) without an α2δ (black bars), with α2δ-1 (red) or with α2δ-3 (blue), and additionally with Nrxn1α or SynCAM1 (SCAM) as indicated below bars. Data are mean ± SEM. n = number of cells as indicated in bars from at least four independent experiments. *** p
    Figure Legend Snippet: Nrxn1α in combination with α2δ-1 facilitates Ca 2+ currents through recombinant Ca V 2.1 channels. A , Representative Ca V 2.1-mediated Ca 2+ current traces recorded from heterologous tsA201 cells expressing α1 A , β3 and α2δ-1 subunits alone (black) or together with Nrxn1α (red). Step potentials as shown (right) were used to elicit Ca 2+ currents. B , I–V relationships of Ca V 2.1/β3/α2δ-1 alone (black) or in combination with Nrxn1α (red). C , Similar analysis as in B but using α2δ-3; trace in combination with Nrxn1α in blue. D , Summary of maximum current densities for cells expressing Ca V 2.1(α1 A /β3) without an α2δ (black bars), with α2δ-1 (red) or with α2δ-3 (blue), and additionally with Nrxn1α or SynCAM1 (SCAM) as indicated below bars. Data are mean ± SEM. n = number of cells as indicated in bars from at least four independent experiments. *** p

    Techniques Used: Recombinant, Expressing

    Biophysical properties of recombinant Ca V 2.1 are not altered by Nrxn1α. A , Voltage dependence of steady-state inactivation of Ca V 2.1 channels tested by a pre-pulse protocol in tsA201 cells expressing α1 A , β3, and α2δ-1 subunits alone (black) or together with Nrxn1α (red). B , Analysis as in A expressing α1 A , β3, and α2δ-3 subunits alone (black) or together with Nrxn1α (blue). C , Tail current amplitude at −40 mV after a 10 ms voltage step to the given pre-potential, recorded in tsA201 cells expressing Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red). D , Slope factor of the voltage dependence of the channel activation of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.204, by unpaired t test, t (21) = 1.32. E , Half-activation voltage of the voltage dependence of activation of Ca V 2.1/α2δ-1 tail current (as given in C ) without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.812, by unpaired t test, t (21) = 0.24. F , I–V curves of tail currents of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red). G , Analysis of tail current deactivation time constant at −20 mV of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.199 by unpaired t test, t (17) = 1.34. Data are mean ± SEM. N = number of cells as shown in bars or in brackets from at least four independent experiments.
    Figure Legend Snippet: Biophysical properties of recombinant Ca V 2.1 are not altered by Nrxn1α. A , Voltage dependence of steady-state inactivation of Ca V 2.1 channels tested by a pre-pulse protocol in tsA201 cells expressing α1 A , β3, and α2δ-1 subunits alone (black) or together with Nrxn1α (red). B , Analysis as in A expressing α1 A , β3, and α2δ-3 subunits alone (black) or together with Nrxn1α (blue). C , Tail current amplitude at −40 mV after a 10 ms voltage step to the given pre-potential, recorded in tsA201 cells expressing Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red). D , Slope factor of the voltage dependence of the channel activation of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.204, by unpaired t test, t (21) = 1.32. E , Half-activation voltage of the voltage dependence of activation of Ca V 2.1/α2δ-1 tail current (as given in C ) without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.812, by unpaired t test, t (21) = 0.24. F , I–V curves of tail currents of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red). G , Analysis of tail current deactivation time constant at −20 mV of Ca V 2.1/α2δ-1 without (black) or with Nrxn1α (red); n.s. = not significant, p = 0.199 by unpaired t test, t (17) = 1.34. Data are mean ± SEM. N = number of cells as shown in bars or in brackets from at least four independent experiments.

