Journal: eLife

Article Title: R7 photoreceptor axon targeting depends on the relative levels of lost and found expression in R7 and its synaptic partners

doi: 10.7554/eLife.65895

Figure Lengend Snippet:

Article Snippet: Guinea pig anti-Runt was made by GenScript using the full-length protein as an antigen.

Techniques: CRISPR, Recombinant, Sequencing, Software

Journal: bioRxiv

Article Title: Antiviral Drug Screen of Kinase inhibitors Identifies Cellular Signaling Pathways Critical for SARS-CoV-2 Replication

doi: 10.1101/2020.06.24.150326

Figure Lengend Snippet: (A) Bright field images of Vero-E6 cells infected with SARS-CoV-2 virus. Viral cytopathic effects are noted in the infected culture. 10X magnification. (B) IFA images of infected cells (48 hpi) stained for SARS-CoV-2. Mouse monoclonal antibody (MS Ab) targeting Spike and a guinea pig polyclonal SARS-CoV antibody (GP) were used. Mock infected cells were included as negative control. 20X magnification. (C) IFA images show compounds with antiviral activity. Images depict that hydroxychloroquine (HQ) drug efficiently inhibited SARS-CoV-2 infection. The mock and infected cells were immunostained with dsRNA antibody, which recognizes double stranded genomic RNA generated during viral replication. 20X magnification. Representative data from three or more independent experiments are presented.

Article Snippet: At 48 hours post infection (hpi), viral infection was examined by immunocytochemistry (ICC) analysis using SARS-CoV Spike (S) antibodies [BEI Resources: NR-10361 Polyclonal Anti-SARS Coronavirus (antiserum, Guinea Pig), and NR-616 Monoclonal Anti-SARS-CoV S Protein (Similar to 240C) SARS coronavirus].

Techniques: Infection, Staining, Negative Control, Activity Assay, Generated

Journal: bioRxiv

Article Title: Antiviral Drug Screen of Kinase inhibitors Identifies Cellular Signaling Pathways Critical for SARS-CoV-2 Replication

doi: 10.1101/2020.06.24.150326

Figure Lengend Snippet: (A) Immunofluorescent images of SARS-CoV-2 (red) infected cells treated with indicated drug compounds at various concentrations. (B) Graphs show percent inhibition of SARS-CoV-2 infectivity by indicated compounds. Note: IC50 of each compound is shown in the graph. Representative data from two independent experiments are presented.

Article Snippet: At 48 hours post infection (hpi), viral infection was examined by immunocytochemistry (ICC) analysis using SARS-CoV Spike (S) antibodies [BEI Resources: NR-10361 Polyclonal Anti-SARS Coronavirus (antiserum, Guinea Pig), and NR-616 Monoclonal Anti-SARS-CoV S Protein (Similar to 240C) SARS coronavirus].

Techniques: Infection, Inhibition

Journal: bioRxiv

Article Title: Antiviral Drug Screen of Kinase inhibitors Identifies Cellular Signaling Pathways Critical for SARS-CoV-2 Replication

doi: 10.1101/2020.06.24.150326

Figure Lengend Snippet: (A) DMSO Vehicle treated Vero-E6 cells had pronounced viral CPE at 48 hpi. Uninfected cells (Mock+vehicle) are included as negative control. (B) Bright field microscopic images (10X) of drug compounds treated SARS-CoV-2 infected cells showing no or reduced level of viral CPE.

Article Snippet: At 48 hours post infection (hpi), viral infection was examined by immunocytochemistry (ICC) analysis using SARS-CoV Spike (S) antibodies [BEI Resources: NR-10361 Polyclonal Anti-SARS Coronavirus (antiserum, Guinea Pig), and NR-616 Monoclonal Anti-SARS-CoV S Protein (Similar to 240C) SARS coronavirus].

Techniques: Negative Control, Infection

Journal: bioRxiv

Article Title: Antiviral Drug Screen of Kinase inhibitors Identifies Cellular Signaling Pathways Critical for SARS-CoV-2 Replication

doi: 10.1101/2020.06.24.150326

Figure Lengend Snippet: IFA images show SARS-CoV-2 (Red) infection in untreated cells (A) and hydroxychloroquine (10 µM) treated cells (B). (C) Complete reduction of SARS-CoV-2 infection in Berzosertib (100nM) treated cells. 20x magnification.

Article Snippet: At 48 hours post infection (hpi), viral infection was examined by immunocytochemistry (ICC) analysis using SARS-CoV Spike (S) antibodies [BEI Resources: NR-10361 Polyclonal Anti-SARS Coronavirus (antiserum, Guinea Pig), and NR-616 Monoclonal Anti-SARS-CoV S Protein (Similar to 240C) SARS coronavirus].

