Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Primary antibodies employed in the study.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques:

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 antibodies validation and RT-PCR analysis. (a) Scheme of human P2RY 12 gene location, transcript variants [ , ], and protein structure, with amino acid epitopes recognized by the used antibodies and highlighted in color ( intra1 , intra2 , and intra fl , red circle; c-ter , green oval). Species conservation for each epitope was calculated by using BLAT tool of UCSC genome browser . (b) Total protein extracts from SH-SY5Y or HEK293 cells expressing Myc-tagged P2Y 12 receptor were subjected to Western blot analysis with the indicated antibodies. (c) Total protein extracted from human, rat and mouse brain, from primary mouse microglia (mMG) and rat oligodendrocyte (OL) cultures were subjected to Western blot analysis with the indicated antibodies. For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (d) RT-PCR using primers specific for P2Y 12 mRNA was performed on total RNA from rat microglia (rMG) and OL. Control lanes show RT-PCR performed without reverse transcriptase enzyme.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in dissociated and organotypic primary cultures. (a) Mouse primary cortical microglia were subjected to immunofluorescence and confocal analysis with phalloidin (green, merged field) and P2Y 12 receptor antibodies (red, insets and merged) and Hoechst (white, merged). Scale bars in insets: 20 μ m. (b) Double immunofluorescence and confocal analysis of primary rat mature (OL) and precursor (OPC) oligodendrocytes was performed with antibodies for P2Y 12 receptor, MBP, NG2 . For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (c) Rat cerebellar organotypic cultures were analyzed by double immunofluorescence and confocal microscopy for intra1 (red) and c-ter (red), highlighting different structures (see also insets), and MBP (green).

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in rat brain tissue. Double immunofluorescence and confocal analysis was performed on sections from rat cerebellum (panels (a), (b), (c), (g), (h), and (i)) and striatum (panels (d), (e), (f), (j), (k), and (l)) with intra1 , intra2 -lots AN01/02/04, intra fl , c-ter (all red), and GFAP (green, inset b1), CD11b (green, insets c1, f1, h1; yellow merged, inset i1; green, panel (k); merged, panel (l)), MBP (yellow merged, inset c2; green, inset e1; green, panels (h) and (i); green, inset j1) antibodies.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Temporal and regional pattern of P2Y 12 expression in SOD1-G93A ALS spinal microglia. (a) Double immunofluorescence and confocal analysis on lumbar spinal cord sections (L3–L5) of wild-type (WT) mice was performed with c-ter antibody (green and yellow, merged and insets), CD11b (left panel, yellow, merged and inset), and CD68 (right panel, red, merged and inset), in both dorsal (DH) and ventral (VH) horns of spinal cord. (b) Double immunofluorescence and confocal analysis on SOD1-G93A lumbar spinal cord sections (L3–L5) at two different stages of ALS disease, that is, 20 weeks, and end stage, was performed with c-ter (green) and CD68 (red) antibodies. (c) Equal amount of total lumbar spinal cord lysates (L3–L5) from WT and SOD1-G93A ( n = 4 for each group) were subjected to Western blotting and immunoreactions with c-ter and CD68 antibodies; anti- β -actin was used for protein normalization. Data represent means ± SEM. Statistical significance was calculated by Student's t -test, * P < 0.05.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in human cortex. Sections from human healthy and SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for the immunoreactive markers c-ter (red, panels (a), (c); insets a1, a2, c1; yellow merged, inset c2), intra1 (red, panels (d), insets d1, d2, d3; yellow merged, panel f, insets f1, f2), MBP (green, panels (b), (c), (e), insets c1, e1; yellow merged, panel (f), inset f1), MHC II (green, inset d3), and integrin α II/ β 3 (green, insets b2, e2; yellow merged, insets c2, f2). The asterisks show decreased P2Y 12 immunoreactivity in proximity to MS lesion.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Regional distribution of P2Y 12 in proximity to MS lesions. Sections from SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for c-ter (red) and MHC II (green) immunoreactivity. In proximity to the demyelinating active cortical lesion expressing augmented positivity for MHC II, microglia gradually lose immunoreactivity for c-ter antibody. Microglia express differential immunoreactivity in the four chosen areas which are found inside (circled b-c) and around (circled a–d) a lesion.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Draw of microglial marker expression as a function of activation. Branched microglia are represented in blue and activated microglia in red. Iba1 , CD11b, and MHC II are mostly expressed in microglia throughout the different morphological states and their expression increases during activation (light blue to red color). P2Y 12 c-ter (light blue) and CD68 (red) are expressed, respectively, in branched or roundish/activated microglia.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Marker, Expressing, Activation Assay