    Techniques Used: Recombinant, Expressing, Mass Spectrometry, Activation Assay

    α2δ-1 auxiliary subunits together with Nrxn1α facilitate presynaptic Ca 2+ influx in TKO neurons. A , Traces of Ca 2+ fluorescence changes determined from TKO neurons cotransfected with Nrxn1α and α2δ-1 subunits. Ca 2+ transients indicated by synGCaMP6f are averaged across multiple boutons in response to 1 (red), 3 (yellow), and 10 (black) APs (arrow: start of stimulation train). Inset, Initial response to a single AP on an enlarged time scale. B , Fluorescence changes of boutons as in A from TKO neurons expressing α2δ-3 subunits together with Nrxn1α. C , Summary of mean peak synGCaMP6f signals (Δ F / F o ) of Ca 2+ transients after single AP stimulation of neurons transfected with different proteins. Data are mean ± SEM. n = ROIs/neurons (in bars), differences to WT and TKO are indicated (dotted lines); significance is given compared with WT above columns (black) and compared with TKO (blue; above dashed line). *** p
    Figure Legend Snippet: α2δ-1 auxiliary subunits together with Nrxn1α facilitate presynaptic Ca 2+ influx in TKO neurons. A , Traces of Ca 2+ fluorescence changes determined from TKO neurons cotransfected with Nrxn1α and α2δ-1 subunits. Ca 2+ transients indicated by synGCaMP6f are averaged across multiple boutons in response to 1 (red), 3 (yellow), and 10 (black) APs (arrow: start of stimulation train). Inset, Initial response to a single AP on an enlarged time scale. B , Fluorescence changes of boutons as in A from TKO neurons expressing α2δ-3 subunits together with Nrxn1α. C , Summary of mean peak synGCaMP6f signals (Δ F / F o ) of Ca 2+ transients after single AP stimulation of neurons transfected with different proteins. Data are mean ± SEM. n = ROIs/neurons (in bars), differences to WT and TKO are indicated (dotted lines); significance is given compared with WT above columns (black) and compared with TKO (blue; above dashed line). *** p

    Techniques Used: Fluorescence, Expressing, Transfection

    αNrxn modulates surface mobility of α2δ-1 and α2δ-3 auxiliary subunits differentially. A , Representative immunofluorescent images of surface α2δ-1 enriched in synaptic boutons, visualized by an antibody against the HA moiety of α2δ-1::HA cotransfected with synGCaMP6f into WT neurons (top) or TKO neurons (bottom). Scale bar, 5 μm. B , Quantification of colocalization between synGCaMP6f and surface α2δ-1-positive puncta in WT and TKO. Data are mean ± SEM; n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.433) by unpaired t test. C , Labeling of the surface population of HA-tagged α2δ-1 ( C 1 ) transfected into WT neurons using an antibody specific to the HA moiety. EGFP was cotransfected to visualize neurites ( C 2 ), merged images ( C 3 ) and an overlay of all trajectories of QD-tracked single α2δ-1 molecules in a subfield as indicated ( C 4 ); sample trajectories of QD-tracked single α2δ-1 molecules ( C 5 ). Scale bars: C 1 – D 3 , 10 μm; C 4 , D 4 , 2 μm; C 5 , D 5 , 0.5 μm. D , Labeling of surface α2δ-1 as in C using TKO neurons. E , Logarithmic distribution of diffusion coefficients for α2δ-1 on axons of WT and TKO neurons, showing more trajectories of higher mobility in TKO (see §) and fewer low mobility trajectories (see #); n = trajectories/cells; error bars (SEM) shown only in outward direction. F , Median and IQR (25–75%) of diffusion coefficients of α2δ-1 shown in E . Numbers of cells from four independent experiments (in bars). * p = 0.0277, by Kruskal–Wallis test with Dunn's post-test. G , Immunofluorescent images of surface α2δ-3 in synaptic boutons as in A . Scale bar, 5 μm. H , Quantification of colocalization between synGCaMP6f and surface α2δ-3-positive puncta in WT and TKO. Data are mean ± SEM. n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.4835), by unpaired t test. I , Logarithmic distribution of diffusion coefficients as in E but for α2δ-3. With α2δ-3, more trajectories of higher mobility occurred in WT (see §), indicating a reverse effect when compared with α2δ-1 ( E ). J , Median and IQR (25–75%) of diffusion coefficients of α2δ-3 shown in I . Numbers of cells from four independent experiments (in bars). * p = 0.0347, by Kruskal–Wallis test with Dunn's post-test.
    Figure Legend Snippet: αNrxn modulates surface mobility of α2δ-1 and α2δ-3 auxiliary subunits differentially. A , Representative immunofluorescent images of surface α2δ-1 enriched in synaptic boutons, visualized by an antibody against the HA moiety of α2δ-1::HA cotransfected with synGCaMP6f into WT neurons (top) or TKO neurons (bottom). Scale bar, 5 μm. B , Quantification of colocalization between synGCaMP6f and surface α2δ-1-positive puncta in WT and TKO. Data are mean ± SEM; n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.433) by unpaired t test. C , Labeling of the surface population of HA-tagged α2δ-1 ( C 1 ) transfected into WT neurons using an antibody specific to the HA moiety. EGFP was cotransfected to visualize neurites ( C 2 ), merged images ( C 3 ) and an overlay of all trajectories of QD-tracked single α2δ-1 molecules in a subfield as indicated ( C 4 ); sample trajectories of QD-tracked single α2δ-1 molecules ( C 5 ). Scale bars: C 1 – D 3 , 10 μm; C 4 , D 4 , 2 μm; C 5 , D 5 , 0.5 μm. D , Labeling of surface α2δ-1 as in C using TKO neurons. E , Logarithmic distribution of diffusion coefficients for α2δ-1 on axons of WT and TKO neurons, showing more trajectories of higher mobility in TKO (see §) and fewer low mobility trajectories (see #); n = trajectories/cells; error bars (SEM) shown only in outward direction. F , Median and IQR (25–75%) of diffusion coefficients of α2δ-1 shown in E . Numbers of cells from four independent experiments (in bars). * p = 0.0277, by Kruskal–Wallis test with Dunn's post-test. G , Immunofluorescent images of surface α2δ-3 in synaptic boutons as in A . Scale bar, 5 μm. H , Quantification of colocalization between synGCaMP6f and surface α2δ-3-positive puncta in WT and TKO. Data are mean ± SEM. n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.4835), by unpaired t test. I , Logarithmic distribution of diffusion coefficients as in E but for α2δ-3. With α2δ-3, more trajectories of higher mobility occurred in WT (see §), indicating a reverse effect when compared with α2δ-1 ( E ). J , Median and IQR (25–75%) of diffusion coefficients of α2δ-3 shown in I . Numbers of cells from four independent experiments (in bars). * p = 0.0347, by Kruskal–Wallis test with Dunn's post-test.