Techniques: Infection

Journal: bioRxiv

Article Title: Antiviral Drug Screen of Kinase inhibitors Identifies Cellular Signaling Pathways Critical for SARS-CoV-2 Replication

doi: 10.1101/2020.06.24.150326

Figure Lengend Snippet: (A) Graph shows viral titer (TCID50/25ul) of supernatant collected at indicated timepoints after SARS-CoV-2 infection of drug treated hiPSC-CMs. (B) IFA images of cells undergoing apoptosis assessed by cleaved caspase-3 staining on drug treated hiPSC-CMs at 72 hpi. Scale bar=25 µm. (C) Graph depicts quantification of SARS-CoV-2 and cleaved caspase-3 positive cells. (D) hiPSC-CMs stained for cardiac marker cardiac troponin T (cTnT) shows cells are protected from SARS-CoV-2 infection by drugs Berzosertib (250nM) and HQ (10µM). Scale bar=25 µm. (E). Graph shows beats per minute of SARS-CoV-2 infected hiPSC-CM cells treated with Berzosertib (250nM) (pre- and post -treatment), Remdesivir (10 µM), and HQ (10 µM). Statistical analysis of graphs (C, E): Multiple-comparison one-way analysis of variance (ANOVA) was conducted (***, P < 0.0001; **, P < 0.001). Representative data from two independent experiments are presented.

Article Snippet: At 48 hours post infection (hpi), viral infection was examined by immunocytochemistry (ICC) analysis using SARS-CoV Spike (S) antibodies [BEI Resources: NR-10361 Polyclonal Anti-SARS Coronavirus (antiserum, Guinea Pig), and NR-616 Monoclonal Anti-SARS-CoV S Protein (Similar to 240C) SARS coronavirus].

Techniques: Infection, Staining, Marker

Journal: bioRxiv

Article Title: Antiviral Drug Screen of Kinase inhibitors Identifies Cellular Signaling Pathways Critical for SARS-CoV-2 Replication

doi: 10.1101/2020.06.24.150326

Figure Lengend Snippet: (A) Chemical structure of compound Berzosertib is shown. (B) Western blot analysis shows phosphorylation of key DNA-Damage Response (DDR) proteins: CHK1, CHK2, and H2A.X. (C) Schematic illustration demonstrates the key pathways identified in the kinase inhibitor drug screen having critical role in SARS-CoV-2 infection. Drug compounds and their respective target kinase pathways (mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-Damage Response) are described.

Article Snippet: At 48 hours post infection (hpi), viral infection was examined by immunocytochemistry (ICC) analysis using SARS-CoV Spike (S) antibodies [BEI Resources: NR-10361 Polyclonal Anti-SARS Coronavirus (antiserum, Guinea Pig), and NR-616 Monoclonal Anti-SARS-CoV S Protein (Similar to 240C) SARS coronavirus].

Techniques: Western Blot, Infection

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Sound Rhythms Are Encoded by Postinhibitory Rebound Spiking in the Superior Paraolivary Nucleus

doi: 10.1523/JNEUROSCI.2450-11.2011

Figure Lengend Snippet: SPON neurons express HCN1 and HCN2 channels and have large Ih inwardly rectifying (IR) currents. A, HCN1 immunoreactivity in the superior olivary complex of an adult mouse. The low-magnification micrograph illustrates that the lateral superior olive expresses the strongest HCN1-like immunostaining, the SPON displays moderate levels of expression, and the MNTB shows the weakest immunoreactivity. At higher magnification, it is evident that both somata (arrowheads) and dendrites (arrow) of SPON neurons are immunopositive. Asterisks denote landmarks in slice. Scale bars: left, 200 μm; right, 50 μm. B) HCN2 is most strongly expressed in the MNTB, whereas expression in the LSO is weakest. At higher magnification, it is evident that SPON neurons are also immunopositive. Asterisks denote landmarks in slice. Scale bars: left, 200 μm; right, 50 μm. C, Current traces recorded from an SPON neuron induced by hyperpolarization from a holding current of −62 to −122 mV, in −10 mV voltage steps, under control conditions (top) and during pharmacological blockade of Ih with 20 μM ZD7288 (bottom). D, The size of the IR current was measured at steady state (i.e., ~1.35 s after induction) at each hyperpolarizing voltage (n = 42). The IR currents were significantly diminished by ZD7288 (n = 6) in the SPON neurons. ***p < 0.001, Student’s t test. E, Average activation time constants of the IR currents in SPON neurons were obtained by fitting a single-exponential function to the current traces.