Journal: STAR Protocols

Article Title: Protocol for modulating the noradrenergic pathway from locus coeruleus to heart to prevent sudden unexpected death in epilepsy in mouse models

doi: 10.1016/j.xpro.2023.102403

Figure Lengend Snippet: Key procedures of verifying the distribution of β1-AR and the content of β1-AR and p-β1-AR in each heart chamber (A) 3.5% chloral hydrate intraperitoneally injected to anesthetize mice (for 20 g male DBA/1 mice, 8 weeks old, 0.30 mL of 3.5% chloral hydrate, 15 mL/kg body weight is recommended). (B) Key procedures for verifying the distribution and expression of β1-AR in RA, RV, LA, and LV: a. Perfuse the heart with cold PBS to rinse the residual blood, and then perfuse the heart with 4% PFA. b. Harvest the heart and saturate it in a 30% sucrose solution for 24 h c. Slice the heart into 15-μm-thick coronal slices with a freezing microtome. d. Immunohistochemically stained slices were photographed using an Olympus microscope and a laser confocal microscope. e. Representative image shows that β1-AR is preferentially distributed in RA, RV, LA, and LV. (C) Key procedures for measuring the content of β1-AR and p-β1-AR in H, RA, RV, LA, and LV: a. Perfuse the heart with cold PBS to rinse the residual blood. b. The heart of a mouse is carefully divided into four parts using a scalpel: RA, RV, LA, and LV. c. Use ophthalmic scissors to crush the heart tissue (H, RA, RV, LA, and LV) separately, then whip the solution with an ultrasonic knife until the solution is uniform. d. The obtained tissue homogenates from H, RA, RV, LA, and LV are centrifuged for 20 min (12000 rpm) to get supernatant. The supernatant is used for the measurement of the content of β1-AR and p-β1-AR. PBS, phosphate-buffered saline; PFA, paraformaldehyde; RA: the right atrium. RV: the right ventricle. LA: the left atrium. LV: the right ventricle. H: the whole heart. β1-AR: Beta receptor1. p-β1-AR: phospho-Beta receptor1.

Article Snippet: Rabbit anti-β1-AR , Alomone , AAR-023(1:400).

Techniques: Injection, Expressing, Staining, Microscopy

Journal: STAR Protocols

Article Title: Protocol for modulating the noradrenergic pathway from locus coeruleus to heart to prevent sudden unexpected death in epilepsy in mouse models

doi: 10.1016/j.xpro.2023.102403

Figure Lengend Snippet:

Article Snippet: Rabbit anti-β1-AR , Alomone , AAR-023(1:400).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software, Labeling

Journal: PLoS ONE

Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

doi: 10.1371/journal.pone.0046012

Figure Lengend Snippet: (A) Representative images of clustering of NR2B in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. Higher magnification views are shown of the dendritic branches studded with numerous clusters enclosed in white boxes. Scale bar, 50 µm. (B) and (C) show the quantification of (A). The numbers of NR2B clusters were 25.1±1.0 (control, n = 37), 25.5±1.0 (NBQX+nimodipine, n = 36), and 25.7±1.0 (Zn+NBQX+nimodipine, n = 37) per 100 µm dendrite length. The mean intensity of NR2B clusters was 122.4±3.3 (control, n = 25), 133.0±4.5 (NBQX+nimodipine, n = 25) and 131.3±3.6 (Zn+NBQX+nimodipine, n = 23). (D) Representative NR2B currents in response to co-application with the agonist plus NVP-AAM077. (E) Statistics of NR2B currents. Averaged peak currents were 342.7±71.7 pA (control, n = 38), 327.0±67.3 pA (NBQX+nimodipine, n = 20) and 320.0±48.5 pA (Zn+NBQX+nimodipine, n = 20).

Article Snippet: Rabbit polyclonal NR2B antibody was from Alomone Labs (Jerusalem, Israel).