    Techniques Used: Labeling, Transfection, Diffusion-based Assay

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    Alomone Labs rabbit anti α2δ 1
    <t>α2δ−1</t> 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
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    Alomone Labs anti α1a adrenergic receptor
    Expression of adrenergic receptors in FEF. From left to right: expression of <t>α1A,</t> α2A, β1, and β2 adrenergic receptors (α1AR, α2AR, β1R, and β2R, respectively) in macaque FEF. Images show a cross-section of all layers of cortex and are oriented with the pial surface at the top and white matter at the bottom. The α2A and β2 adrenergic receptors had strong, punctate staining of cell bodies, with little to no background labeling of processes. While the α1A and β1 adrenergic receptors also had strong, punctate staining of cell bodies, there was also staining of the surrounding processes (dendrites and axons), which resulted in a higher amount of background signal. Scale bar = 100 μm for all panels.
    Anti α1a Adrenergic Receptor, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti α1a adrenergic receptor - by Bioz Stars, 2022-01
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    Image Search Results


    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

    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

    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.

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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:

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    Expression of adrenergic receptors in FEF. From left to right: expression of α1A, α2A, β1, and β2 adrenergic receptors (α1AR, α2AR, β1R, and β2R, respectively) in macaque FEF. Images show a cross-section of all layers of cortex and are oriented with the pial surface at the top and white matter at the bottom. The α2A and β2 adrenergic receptors had strong, punctate staining of cell bodies, with little to no background labeling of processes. While the α1A and β1 adrenergic receptors also had strong, punctate staining of cell bodies, there was also staining of the surrounding processes (dendrites and axons), which resulted in a higher amount of background signal. Scale bar = 100 μm for all panels.

    Journal: Frontiers in Neuroanatomy

    Article Title: Differences in Noradrenaline Receptor Expression Across Different Neuronal Subtypes in Macaque Frontal Eye Field

    doi: 10.3389/fnana.2020.574130

    Figure Lengend Snippet: Expression of adrenergic receptors in FEF. From left to right: expression of α1A, α2A, β1, and β2 adrenergic receptors (α1AR, α2AR, β1R, and β2R, respectively) in macaque FEF. Images show a cross-section of all layers of cortex and are oriented with the pial surface at the top and white matter at the bottom. The α2A and β2 adrenergic receptors had strong, punctate staining of cell bodies, with little to no background labeling of processes. While the α1A and β1 adrenergic receptors also had strong, punctate staining of cell bodies, there was also staining of the surrounding processes (dendrites and axons), which resulted in a higher amount of background signal. Scale bar = 100 μm for all panels.

