Journal: British Journal of Pharmacology

Article Title: Selective block of adenosine A 2A receptors prevents ischaemic‐like effects induced by oxygen and glucose deprivation in rat medium spiny neurons

doi: 10.1111/bph.15922

Figure Lengend Snippet: The A 2A receptor antagonist SCH58261 delayed the appearance of anoxic depolarization (AD) induced by OGD in rat striatal medium spiny neurons. (a–e) . Pooled data (median ± 95% confidence interval: CI) of AD latency (measured as the time needed for I h to change more that 20% from OGD start: (a) ), AD amplitude (measured as the difference between pre‐OGD I h value and the AD peak: (b) ), E rev changes during OGD (measured as the difference between pre‐OGD E rev value and the value reached during the AD peak: (c) ), the latency to initiate the spontaneous action potential (AP) burst (when detected) (d) and the latency between I h change (more than 20%) and AD peak (e) in striatal medium spiny neurons (MSNs) in different experimental conditions: untreated OGD slices (ctrl; blue circles) or slices subjected to OGD in the presence of different pharmacological treatments: the A 2A receptor antagonist SCH58261 (SCH, 10 μM; yellow circles); the A 2A receptor agonist CGS21680 (CGS, 1 μM; orange circles); the K + channel blocker Ba 2+ (2 mM; purple circles) or Na + channel blocker tetrodotoxin (TTX, 1 μM; green circles). Each compound was added at least 10 min before OGD and throughout the insult. Note that the proportion of MSNs presenting the burst of APs during OGD is reported below each column in panel (d) . (f, g) Averaged time courses of I h ( f ) or E rev ( g ) before or during OGD performed in different experimental groups. * P < 0.05; Dunn's multiple comparisons test

Article Snippet: Tetrodotoxin (TTX) was purchased from Ascent Scientific (Bristol, UK) or Alomone Labs (Jerusalem, Israel).

Techniques:

Journal: British Journal of Pharmacology

Article Title: Selective block of adenosine A 2A receptors prevents ischaemic‐like effects induced by oxygen and glucose deprivation in rat medium spiny neurons

doi: 10.1111/bph.15922

Figure Lengend Snippet: Oxygen and glucose deprivation (OGD) caused a decrease in the frequency of spontaneous, but not miniature, excitatory post‐synaptic currents in medium spiny neurons, an effect prevented by K + channel block and by the selective stimulation of A 2A receptors. (a) Averaged time courses of spontaneous or miniature EPSCs (sEPSCs or mEPSCs, respectively) event frequency recorded in MSNs subjected to OGD in different experimental conditions: in control conditions (ctrl: blue circles; n = 12); in the presence of the A 2A receptor antagonist SCH58261 (SCH, 10 μM: yellow circles; n = 5); in the presence of the A 2A receptor agonist CGS21680 (CGS, 1 μM: orange circles; n = 5); in Ba 2+ (2 mM: purple circles; n = 5) or in tetrodotoxin (TTX, 1 μM: green circles; n = 5). (b, c) Pooled data (median ± 95% confidence interval: CI) of event frequency (b) or amplitude (c) measured during the last 2 min before OGD (pre‐OGD) or between 3 and 5 min OGD (OGD) in different experimental groups. * P < 0.05; Wilcoxon test

Article Snippet: Tetrodotoxin (TTX) was purchased from Ascent Scientific (Bristol, UK) or Alomone Labs (Jerusalem, Israel).

Techniques: Blocking Assay, Control

Journal: Neuron

Article Title: Dopamine Triggers the Maturation of Striatal Spiny Projection Neuron Excitability during a Critical Period