Techniques:

Journal: PLoS ONE

Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

doi: 10.1371/journal.pone.0046012

Figure Lengend Snippet: (A) Hippocampal neurons derived from control, NBQX+nimodipine and Zn+NBQX+nimodipine groups were incubated in 35 µg/ml CHX. Total expressions of NR1, NR2A and NR2B were measured at 0 hr, 12 hr or 18 hr in the CHX treatment. (B) shows the quantification of (A). (C) Surface NR1, NR2A and NR2B derived from cultured hippocampal neurons were isolated by biotinylation assay and detected by anti-NR1, anti-NR2A and anti-NR2B antibodies. Surface NR1 and NR2A, but not NR2B, were notably lower in the Zn+NBQX+nimodipine group, while total NR1, NR2A and NR2B did not change. (D) Quantifications of (C). Percentage changes of surface signal intensities versus control were: NR1, 97.6±13.8 (NBQX+nimodipine) and 69.4±8.0 (Zn+NBQX+nimodipine); NR2A, 87.7±4.9 (NBQX+nimodipine) and 57.0±14.3 (Zn+NBQX+nimodipine); and NR2B, 99.0±19.0 (NBQX+nimodipine) and 101.0±21.8 (Zn+NBQX+nimodipine). All experiments were performed at least 3 times (N > = 3). *, P<0.05.

Article Snippet: Rabbit polyclonal NR2B antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Derivative Assay, Incubation, Cell Culture, Isolation, Cell Surface Biotinylation Assay

Journal: PLoS ONE

Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

doi: 10.1371/journal.pone.0046012

Figure Lengend Snippet: (A) Lysates were immunoprecipitated with anti-PSD-95 antibody in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. The immunoprecipitates were probed with the anti-NR2A (NR2A), anti-NR1 (NR1), anti-NR2B (NR2B), anti-Src (Src) and anti-Fyn (Fyn) antibodies. The Input lane was loaded with total protein (20 µg) derived from cultured hippocampal neurons. (B) Supernatant samples were immunoprecipitated with anti-PSD-95 antibody in different groups, which showed the clearance of PSD95 after Co-IP experiment. All experiments were performed at least 3 times (N > = 3) and the summarized data are shown in (C) and (D). Y axes in (C) and (D) represent the percentage change of each co-precipitated protein (labeled on X axes) relative to the corresponding control. *, P<0.05.

Article Snippet: Rabbit polyclonal NR2B antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Immunoprecipitation, Derivative Assay, Cell Culture, Co-Immunoprecipitation Assay, Labeling

Journal: Physiological Reports

Article Title: Positive chronotropic action of HCN channel antagonism in human collecting lymphatic vessels

doi: 10.14814/phy2.15401

Figure Lengend Snippet: Primer sequences for RT‐PCR expression analysis of HCN channels

Article Snippet: Thereafter, tissue was permeabilized with 0.25% Triton X‐100 in PBS for 10 min following by blocking with 2% bovine serum albumin (BSA) in PBS for 20 min. Tissue was incubated overnight in a dark, humidified chamber at 4°C with 1:50 rabbit polyclonal anti‐HCN2 (APC‐030‐AR; Alomone, Israel) preconjugated to ATTO‐594 fluorophore.

Techniques: Expressing, Amplification, Sequencing

Journal: Physiological Reports

Article Title: Positive chronotropic action of HCN channel antagonism in human collecting lymphatic vessels

doi: 10.14814/phy2.15401

Figure Lengend Snippet: Human lymphatic vessels predominantly express HCN2. (a) RT‐PCR analysis of HCN2 (229 base pairs) and HCN3 (589 bp) amplified from thoracic duct (TD) and mesenteric lymphatic vessels (ML); HCN1 (597 bp) and HCN4 (232 bp) were consistently amplified from the control human RNA, dorsal root ganglia (DRG) and heart, respectively, while all lymphatic samples were negative. Samples are presented pairwise as reverse transcriptase positive (+) followed by reverse transcriptase negative (−). HCN2 immunoreactivity (red fluorescence, left panels) was observed in smooth muscle cells of (b) human thoracic duct and (c) mesenteric lymphatic vessels. (d) Antibody specificity was confirmed by the absence of staining when the antibody was preincubated with peptide control (green fluorescent nuclear stain). Scale bars denote 50 μm (top) and 100 μm (middle and bottom), * indicates lumen, and right panels present differential interference contrast (DiC) images of the same section.

Article Snippet: Thereafter, tissue was permeabilized with 0.25% Triton X‐100 in PBS for 10 min following by blocking with 2% bovine serum albumin (BSA) in PBS for 20 min. Tissue was incubated overnight in a dark, humidified chamber at 4°C with 1:50 rabbit polyclonal anti‐HCN2 (APC‐030‐AR; Alomone, Israel) preconjugated to ATTO‐594 fluorophore.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Fluorescence, Staining

Journal: Physiological Reports

Article Title: Positive chronotropic action of HCN channel antagonism in human collecting lymphatic vessels

doi: 10.14814/phy2.15401

Figure Lengend Snippet: HCN transcript expression profile for human thoracic duct (TD) and mesenteric lymphatic vessels (MLV)

Article Snippet: Thereafter, tissue was permeabilized with 0.25% Triton X‐100 in PBS for 10 min following by blocking with 2% bovine serum albumin (BSA) in PBS for 20 min. Tissue was incubated overnight in a dark, humidified chamber at 4°C with 1:50 rabbit polyclonal anti‐HCN2 (APC‐030‐AR; Alomone, Israel) preconjugated to ATTO‐594 fluorophore.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: α3β4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells

doi: 10.3390/ijms23169101

Figure Lengend Snippet: Peripheral distribution of endogenous α3 or β4 nAChR subunits to EGFP-SNAP25. ( a ) Cortical layer (TOP) of a cell expressing EFGP-SNAP25 (green) and inmunolabeled with antibody anti-β4 Atto-594 (red). ( b ) Similar image of a cell expressing EFGP-SNAP25 (green), but immunolabeled with the anti-α3 antibody and revealed with the appropriate secondary antibody Alexafluor-546 (red). ( c ) A comparison of the average distance between the centroids of each immunologically identified α3 or β4 nAChR subunits and the centroids of the nearest EGFP-SNAP25. All of the records were obtained using sequential laser excitation and acquisition. Bars indicate 1 µm.

Article Snippet: We revealed the immunological location of the endogenous β4 subunits using a rabbit polyclonal antibody anti-Nicotinic Acetylcholine Receptor β4 (extracellular)-Atto-594 (alomone Labs, Cat #: ANC-014-AR; Lot: ANC014ARAN0150, Jerusalem, Israel).

Techniques: Expressing, Immunolabeling

Journal: International Journal of Molecular Sciences

Article Title: α3β4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells

doi: 10.3390/ijms23169101

Figure Lengend Snippet: Membrane proximity between endogenous (real) or random simulated α3β4 nAChRs (red) and DBH exocytosis patches (green) upon K + or ACh secretion stimulus. α3β4 nAChR real structures are apparently in contact with DBH exocytotic points more than what would be expected for a random distribution. ( a – d ) A chromaffin cell with both green DBH ( a ) and red α3β4 nAChR ( b ) immunolabeling in a secretion stimulus assay using depolarization with high K + (see ). Many of the real α3β4 nAChR (red) structures seem to have at least one pixel that colocalizes with DBH green patches ( c ). The random distributions of the same number of red simulated α3β4 nAChR patches are compared ( d ). ( e – h ) A similar experiment but using ACh as the secretion stimulus (see ). Real ( g ) and random simulated ( h ) α3β4 nAChR structures are compared. Many of the red α3β4 nAChR structures ( f ) seem to have at least one pixel that colocalizes with DBH green patches ( g ). Significant differences are seen in the mean distance between each patch centroid and the nearest DBH centroid ( i ) (one-way ANOVA Kruskal-Wallis test (non-parametrical), K + to ACh: p * < 0.05 moderately significative; K + to RND and ACh to RND p *** < 0.0001 highly significative). The distribution of XY distances confirms these tendencies in each condition ( j ): under K+ stimulation (grey fill), 323 real α3β4 nAChR structures from 24 cells are measured. Under ACh stimulation (white fill), 325 real α3β4 nAChR structures from 30 cells are measured. Similar number (306) of simulated α3β4 nAChRs are measured to obtain the random distribution. Bars represent 2 μm.

Article Snippet: We revealed the immunological location of the endogenous β4 subunits using a rabbit polyclonal antibody anti-Nicotinic Acetylcholine Receptor β4 (extracellular)-Atto-594 (alomone Labs, Cat #: ANC-014-AR; Lot: ANC014ARAN0150, Jerusalem, Israel).