Article Snippet: Sections were incubated in 2% normal donkey serum in blocking solution overnight at 4°C with one of the following primary antibodies: polyclonal rabbit α -HCN1 (1:250, lot number AN-10; Alomone Labs), which is directed against amino acid residues 6–24 of the intracellular N terminus of rat HCN1 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9JKB0","term_id":"29840774","term_text":"Q9JKB0"}} Q9JKB0 ); polyclonal rabbit α -HCN2 (1:400, lot number AN-08; Alomone Labs), directed against amino acids 147–161 of the intracellular N terminus of human HCN2 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9UL51","term_id":"108935843","term_text":"Q9UL51"}} Q9UL51 ); or monoclonal mouse α -HCN2 (1:400, clone N71/37; NeuroMab, UC Davis/NIH NeuroMab Facility) directed against amino acids 761–863 of the C terminus of rat HCN2 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9JKA9","term_id":"83303515","term_text":"Q9JKA9"}} Q9JKA9 ) used in combination with a mouse-on-mouse kit (Vector Laboratories).

Techniques: Immunostaining, Expressing, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: The D1R–NMDAR interaction bidirectionally regulates the surface distribution and dynamics of D1R and NMDAR. (A) Immunostaining of surface D1R-CFP (green) and GluN1 subunit (red) in hippocampal neurons. The yellow arrow shows overlay. (B) Immunostaining of surface D1R-CFP in control or after D1/5R agonist, TAT-t2, or TAT-[N2A15] application. (Scale bar, 250 nm.) (C) Normalized measures of D1R-CFP clusters intensity in control (n = 32 neuronal fields), D1/5R agonist-treated (n = 24 neuronal fields; *P < 0.05 compared with control), TAT-NSt2–treated (non-sense of TAT-t2, n = 19 neuronal fields), TAT-t2–treated (n = 21 neuronal fields; **P < 0.01 compared with TAT-NSt2), TAT-NSt3–treated (non-sense of TAT-t3, n = 11 neuronal fields), TAT-t3–treated (n = 12 neuronal fields; P > 0.05 compared with TAT-NSt3), TAT-[NS15]–treated (n = 27 neuronal fields; P > 0.05), or TAT-[N2A15]–treated (n = 21 neuronal fields, *P < 0.05 compared with TAT-[NS15]) conditions. (D) Representative trajectories (1,000 frames, 20-Hz acquisition rate) of surface single D1R-CFP (Left) (green) (scale bar, 400 nm) and GluN1-NMDAR (Right) (blue) (scale bar, 300 nm) in the absence and presence of either D1/5R agonist (10 µM, 15 min) or TAT-t2 (10 µM, 15 min). Bold dotted line, perisynaptic area; thin dotted line, PSD area. (E) Plot of the MSD of surface D1R-CFP (Upper) (green) and GluN1-NMDAR (Lower) (blue) versus time in presence of TAT-NS or TAT-t2 peptides (10 µM, 15 min). The SEM is included for each data point (D1R: TAT-NS, n = 986 trajectories, and TAT-t2, n = 1,326; GluN1-NMDAR: TAT-NS, n = 198, and TAT-t2, n = 134). (F and G) Representative surface distributions of single D1R-CFP (green) (F) and GluN1-NMDAR (blue) (G) in the synaptic area (PSD + perisynaptic area) in control, D1/5R agonist, and TAT-t2 conditions. Each dot represents the detection of a single receptor during a frame. Comparisons of the time spent in the synaptic area (dwell time) by single D1R-CFP (control, n = 173 trajectories; D1/5R agonist, n = 142, **P < 0.01; TAT-t2, n = 752, *P < 0.05) (F) and GluN1-NMDAR (control, n = 189 trajectories; D1/5R agonist, n = 157, *P < 0.05; TAT-t2, n = 134, **P < 0.01) (G) and the synaptic fraction of detected single D1R-CFP (control, n = 14 neuronal fields; D1/5R agonist, n = 19, **P < 0.01; D1/5R agonist in the presence of dynasore, n = 47, **P < 0.01; TAT-t2, n = 15, ***P < 0.001) (F), D5R-CFP (n = 16, P > 0.05) (F), and GluN1-NMDAR (control, n = 11; D1/5R agonist, n = 15, *P < 0.05; TAT-t2, n = 14, *P < 0.05) (G). Dyn., dynasore; D1/5 ago., D1/5 receptor agonist SKF-38393.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: D1R activation or D1R/GluN1-NMDAR interaction blockade increases synaptic NMDAR content and favors AMPAR synaptic long-term potentiation. (A) (Left) Excitatory postsynaptic current traces recorded at −70 mV and +40 mV from a representative hippocampal CA1 pyramidal cell, before and 10 min after exposure to D1/5R agonist. (Right) Relative change over time of the AMPA/NMDA ratio at CA1 synapses in the absence or presence of D1/5R agonist (n = 13, *P < 0.05 10 min after agonist) and in the absence or presence of vehicle (n = 7, P > 0.05). (B) Surface imaging of GluN1-SEP in neurons incubated with either TAT-NS or TAT-t2 (10 µM). (Scale bar, 5 µm.) (Right) Average value of GluN1-SEP content in the synaptic area after TAT-NS or TAT-t2 application (n = 8 neurons per group, **P < 0.01). (C) Dendritic fragment of a hippocampal neuron expressing Homer 1c-DsRed (Upper) and GluA1-SEP (Lower). SEP only fluoresces at neutral pH when receptors are inserted at the plasma membrane. Ten minutes after chemical LTP induction (cLTP), the GluA1-SEP fluorescence intensity increased in postsynaptic clusters. (Insets) High magnification of a synaptic GluA1-SEP cluster. (Scale bar, 2 µm.) (D) Comparison of the synaptic GluA1-SEP fluorescence intensity before and after cLTP with prior TAT-NS (n = 198 synapses, *P < 0.05) or TAT-t2 (n = 215 synapses, *P < 0.05) (TAT-NS versus TAT-t2; *P < 0.05) application. (E) Schematic model of the D1R–NMDAR surface interplay in hippocampal neurons. D1Rs are highly diffusive at the neuronal surface and are dynamically retained in clusters in the vicinity of glutamate synapses where they interact with NMDAR. Dopamine release disrupts this interaction and favors the lateral redistribution of both receptors: D1Rs freely explore extrasynaptic areas, whereas NMDARs laterally reach the PSD where they impact on the long-term plasticity of glutamate synapses.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Activation Assay, Imaging, Incubation, Expressing, Fluorescence