doi: 10.1016/j.neuron.2018.06.044

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-Tyrosine Hydroxylase monoclonal Millipore Cat # MAB5280 Rabbit anti-Red fluorescent protein polyclonal Rockland Cat # 600-401-379 Rabbit anti-DARPP32 monoclonal Cell Signaling Technology Cat # 2306S Mouse anti-beta actin monoclonal Novus Biologicals Cat # NB600-501 Mouse anti-Kir2.1 monoclonal Antibodies Incorporated Item # 73-210; RRID: AB_11000720 Mouse anti-Kir2.3 monoclonal Antibodies Incorporated Item # 75-069; RRID: AB_2130742 Mouse anti-K v 1.2 monoclonal Antibodies Incorporated Item # 75-008; RRID: AB_2296313 Rabbit anti- Phospho-(Ser/Thr) PKA Substrate Antibody polyclonal Cell Signaling Technology Cat # 9621S Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa 488 Invitrogen Cat # A-21202 Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa 594 Invitrogen Cat # A-21207 Donkey anti-Rabbit IgG (H+L) Secondary IRDye 680LT LI-COR P/N 925-68023 Goat anti-Mouse IgG (H+L) Secondary IRDye 800CW LI-COR P/N 925-32210 Streptavidin, Alexa 488 conjugate Invitrogen {"type":"entrez-protein","attrs":{"text":"S11223","term_id":"112468","term_text":"pir||S11223"}} S11223 Donkey anti-Mouse IgG (H+L) conjugated to HRP Jackson ImmunoResearch Code: 715-035-151 Chemicals, Peptides, and Recombinant Proteins Tetrodotoxin citrate Tocris Cat # 1069 Forskolin Tocris Cat # 1099/10 diC8-PIP2 Echelon Biosciences P-4508 Picrotoxin Tocris Cat # 1128/1G D-AP5 Tocris Cat # 0106 G418 disulfate salt (Neomycin) Tocris Cat # 4131/100 Neurobiotin tracer Vector Laboratories Cat # SP-1120 CNQX disodium salt Tocris Cat # 1045/1 Critical Commercial Assays BCA Protein Assay Kit Thermo Fisher Scientific Prod # 23227 Immobilon Western Chemiluminescent HRP Substrate Millipore Cat # WBKLS0500 Experimental Models: Organisms/Strains Mouse: Pitx3ak/2J The Jackson Laboratory Stock No: 000942 Mouse: B6.Cg-Tg(Drd1a-tdTomato)6Calak/J The Jackson Laboratory RRID: IMSR_JAX:016204 Mouse: WT: C57BL/6J The Jackson Laboratory RRID: IMSR_JAX:000664 Mouse: DAT KO Courtesy of Marc Caron Giros et al., 1996 Software and Algorithms GraphPad Prism GraphPad RRID: SCR_002798 Igor Wavemetrics RRID: SCR_000325 pClamp Molecular Devices RRID: SCR_011323 Image Studio Lite LI-COR RRID: SCR_014211 ImageJ NIH RRID: SCR_003070 Open in a separate window KEY RESOURCES TABLE Striatal dopamine release and projection neuron excitability mature postnatally Dopamine is required for the maturation of direct pathway SPN intrinsic excitability Maturation of direct pathway SPN excitability arises from PIP 2 -Kir2 interactions Dopamine must act during a critical period in development for this maturation

Techniques: Recombinant, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Western Blot, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor

doi: 10.3389/fcell.2020.598634

Figure Lengend Snippet: Effect of lssDNA 3′ homology arm length on knock-in efficiency. (A) Donor lssDNA templates with different 3′ homology arm lengths used for comparison. (B) Schematic illustration of the sox3 knock-in allele and knock-in allele-specific PCRs for 5′ and 3′ junctions. Each lssDNA was microinjected with 1.5 fmol of the RNP complex into the cytoplasm of one-cell stage zebrafish embryos, and genomic DNA was extracted from 20-embryo pools. (C) Agarose gel image showing the PCR amplicons of knock-in allele-specific PCRs and the β -actin2 gene-specific PCR (control) to confirm DNA integrity. (D) Knock-in allele-specific qPCRs for 5′ and 3′ junctions using the hydrolysis probes shown in (B) . The vertical bars represent the means of 8–10 replicates, each of which consists of a pooled sample of 10 injected embryos and is shown as a colored circle.

Article Snippet: The standard PCR conditions were the following: 95°C for 30 s; 30 cycles of 95°C for 15 s, 55–62°C for 30 s, and 68°C for 1 min/kb; and 68°C for 5 min. Knock-in allele-specific qPCR was performed using the primer pairs and hydrolysis probes listed in using the Hot Start TTx (DNA) Kit (Toyobo). qPCR for the hesx1 promoter region was performed with Luna Universal Probe qPCR Master Mix (New England BioLabs) and was used for normalization.

Techniques: Knock-In, Comparison, Agarose Gel Electrophoresis, Control, Injection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor

doi: 10.3389/fcell.2020.598634

Figure Lengend Snippet: Effect of lssDNA strand choice and 3′ homology arm length on composite tag knock-in into the sox11a and pax6a genes. In these knock-in designs, ∼200-nt-long composites that contain the HBH (His6-Bio-His6) tag followed by a TEV protease cleavage site and FLAG epitope tag in its trimeric form were knocked into the 5′ end of the coding sequence of the sox11a (A) and pax6a (B) genes. (a) Sequences and locations of crRNAs for DSB induction of sox11a and pax6a . (b) Target and non-target strands of lssDNA with different 3′ homology arm lengths were used as donor templates. Each lssDNA was microinjected with 1.5 fmol of the RNP complex into one-cell stage zebrafish embryos, and genomic DNA was extracted from 10-embryo pools. (c) Schematic illustration of the sox11a and pax6a knock-in alleles and knock-in allele-specific PCRs. (d) Knock-in allele-specific qPCRs for 5′ and 3′ junctions using the hydrolysis probes shown in panel (c) . The vertical bars represent the means of 7–11 replicates, each of which consists of a pooled sample of 10 injected embryos and is shown as a colored circle.

Article Snippet: The standard PCR conditions were the following: 95°C for 30 s; 30 cycles of 95°C for 15 s, 55–62°C for 30 s, and 68°C for 1 min/kb; and 68°C for 5 min. Knock-in allele-specific qPCR was performed using the primer pairs and hydrolysis probes listed in using the Hot Start TTx (DNA) Kit (Toyobo). qPCR for the hesx1 promoter region was performed with Luna Universal Probe qPCR Master Mix (New England BioLabs) and was used for normalization.

Techniques: Knock-In, FLAG-tag, Sequencing, Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Isolation and transfection of myenteric neurons from mice for patch-clamp applications

doi: 10.3389/fnmol.2022.1076187

Figure Lengend Snippet: Voltage-clamp recordings of Na V channels endogenously expressed in murine enteric neurons. (A) Families of current traces obtained from representative enteric neurons in the absence (I Na + total, black) or presence (I Na + TTXr, blue) of 1 μM TTX in response to a series of depolarizing test pulses (top) increasing from –127 to 23 mV in steps of 10 mV. Traces shown in red correspond to a test pulse voltage of –37 mV. (B) Peak current densities (open circles) recorded in the absence (I Na + total, black) or presence (I Na + TTXr, blue) of 1 μM TTX as a function of test pulse voltage, obtained from experiments as shown in panel (A) . Continuous curves are superimposed fits according to Eq. 1 describing the voltage dependence of activation of Na V channels. To facilitate comparison of both conditions, the data fit to currents in the presence of TTX is shown as blue dashed line in the left panel. (C) Families of current traces obtained from representative enteric neurons in the absence (I Na + total, black) or presence (I Na + TTXr, blue) of 1 μM TTX with test pulses to –37 mV, applied before (I 0 ) and after (I 500 ) a 500-ms conditioning period at voltages ranging from –147 to –7 mV in steps of 10 mV (top). (D) Ratio of peak currents measured before and after 500-ms conditioning (I 500 /I 0 ) as a function of conditioning voltage, obtained from experiments as shown in panel (C) to characterize the voltage dependence of steady-state inactivation of Na V channels. Superimposed fits are Boltzmann functions according to Eq. 2. In the presence of 1 μM TTX (I Na + TTXr, blue), inactivation of Na V channels displayed two components reflecting inactivation properties of different TTXr channel subtypes such as Na V 1.5 and Na V 1.9 , both of which are endogenously expressed in enteric neurons. In panels (A–D) the holding potential was –137 mV. Data points in panels (B,D) are means ± s.e.m. with numbers of experiments given in parentheses. Significance between pairs of data was tested with a two-sided Student’s t -test: *** P < 0.001, ** P < 0.01.

Article Snippet: Where indicated, the bath solution was further supplemented with 1 μM Tetrodotoxin (TTX) (Alomone Labs, Jerusalem, Israel) to block endogenous TTX-sensitive Na V channels.

Techniques: Activation Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Isolation and transfection of myenteric neurons from mice for patch-clamp applications

doi: 10.3389/fnmol.2022.1076187

Figure Lengend Snippet: Expression of recombinant human Na V 1.9 wild type and mutant channels in isolated mouse enteric neurons. (A) Representative families of current traces obtained from enteric neurons transfected with cDNA constructs coding for wild type Na V 1.9 (black) or Na V 1.9-L396P mutant channels (blue) in response to a series of depolarizing test pulses (top) increasing from –127 to 23 mV in steps of 10 mV. Red traces were recorded at –37 mV illustrating the different inactivation kinetics of wild type and mutant channels. (B) Peak current densities (open circles) recorded from neurons transfected with wild type Na V 1.9 (black) or Na V 1.9-L396P mutant channels (blue) as a function of test pulse voltage, obtained from experiments as shown in panel (A) . Continuous curves are superimposed fits according to Eq. 1 describing the voltage dependence of channel activation. For better comparison, the data fit to currents generated by neurons expressing mutant channels (blue dashed line) is superimposed in the left panel. (C) Families of current traces obtained from representative enteric neurons transfected with type Na V 1.9 (black) or Na V 1.9-L396P mutant channels (blue) in response to test pulses to –37 mV which were applied before (I 0 ) and after (I 500 ) a 500-ms conditioning period at voltages ranging from –147 to –7 mV in steps of 10 mV (top). (D) Ratio of peak currents measured before and after 500-ms conditioning (I 500 /I 0 ) as a function of conditioning voltage, obtained from experiments as shown in panel (C) to characterize the voltage dependence of steady-state inactivation of Na V channels. Superimposed fits are Boltzmann functions according to Eq. 2. Note, currents generated by cells expressing Na V 1.9-L396P mutant channels exhibit a biphasic voltage dependence of inactivation along with a pronounced non-inactivating component. All recordings were obtained in presence of 1 μM TTX, data points in (B,D) are means ± s.e.m. with numbers of experiments provided in parentheses. In panel (D) , significance between pairs of data was tested with a two-sided Student’s t -test: ** P < 0.01, * P < 0.05.

Article Snippet: Where indicated, the bath solution was further supplemented with 1 μM Tetrodotoxin (TTX) (Alomone Labs, Jerusalem, Israel) to block endogenous TTX-sensitive Na V channels.

Techniques: Expressing, Recombinant, Mutagenesis, Isolation, Transfection, Construct, Activation Assay, Generated