    Article Snippet: Membranes were blocked with Intercept Blocking Buffer (LiCor 927-70001) and then incubated overnight with anti-α1A adrenergic receptor (Alomone Labs AAR-015), anti-α2A adrenergic receptor (Alomone Labs AAR-020), anti-β1 adrenergic receptor (Alomone Labs AAR-023), or anti-β2 adrenergic receptor (Alomone Labs AAR-016) pre-incubated with peptide plus 1% BSA, or pre-incubated with 1% BSA alone.

    Techniques: Expressing, Staining, Labeling

    Expression of adrenergic receptors across cell types and layers. (A) The number of different classes of cell types per mm 2 across FEF layers. General classes of pyramidal neurons (neurogranin and RP) are more abundant than any other class of neuron across layers II through VI. There is low expression of all receptor classes in layer I where there are few neurons. (B) For each of the four adrenergic receptors (pale to dark: α1A, α2A, β1, β2) we quantified the proportion of each cell type that expressed that receptor across all cortical layers in the FEF. We found that expression was very consistent for any given receptor/cell type pair.

    Journal: Frontiers in Neuroanatomy

    Article Title: Differences in Noradrenaline Receptor Expression Across Different Neuronal Subtypes in Macaque Frontal Eye Field

    doi: 10.3389/fnana.2020.574130

    Figure Lengend Snippet: Expression of adrenergic receptors across cell types and layers. (A) The number of different classes of cell types per mm 2 across FEF layers. General classes of pyramidal neurons (neurogranin and RP) are more abundant than any other class of neuron across layers II through VI. There is low expression of all receptor classes in layer I where there are few neurons. (B) For each of the four adrenergic receptors (pale to dark: α1A, α2A, β1, β2) we quantified the proportion of each cell type that expressed that receptor across all cortical layers in the FEF. We found that expression was very consistent for any given receptor/cell type pair.

    Article Snippet: Membranes were blocked with Intercept Blocking Buffer (LiCor 927-70001) and then incubated overnight with anti-α1A adrenergic receptor (Alomone Labs AAR-015), anti-α2A adrenergic receptor (Alomone Labs AAR-020), anti-β1 adrenergic receptor (Alomone Labs AAR-023), or anti-β2 adrenergic receptor (Alomone Labs AAR-016) pre-incubated with peptide plus 1% BSA, or pre-incubated with 1% BSA alone.

    Techniques: Expressing

    Expression of adrenergic receptors on pyramidal neurons. (A) Panels show expression of α1A, α2A, β1, and β2 adrenergic receptors (α1AR, α2AR, β1R, and β2R, respectively) from top to bottom with pyramidal neuron markers (RP, neurogranin, and SMI-32) from left to right. RP and neurogranin are both putative general markers of pyramidal neurons and SMI-32 is a marker for putative long-range projecting pyramidal neurons. All adrenergic receptors are labeled in green, and all pyramidal neurons are labeled in magenta. (B) Quantification of the proportion of each neuron class that expressed each receptor class. Chi-squared tests were performed using pooled neuron counts across all animals. All four adrenergic receptors were expressed significantly more highly on long-range projecting pyramidal neurons than either class of general pyramidal neuron. Significance levels are noted as *** p

    Journal: Frontiers in Neuroanatomy

    Article Title: Differences in Noradrenaline Receptor Expression Across Different Neuronal Subtypes in Macaque Frontal Eye Field

    doi: 10.3389/fnana.2020.574130

    Figure Lengend Snippet: Expression of adrenergic receptors on pyramidal neurons. (A) Panels show expression of α1A, α2A, β1, and β2 adrenergic receptors (α1AR, α2AR, β1R, and β2R, respectively) from top to bottom with pyramidal neuron markers (RP, neurogranin, and SMI-32) from left to right. RP and neurogranin are both putative general markers of pyramidal neurons and SMI-32 is a marker for putative long-range projecting pyramidal neurons. All adrenergic receptors are labeled in green, and all pyramidal neurons are labeled in magenta. (B) Quantification of the proportion of each neuron class that expressed each receptor class. Chi-squared tests were performed using pooled neuron counts across all animals. All four adrenergic receptors were expressed significantly more highly on long-range projecting pyramidal neurons than either class of general pyramidal neuron. Significance levels are noted as *** p

    Article Snippet: Membranes were blocked with Intercept Blocking Buffer (LiCor 927-70001) and then incubated overnight with anti-α1A adrenergic receptor (Alomone Labs AAR-015), anti-α2A adrenergic receptor (Alomone Labs AAR-020), anti-β1 adrenergic receptor (Alomone Labs AAR-023), or anti-β2 adrenergic receptor (Alomone Labs AAR-016) pre-incubated with peptide plus 1% BSA, or pre-incubated with 1% BSA alone.