Journal: Journal of Neuroendocrinology

Article Title: Vasoactive Intestinal Polypeptide Contacts on Gonadotropin-Releasing Hormone Neurones Increase Following Puberty in Female Rats

doi:

Figure Lengend Snippet: High-power light photomicrographs of representative interactions between vasoactive intestinal polypeptide (VIP) and gonadotropin-releasing hormone (GnRH) neurones in both (A) prepubertal and (B) adult female rats (section thickness=30 μm). Confocal microscopy photomicrographs of 0.5 μm optical sections of representative interactions between VIP and GnRH neurones in both (C) prepubertal and (D) adult female rats. Red (GnRH) and Green (VIP) channels are separated in the confocal image (and shown together) to clearly indicate the extent of VIP fibres on the same 0.5 μm plane as the GnRH cell body. Green=VIP; Red=GnRH in all images.

Article Snippet: For visualization of GnRH, sections were washed in PBS, incubated in 1% H 2 O 2 , and then incubated in normal donkey serum in 0.1% Triton X-100 (PBT) for 1 h. Sections were then incubated for 48 h at 4 °C in guinea-pig anti-mammalian GnRH (antigenic determinants are amino acids 6–10; Advanced Chemtech, Louisville, KY, USA) diluted at 1 : 10 000 with 0.1% PBT.

Techniques: Confocal Microscopy

Journal: Journal of Neuroendocrinology

Article Title: Vasoactive Intestinal Polypeptide Contacts on Gonadotropin-Releasing Hormone Neurones Increase Following Puberty in Female Rats

doi:

Figure Lengend Snippet: Mean±SEM percentage of gonadotropin-releasing hormone (GnRH) neurones contacted by vasoactive intestinal polypeptide (VIP)-immunoreactive (ir) axons in prepubertal female rats and adult females in diestrus. (A) Total percentage of brain GnRH-ir neurones contacted by VIP-ir axons and (B) regional distribution of the percentage of GnRH-ir cells contacted. *P<0.05, †P<0.01 prepubertal.

Article Snippet: For visualization of GnRH, sections were washed in PBS, incubated in 1% H 2 O 2 , and then incubated in normal donkey serum in 0.1% Triton X-100 (PBT) for 1 h. Sections were then incubated for 48 h at 4 °C in guinea-pig anti-mammalian GnRH (antigenic determinants are amino acids 6–10; Advanced Chemtech, Louisville, KY, USA) diluted at 1 : 10 000 with 0.1% PBT.

Techniques:

Journal: Journal of Neuroendocrinology

Article Title: Vasoactive Intestinal Polypeptide Contacts on Gonadotropin-Releasing Hormone Neurones Increase Following Puberty in Female Rats

doi:

Figure Lengend Snippet: Mean±SEM number of gonadotropin-releasing hormone (GnRH) cells in prepubertal and adult females rats. (A) Total number of brain GnRH-immunoreactive (ir) neurones and (B) regional distribution of the number of GnRH-ir cells.

Article Snippet: For visualization of GnRH, sections were washed in PBS, incubated in 1% H 2 O 2 , and then incubated in normal donkey serum in 0.1% Triton X-100 (PBT) for 1 h. Sections were then incubated for 48 h at 4 °C in guinea-pig anti-mammalian GnRH (antigenic determinants are amino acids 6–10; Advanced Chemtech, Louisville, KY, USA) diluted at 1 : 10 000 with 0.1% PBT.

Techniques: