Journal: Neuron

Article Title: Reducing astrocyte calcium signaling in vivo alters striatal microcircuits and causes repetitive behavior

doi: 10.1016/j.neuron.2018.08.015

Figure Lengend Snippet: (A) Current recordings in voltage-clamp (−60 mV) from MSNs in control and hPMCA2w/b-expressing mice. Dashed lines and arrows indicate the changes of baseline induced by bicuculline application (BIC; 25 μM). Histograms show the shift in baseline currents recorded during BIC application. (B) Tonic GABA currents were reduced in MSNs from hPMCA2w/b-expressing mice, and this effect was rescued by GAT-3 inhibitor SNAP5114 (40 μM). (C) Left, images of GAT-3 IHC in astrocytes expressing control protein or hPMCA2w/b. Right, quantification of GAT-3 fluorescence intensity in hPMCA2w/b-expressing astrocytes relative to those expressing tdTomato (Mann-Whitney test). (D-E) As in C, but for Kir4.1 and S100β immunostaining. (F) Western blot analysis of striatum membrane protein fractions showing for GAT-3 and GAT-1 levels in hPMCA2w/b groups relative to controls. The gel shown has been cropped to fit in the figure and the crop lines are shown as a solid border. The data were normalized to tubulin as a loading control. (G, H) Tonic GABA inhibition was decreased in D1-MSNs (G), but not D2-MSNs (H) in hPMCA2w/b-expressing mice (assessed with Student’s t or Mann-Whitney tests). The MSNs are colored, but were from Drd1-cre and Adora2a-cre mice with FLEX AAV tdTomato injections. (I) Behavioral traces of control and hPMCA2w/b-expressing mice under baseline and SNAP5114 treatment showing self-grooming and non-grooming episodes over 10 min. (J) Increased self-grooming behaviors in hPMCA2w/b-expressing mice were rescued by SNAP5114 (50 μmol/kg; data were assessed with One-way ANOVA followed by post hoc Bonferroni tests). (K) AAV2/5 overexpressing GAT-3 in astrocytes and GAT-3HA detection in S100β positive astrocytes. (L) Traces for self-grooming. (M) Average data for self-grooming duration and bouts for controls and GAT-3HA overexpressing mice. Data were assessed with Student’s t test. The average data are shown as mean ± SEM.

Article Snippet: Serial 40 μm sections were collected and incubated with the following primary antibodies overnight at 4°C: rabbit anti-S100β (1:1,000; Abcam, ab41548), mouse anti-NeuN (1:1,000; Millipore, MAB377), chicken anti-GFP (1:1,000; Abcam, ab13970), rabbit anti-RFP (1:1,000; Rockland, 600–401-379), chicken anti-RFP (1:1,000; a gift from Dr. Brecha’s laboratory at UCLA), rabbit anti-GAT-3 (1:500; a gift from Dr. Brecha’s laboratory at UCLA), rabbit antiKir4.1 (1:1,500; Alomone, APC-035), rabbit anti-DARPP-32 (1:200; Abcam, ab40801) or mouse anti-HA (1:1,000; Covance, MMS-101R).

Techniques: Expressing, Fluorescence, MANN-WHITNEY, Immunostaining, Western Blot, Inhibition

Journal: Neuron

Article Title: Reducing astrocyte calcium signaling in vivo alters striatal microcircuits and causes repetitive behavior

doi: 10.1016/j.neuron.2018.08.015

Figure Lengend Snippet: (A) Hierarchical cluster dendrogram with colors underneath denoting three distinct modules of co-expressed transcripts in RiboTag mice RNA-seq data. (B) Heat map showing module-trait relationships, each row representing a module. Correlation coefficients between a Module Eigengene (ME) and trait (coded from −1 to 1) and corresponding P values are shown in each cell. The color indicates the level of correlation (positive correlation in red and negative correlation in green). Individual plots showing the expression of the module Eigengene across sample categories with significantly enriched terms for biological processes revealed by gene ontology enrichment analysis (FDR < 0.05). (C) Cluster dendrogram identified 16 co-expressed WGCNA modules for RiboTag AAV RNA-seq data. (D) Module-trait relationships revealed modules correlated with hPMCA2w/b expression in striatal astrocytes. Green module was composed of genes regulating protein transport, which were down-regulated in striatal astrocytes expressing hPMCA2w/b. Pink module was enriched in genes regulating chromatin silencing and transcription, which were upregulated in striatal astrocytes expressing hPMCA2w/b. (E) Expression levels of GABA transporters: Slc6a1 (GAT-1), Slc6a13 (GAT-2), Slc6a11 (GAT-3) and Slc6a12 (GAT-4) in RiboTag mice and RiboTag AAV RNA-seq data sets. (F) Expression levels of potential GABA transporter regulators suggested by previous work in RiboTag mice and RiboTag AAV RNA-seq data. (G) Changes in gene expression of striatal astrocytes expressing hPMCA2w/b, expressed as log2 (fold-change), in RiboTag mice and RiboTag AAV RNA-seq data. Asterisk (*) indicates differential expression between hPMCA2w/b IP and control IP using edgeR analysis on combined RNA-seq datasets (FDR < 0.1). (H-I) Cartoon summary of the main findings at synaptic (H) and in vivo levels (I). (J) Descriptive model based on our work (see discussion). Average data shown as mean ± SEM from 4 mice in each group.