Techniques: Immunolabeling

Journal: International Journal of Molecular Sciences

Article Title: α3β4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells

doi: 10.3390/ijms23169101

Figure Lengend Snippet: Membrane colocalization and overlapping between α3β4 nAChR (red) and DBH (green) after secretion stimulus: in each image we individually selected several regions of interest (ROIs) ( a , b ) and proceeded to analyze them using ImageJ JACoP complement (See ), obtaining the values of the Pearson’s and Manders coefficients, whose averages are shown in , as well as their corresponding scatterplot graphs, whose examples are shown ( d ). The averaged Pearson’s coeficients for 50% threshold images show a significant colocalization between endogenous α3β4 nAChRs structures and the DBH sites in both stimulation conditions to those obtained for randomly simulated α3β4 nAChRs structures ( ( c )). Furthermore, the averaged coefficients obtained for the stimulus with ACh are significantly higher than those obtained when stimulating with a high K + ( p value *** < 0.0001; ). ( d ) A scatterplot or fluorogram for pixel colocalization of the red channel (AChR structures) and green channel (DBH) comparing three examples of the images obtained in each of the three analyzed conditions. Bars represent 1 µm.

Article Snippet: We revealed the immunological location of the endogenous β4 subunits using a rabbit polyclonal antibody anti-Nicotinic Acetylcholine Receptor β4 (extracellular)-Atto-594 (alomone Labs, Cat #: ANC-014-AR; Lot: ANC014ARAN0150, Jerusalem, Israel).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: α3β4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells

doi: 10.3390/ijms23169101

Figure Lengend Snippet: Results of the simulation of a pulse lasting 1 s. ( a ) Calcium current that enters the cell through nAChRs during the pulse ( b ). Average calcium concentration obtained at distances between 0–70 nm to the cell membrane ( c ). Time course of the accumulated secretory response (as a percentage) obtained with random and colocalized configurations of nAChR secretory vesicles.

Article Snippet: We revealed the immunological location of the endogenous β4 subunits using a rabbit polyclonal antibody anti-Nicotinic Acetylcholine Receptor β4 (extracellular)-Atto-594 (alomone Labs, Cat #: ANC-014-AR; Lot: ANC014ARAN0150, Jerusalem, Israel).

Techniques: Concentration Assay

Journal: Frontiers in Behavioral Neuroscience

Article Title: Alpha 1 -adrenergic receptor blockade in the ventral tegmental area attenuates acquisition of cocaine-induced pavlovian associative learning

doi: 10.3389/fnbeh.2022.969104

Figure Lengend Snippet: Effects of α 1 -AR blockade in the VTA on the acquisition of cocaine-evoked CPP. (A,F) The experimental time-line and schedule of intra-VTA microinfusions during the acquisition of CPP induced by one (A) or two (F) cocaine (20 mg/kg, i.p.) conditionings. (B) Graphical representation of the two methods of calculating conditioned responses in the CPP paradigm. CPP score is defined as a difference between time spent (t 2 ) in the cocaine-paired chamber minus time spent in (t 3 ) control chamber (saline-paired) during post-conditioning (post-test). Delta score is defined as a difference between time spent (t 2 ) in the cocaine-paired chamber during post-conditioning minus time spent (t 1 ) in the same chamber but during pre-conditioning (pre-test). (C,G) There were no pre-existing differences in the time spent in the cocaine- and saline-paired chambers during pre-test between future vehicle (Veh)- and prazosin (Praz)-treated subjects. (D,E) Intra-VTA microinfusion of prazosin attenuated the acquisition of CPP induced by one ( D for CPP score and E for delta score) and two ( H for CPP score and I for delta score) cocaine (20 mg/kg, i.p.) conditionings. (J) Localization of histologically verified cannula placements. Data are presented as individual data points as well as the mean and SEM. ** p < 0.01, *** p < 0.001, Newman–Keuls post-hoc test vs. Praz 0 μg.

Article Snippet: The specificity of anti-α 1 -AR antibody was tested by pre-incubation with manufacturer-supplied control antigen peptides (1:125, AK-490, Alomone Labs, Jerusalem, Israel) and comparing the strength of the signal between the control and blocked antibodies.

