rabbit anti α2δ 1  (Alomone Labs)

Journal: Journal of neurochemistry

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

doi: 10.1111/jnc.14627

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

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

Techniques: Injection

Journal: Journal of neurochemistry

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

doi: 10.1111/jnc.14627

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

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

Techniques: Knock-Out, Mouse Assay

Journal: Journal of neurochemistry

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

doi: 10.1111/jnc.14627

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

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

Techniques: Activation Assay, Whisker Assay

Journal: Journal of neurochemistry

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

doi: 10.1111/jnc.14627

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

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

Techniques: Activation Assay, Whisker Assay

Journal: Journal of neurochemistry

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

doi: 10.1111/jnc.14627

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

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

Techniques: Activation Assay, Whisker Assay

Journal: Journal of neurochemistry

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

doi: 10.1111/jnc.14627

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

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

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

Journal: Journal of neurochemistry

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

doi: 10.1111/jnc.14627

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

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

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

Journal: Journal of neurochemistry

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

doi: 10.1111/jnc.14627

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

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

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

Journal: Journal of neurochemistry

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

doi: 10.1111/jnc.14627

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

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

Techniques: Activation Assay, Whisker Assay

Journal: Anesthesiology

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

doi: 10.1097/ALN.0000000000002648

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

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

Techniques:

Journal: Anesthesiology

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

doi: 10.1097/ALN.0000000000002648

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

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

Techniques: Activation Assay, Mouse Assay

Journal: Anesthesiology

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

doi: 10.1097/ALN.0000000000002648

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

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

Techniques: Activity Assay, Mouse Assay

Journal: Anesthesiology

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

doi: 10.1097/ALN.0000000000002648

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

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

Techniques: Activity Assay, Incubation

Journal: Anesthesiology

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

doi: 10.1097/ALN.0000000000002648

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

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

Techniques: Activity Assay

Journal: Anesthesiology

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

doi: 10.1097/ALN.0000000000002648

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

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

Techniques: Activation Assay

Journal: Anesthesiology

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

doi: 10.1097/ALN.0000000000002648

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

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

Techniques: Mouse Assay, Injection

Journal: Anesthesiology

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

doi: 10.1097/ALN.0000000000002648

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

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

Techniques: Activity Assay

Journal: The Journal of Neuroscience

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

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend 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

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: , AF3074, R & D Systems], goat-anti-TSP2 1:200 [RRID: , AF1635, R & D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: , AB5535, Millipore], goat anti-ChAT[RRID: , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: Transplantation Assay, Labeling, Staining

Journal: The Journal of Neuroscience

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

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend 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

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: , AF3074, R & D Systems], goat-anti-TSP2 1:200 [RRID: , AF1635, R & D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: , AB5535, Millipore], goat anti-ChAT[RRID: , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: In Vivo, Labeling

Journal: The Journal of Neuroscience

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

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend 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

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: , AF3074, R & D Systems], goat-anti-TSP2 1:200 [RRID: , AF1635, R & D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: , AB5535, Millipore], goat anti-ChAT[RRID: , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: Expressing, Staining, Labeling

Journal: The Journal of Neuroscience

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

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend 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

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: , AF3074, R & D Systems], goat-anti-TSP2 1:200 [RRID: , AF1635, R & D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: , AB5535, Millipore], goat anti-ChAT[RRID: , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: Transplantation Assay, Labeling