Article Snippet: Serial 40 μm sections were collected and incubated with the following primary antibodies overnight at 4°C: rabbit anti-S100β (1:1,000; Abcam, ab41548), mouse anti-NeuN (1:1,000; Millipore, MAB377), chicken anti-GFP (1:1,000; Abcam, ab13970), rabbit anti-RFP (1:1,000; Rockland, 600–401-379), chicken anti-RFP (1:1,000; a gift from Dr. Brecha’s laboratory at UCLA), rabbit anti-GAT-3 (1:500; a gift from Dr. Brecha’s laboratory at UCLA), rabbit antiKir4.1 (1:1,500; Alomone, APC-035), rabbit anti-DARPP-32 (1:200; Abcam, ab40801) or mouse anti-HA (1:1,000; Covance, MMS-101R).

Techniques: RNA Sequencing Assay, Expressing, In Vivo

Journal: Experimental neurology

Article Title: A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function

doi: 10.1016/j.expneurol.2019.112973

Figure Lengend Snippet: (A) Schematic representation of GAT-1 protein topology and locations of GAT-1 variants identified in patients associated with a spectrum of epilepsy syndromes. It is predicted that GAT-1 contains 12 transmembrane domains. G234S is located at the junction of the second intracellular loop and the 5th transmembrane domain of the GAT-1 protein. The positions of variants are based on the published LeuT crystal structure. (B) Amino acid sequence homology shows that glycine (G) at residue 234 is highly conserved in SCL6A1 in human (Accession NO. NP_003033.3) and across species. (C) Tertiary structures of both the wildtype and G234S mutant protein GAT-1 are predicted by I-TASSER. Residue 234 is highlighted as red and Glycine is mutated to Serine. (D) From interatomic interactions predictions by DynaMut, wild-type (upper) and G234S mutation (bottom) residues are colored in light-green and are also represented as sticks alongside with the surrounding residues. Halogen bonds are depicted in blue. Hydrogen bonds are colored in red. Machine learning methods as Supplementary Table 1 predicted this mutation destabilized the global conformation of the GAT-1 protein.

Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone, 1:200) or mouse monoclonal GFP (Millipore, Billerica, MA, 1:200).

Techniques: Sequencing, Mutagenesis

Journal: Experimental neurology

Article Title: A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function

doi: 10.1016/j.expneurol.2019.112973

Figure Lengend Snippet: A-B. HEK293T cells were transfected with GAT-1YFP (3μg) for 48 hrs. (A) Total lysates were analyzed by SDS-PAGE and western blot. The membranes were blotted with mouse anti-GFP antibody. (B) Rat cortical neurons were transfected with the wildtype or the mutant GAT-1(G234S)YFP cDNAs at day 7 days old in cultured dish. The total lysates were harvested from rat cortical neurons expressing the wildtype GAT-1YFP (wt) or mutant GAT-1(G234S)YFP (G234S) transporters after 8 days of transfection. The total lysates were then analyzed by SDS-PAGE. In HEK 293T cells (A), three protein bands were detected in both the wildtype and the mutant conditions. In rat cortical neurons (B), only a single strong band was detected in both the wildtype and the mutant conditions. In A and B, 1:500 means the ratio of the GFP antibody in buffer (1μg of GFP in 500 μl 1XPBS). (C, D). The total protein integrated density values (IDVs) were measured. The abundance of the mutant (GAT-1(G234S) transporter was normalized to the wildtype condition. In C, the total protein abundance was measured by adding up all the three bands run between 90–110 KDa. In both C and D, the total protein IDVs of either the wildtype or the mutant was normalized to its loading control. The abundance of the mutant transporter was then normalized to the wildtype. (***p < 0.001 vs. wt, n=4 different transfections).

Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone, 1:200) or mouse monoclonal GFP (Millipore, Billerica, MA, 1:200).

Techniques: Transfection, SDS Page, Western Blot, Mutagenesis, Cell Culture, Expressing

Journal: Experimental neurology

Article Title: A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function

doi: 10.1016/j.expneurol.2019.112973

Figure Lengend Snippet: (A) The flow cytometry histograms depict surface expression levels of GAT-1 from HEK293T cells were transfected with wildtype GAT-1YFP (wt), or the mutant GAT-1(G234S)YFP cDNAs or control (insert). Cell surface wild type and mutant GAT-1 stained with polyclonal anti-GAT-1 antibody that was fluorescently conjugated with Alexa Fluor-555. (B) Surface protein was isolated by biotinylating live cells expressing wildtype GAT-1YFP (wt), or the mutant GAT-1(G234S)YFP and analyzed by SDS-PAGE. The membranes were visualized by polyclonal anti-GAT-1 protein. (C) The normalized relative fluorescence intensity (FI) (C) or the surface protein intensity values (IDVs) (D) were normalized to those obtained with wildtype GAT-1 while the FI or protein IDVs in the wildtype was arbitrarily taken as 1 in each experiment (***p < 0.001 vs. wt, n=4 different transfections). (E). The GABA uptake assay was carried out with 3H radioactive GABA transport in HeLa cells transiently expressing the wildtype or the mutant GAT-1YFP for 48 hrs. GABA flux was measured after 15 min transport at room temperature (**p < 0.01 vs. wt, n=6 for wildtype and 7 for the mutant which represents number of transfections).

Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone, 1:200) or mouse monoclonal GFP (Millipore, Billerica, MA, 1:200).

Techniques: Flow Cytometry, Expressing, Transfection, Mutagenesis, Staining, Isolation, SDS Page, Fluorescence

Journal: Stem Cells International

Article Title: Purinergic Signaling Pathway in Human Olfactory Neuronal Precursor Cells

doi: 10.1155/2019/2728786

Figure Lengend Snippet: Detection of P2 receptors in the clonal culture of the hOE. Cloned cells were scraped with a RIPA buffer, and cell lysates were assayed by Western blot to immunodetect P2 receptor subtypes. Representative chemiluminescent bands corresponding to P2X receptors are shown in (a) and to P2Y receptors in (b). (c) shows antibody specificity controls assessed by omission of primary antibody (P2X3) or by preadsorption of anti-P2X5 and anti-P2Y2 with the corresponding blocking peptides. The molecular weights of sample bands were corroborated using biotinylated molecular weight standards. (d) shows immunodetection of P2Y2 and P2Y4 receptors in both the fresh exfoliate (FE) sample and the clonal culture (CC) from the hOE.

Article Snippet: The primary antibodies were as follows: P2X1 (PVDF; Abcam ab81122; 1 : 1500), P2X2 (PVDF; Abcam ab10266; 1 : 500), P2X3 (PVDF; Abcam ab90905; 1 : 1000), P2X4 (PVDF; Alomone APR-002; 1 : 1000), P2X5 (NITRO; Alomone APR-027; 1 : 500), P2X7 (NITRO; Alomone APR-004; 1 : 500), P2Y2 (PVDF; Alomone APR-010; 1 : 1000), P2Y4 (PVDF; Alomone APR-006; 1 : 1000), P2Y6 (NITRO; Alomone APR-011; 1 : 500), and P2Y11 (NITRO; Santa Cruz sc-98600; 1 : 500).

Techniques: Clone Assay, Western Blot, Blocking Assay, Molecular Weight, Immunodetection

Journal: Molecular Pain

Article Title: Systemic administration of a β2-adrenergic receptor agonist reduces mechanical allodynia and suppresses the immune response to surgery in a rat model of persistent post-incisional hypersensitivity