    Techniques: Expressing, Marker, Labeling

    Expression of adrenergic receptors on inhibitory interneurons. (A) Panels show expression of α1A, α2A, β1, and β2 adrenergic receptors (α1AR, α2AR, β1R, and β2R, respectively) from top to bottom with inhibitory interneuron markers (parvalbumin, calbindin and calretinin) from left to right. All adrenergic receptors are labeled in green, and all inhibitory interneurons are labeled in magenta. (B) Quantification of the proportion of each neuron class that expressed each receptor class. Chi-squared tests were performed using pooled neuron counts across all animals. Lines above the bars show the significance of different comparisons. Black lines indicate significant differences between the expression of different receptors within a neuron class; gray lines indicate significant differences of expression of a specific receptor across different neuron classes. The shade of gray indicates which receptor class is being compared and matches the shading of the bars: from light to dark—α1AR, α2AR, β1R, and β2R. Significance levels are noted as *** p

    Journal: Frontiers in Neuroanatomy

    Article Title: Differences in Noradrenaline Receptor Expression Across Different Neuronal Subtypes in Macaque Frontal Eye Field

    doi: 10.3389/fnana.2020.574130

    Figure Lengend Snippet: Expression of adrenergic receptors on inhibitory interneurons. (A) Panels show expression of α1A, α2A, β1, and β2 adrenergic receptors (α1AR, α2AR, β1R, and β2R, respectively) from top to bottom with inhibitory interneuron markers (parvalbumin, calbindin and calretinin) from left to right. All adrenergic receptors are labeled in green, and all inhibitory interneurons are labeled in magenta. (B) Quantification of the proportion of each neuron class that expressed each receptor class. Chi-squared tests were performed using pooled neuron counts across all animals. Lines above the bars show the significance of different comparisons. Black lines indicate significant differences between the expression of different receptors within a neuron class; gray lines indicate significant differences of expression of a specific receptor across different neuron classes. The shade of gray indicates which receptor class is being compared and matches the shading of the bars: from light to dark—α1AR, α2AR, β1R, and β2R. Significance levels are noted as *** p

    Article Snippet: Membranes were blocked with Intercept Blocking Buffer (LiCor 927-70001) and then incubated overnight with anti-α1A adrenergic receptor (Alomone Labs AAR-015), anti-α2A adrenergic receptor (Alomone Labs AAR-020), anti-β1 adrenergic receptor (Alomone Labs AAR-023), or anti-β2 adrenergic receptor (Alomone Labs AAR-016) pre-incubated with peptide plus 1% BSA, or pre-incubated with 1% BSA alone.

    Techniques: Expressing, Labeling

    Density of adrenergic receptors across different layers of the FEF. The number of neurons per mm 2 that express a given receptor across FEF layers. α2A adrenergic receptors (α2ARs) and β2 adrenergic receptors (β2Rs) are more abundant than either α1A adrenergic receptors (α1ARs) or β1 adrenergic receptors (β1Rs) across layers II through V. There are no obvious differences in expression across layers other than the predictably low expression of all receptor classes in layer I where there are few neurons.

    Journal: Frontiers in Neuroanatomy

    Article Title: Differences in Noradrenaline Receptor Expression Across Different Neuronal Subtypes in Macaque Frontal Eye Field

    doi: 10.3389/fnana.2020.574130

    Figure Lengend Snippet: Density of adrenergic receptors across different layers of the FEF. The number of neurons per mm 2 that express a given receptor across FEF layers. α2A adrenergic receptors (α2ARs) and β2 adrenergic receptors (β2Rs) are more abundant than either α1A adrenergic receptors (α1ARs) or β1 adrenergic receptors (β1Rs) across layers II through V. There are no obvious differences in expression across layers other than the predictably low expression of all receptor classes in layer I where there are few neurons.

    Article Snippet: Membranes were blocked with Intercept Blocking Buffer (LiCor 927-70001) and then incubated overnight with anti-α1A adrenergic receptor (Alomone Labs AAR-015), anti-α2A adrenergic receptor (Alomone Labs AAR-020), anti-β1 adrenergic receptor (Alomone Labs AAR-023), or anti-β2 adrenergic receptor (Alomone Labs AAR-016) pre-incubated with peptide plus 1% BSA, or pre-incubated with 1% BSA alone.

    Techniques: Expressing