Techniques:

Journal: Frontiers in Behavioral Neuroscience

Article Title: Alpha 1 -adrenergic receptor blockade in the ventral tegmental area attenuates acquisition of cocaine-induced pavlovian associative learning

doi: 10.3389/fnbeh.2022.969104

Figure Lengend Snippet: Effects of α 1 -AR blockade in the VTA on cocaine reinforcement in the cocaine self-administration paradigm. (A) Rats, after intravenous catheter and intra-VTA cannula implantation surgeries and recovery were trained to self-administer cocaine (∼0.5 mg/kg/inf.) for 8 days. On day 9, intra-VTA vehicle (Veh) or prazosin (Praz) microinfusions were performed immediately prior to subsequent cocaine self-administration session. (B–D) There were no pre-existing differences during cocaine self-administration training in the number of (B) active and (C) inactive lever presses as well as in (D) the number of cocaine infusions between future vehicle (Veh)- and prazosin (Praz)-treated subjects. (E) Intra-VTA microinfusion of prazosin (Praz 1 μg/side) on day 9 had no effect on the numbers of active and inactive lever responses and on (F) the number of cocaine infusions. (G) Intra-VTA α 1 -AR blockade had no effects on the numbers of active lever presses over time. Similarly, prazosin treatment on day 9 had no effects on (H) the numbers of active and inactive lever presses and (I) cocaine infusions, 24 h after treatment (on day 10). (J) Finally, prazosin microinfusion on day 9 had no effects on active lever responding during time-out. (K) Localization of histologically verified cannula placements. Data are presented as individual data points as well as the mean and SEM. Act, active lever; Inact, inactive lever.

Article Snippet: The specificity of anti-α 1 -AR antibody was tested by pre-incubation with manufacturer-supplied control antigen peptides (1:125, AK-490, Alomone Labs, Jerusalem, Israel) and comparing the strength of the signal between the control and blocked antibodies.

Techniques:

Journal: Frontiers in Behavioral Neuroscience

Article Title: Alpha 1 -adrenergic receptor blockade in the ventral tegmental area attenuates acquisition of cocaine-induced pavlovian associative learning

doi: 10.3389/fnbeh.2022.969104

Figure Lengend Snippet: Effects of α 1 -AR blockade in the VTA on cocaine-evoked 50-kHz USVs. α 1 -AR blockade in the VTA with 1 μg/side prazosin (Praz) had no effect on the number of appetitive USVs after (A) saline (i.p.) or cocaine administration at (B) 10 mg/kg, i.p. or (C) 20 mg/kg dose. (D) Intra-VTA prazosin infusion had no effect on the cocaine-induced increase in the number of appetitive USVs and (E) did not modulate selected categories USVs after cocaine (20 mg/kg, i.p.) administration. (F) Localization of histologically verified cannula placements. Data are presented as individual data points as well as the mean and SEM. *** p < 0.001, Newman–Keuls post-hoc test vs. Cocaine 0 mg/kg.

Article Snippet: The specificity of anti-α 1 -AR antibody was tested by pre-incubation with manufacturer-supplied control antigen peptides (1:125, AK-490, Alomone Labs, Jerusalem, Israel) and comparing the strength of the signal between the control and blocked antibodies.

Techniques:

Journal: Frontiers in Behavioral Neuroscience

Article Title: Alpha 1 -adrenergic receptor blockade in the ventral tegmental area attenuates acquisition of cocaine-induced pavlovian associative learning

doi: 10.3389/fnbeh.2022.969104

Figure Lengend Snippet: Localization of α 1 -AR-expressing cells in the anterior parts of the VTA of TH-cre + rats. (A) Representative image of the EYFP- and α 1 -AR-expressing cells. (B–E) Magnified cells with α 1 -AR expression (red), EYFP (green), DAPI (blue), and merged channels showing α 1 -AR-immunoreactive and EYFP-expressing cells (white arrows) in the medial part of the anterior VTA. (F–I) Magnified cells with α 1 -AR (red), EYFP (green), DAPI (blue), and merged channels showing co-localized α 1 -AR-positive and EYFP-expressing cells (white arrows) in the lateral part of the anterior VTA. (J–Q) α 1 -AR expression in astrocytes (S100B) and GAD67-positive GABAergic interneurons. (J–M) Magnified images of astrocytes (red), α 1 -AR immunostaining (green), DAPI (blue), and composite images showing co-localization (white arrows) between α 1 -AR and S100B expression. (N–Q) Magnified images of GAD67-positive interneurons (red), α 1 -AR expression (green), DAPI (blue), and composite images showing co-localization between α 1 -AR and GAD67 immunoreactivity (white arrows). PBP, parabrachial pigmented nucleus; RLi, rostral linear nucleus.

Article Snippet: The specificity of anti-α 1 -AR antibody was tested by pre-incubation with manufacturer-supplied control antigen peptides (1:125, AK-490, Alomone Labs, Jerusalem, Israel) and comparing the strength of the signal between the control and blocked antibodies.

Techniques: Expressing, Immunostaining