doi: 10.1177/1744806921997206

Figure Lengend Snippet: Effects of β2 adrenergic receptor (AR) agonist on enhanced spinal microglia activation in a rat model of persistent postoperative pain. Sections of spinal cord were collected from rats 8 days following plantar incision and following treatment with DβH-saporin to deplete spinal noradrenergic terminals or control IgG-saporin. Rats were chronically administered clenbuterol (0.5 mg/kg, 2×/day, i.p.) or saline vehicle 6 days prior to and for 8 days after plantar incision. Depletion of spinal noradrenergic fibers was verified immunohistochemically with an antibody against dopamine β hydroxylase (DβH, (a)–(c)). Representative confocal images of IBA1-IR (blue, (d)–(f)) and phospho-p38 MAPK-IR (purple, (g)–(i)) in the ipsilateral spinal cord of incision rats. Localization of p38 MAPK in microglia was confirmed by colocalization with an antibody against the cell surface antigen CD11b (green, inset in (h)). Quantification of IBA1-IR in ipsilateral and contralateral spinal cord of rats with incision (j). Data represent mean ± SEM, n = 3 rats per group. Two way ANOVA indicated effect of group: p < 0.001 but not side p = 0.184 or interaction: p = 0.59 with SNK pairwise comparisons *p < 0.001 versus Incision+ DβH-saporin+ vehicle. Quantification of phospho-p38 MAPK microglial in the ipsilateral and contralateral spinal cord of rats with incision (k). Data represent mean ± SEM, n = 3 rats per group. Two way ANOVA indicate effect of group: p = 0.002 but not side p = 0.398 or interaction: p = 0.67 with SNK pairwise comparisons * p < 0.005 versus Incision+ DβH-saporin+ vehicle.

Article Snippet: We used a previously characterized antibody against β2 adrenergic receptors (AAR-016, β2-AR, 1:1000, rabbit anti-mouse, Alomone Labs; Jerusalem, Israel).

Techniques: Activation Assay

Journal: Molecular Pain

Article Title: Systemic administration of a β2-adrenergic receptor agonist reduces mechanical allodynia and suppresses the immune response to surgery in a rat model of persistent post-incisional hypersensitivity

doi: 10.1177/1744806921997206

Figure Lengend Snippet: Beta 2-adrenergic receptor immunoreactivity in the spinal cord of rats under naïve conditions and two days following plantar incision. Transverse section of L4 spinal cord of rat reacted with antibody against β2 adrenergic receptor ((a), β2-AR, green). There is a high density of immunoreactivity in cellular profiles throughout dorsal and ventral horn. There is also dense immunoreactivity in axon terminals within the lateral portion of the superficial laminae (arrow) and ependymal cells in the vicinity of the central canal (Arrowhead). Note lack of staining for β2-AR in motor neurons within the ventral horn (asterisk). Higher magnification confocal images show β2-AR-IR ((c), green) is present in a subpopulation of neurons ((d), NeuN, purple) in the dorsal spinal cord. Most β2-AR-IR cellular profiles colocalized with NeuN with the exception of a few non-neuronal profiles with morphology typical of microglia (arrows, (c)–(f)). β2-AR-IR non-neuronal cellular profiles in the spinal cord colocalized with the microglial marker IBA1 (red, (e) and (f)). Arrows in F indicate IBA1 negative neuronal cellular profiles. Representative images of β2 mRNA and DAPI in the dorsal spinal cord (g) with high power image showing colocalization with a subset of nuclei (h).

Article Snippet: We used a previously characterized antibody against β2 adrenergic receptors (AAR-016, β2-AR, 1:1000, rabbit anti-mouse, Alomone Labs; Jerusalem, Israel).

Techniques: Staining, Marker

Journal: Molecular Pain

Article Title: Systemic administration of a β2-adrenergic receptor agonist reduces mechanical allodynia and suppresses the immune response to surgery in a rat model of persistent post-incisional hypersensitivity

doi: 10.1177/1744806921997206

Figure Lengend Snippet: β2-adrenergic receptor immunoreactivity (β2AR-IR) in hindpaw of rats under naïve conditions and following plantar incision. Skin sections were obtained from the hind paw of naïve rats and incision rats two days following surgery. Sixteen-μm-thick sections were stained with antibodies against β2AR-IR (green, (a)–(c)), IBA1 (red, (d)–(f)) to label all monocytes/and macrophage, CD68 (blue, (g)–(i)) for activated M1 macrophage) and DAPI ((j) and (k)) to label all nuclei. β2-AR IR was present in keratinocytes of both naïve and incision rats. Two days following plantar incision there were increased β2-AR IR cellular profiles in predominantly the dermal layers of the skin. Higher magnification confocal images ((c), (f), (i), and (l)) indicate colocalization of β2-AR in IBA1 + cells and a subset of which express CD68-IR. Note in naïve skin IBA1-IR was primarily present at the epidermal/dermal interface and had reduced dermal cellularity (DAPI + cells) compared to skin adjacent to the wound in incision rats.

Article Snippet: We used a previously characterized antibody against β2 adrenergic receptors (AAR-016, β2-AR, 1:1000, rabbit anti-mouse, Alomone Labs; Jerusalem, Israel).

Techniques: Staining