D ap5

Manufactured by Merck Group

Sourced in United Kingdom, United States

D-AP5 is a chemical compound used in scientific research. It functions as an N-methyl-D-aspartate (NMDA) receptor antagonist. D-AP5 is a tool used by researchers to study the role of NMDA receptors in various biological processes.

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23 protocols using d ap5

Pharmacological Modulation of Synaptic Plasticity

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NMDAR antagonist D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5, Cat#-0106), GABA type A receptor (GABA_AR) antagonist bicuculline (BIC; Cat#-0130), and positive mGluR5 allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB, Cat#-3235) were purchased from Tocris Bioscience (Minneapolis, MN). Negative mGluR5 allosteric modulator 2-chloro-4-([2,5-dimethyl-1-(4-[trifluoromethoxy]-phenyl)-1H-imidazol-4-yl]ethynyl)pyridine (CTEP,^{23 (link)} Cat#-B1633) was purchased from ApexBio Technology (Boston, MA), and mTOR inhibitor rapamycin (Cat#-R-5000) was purchased from LC Laboratories (Woburn, MA). Protein synthesis inhibitor cycloheximide (CHX, Cat#-01810) was purchased from Sigma (St Louis, MO).
The half-maximal inhibitory concentration of D-AP5 to inhibit NMDAR-mediated LTD is 0.45μM,^{24 (link)} and we used 50μM D-AP5.^{22 (link),25 (link)} We used 25μM BIC, at which concentration it reliably blocks GABA_ARs.^{26 (link)}

Sun Y., Lipton J.O., Boyle L.M., Madsen J.R., Goldenberg M.C., Pascual-Leone A., Sahin M, & Rotenberg A. (2016). Direct Current Stimulation Induces mGluR5-Dependent Neocortical Plasticity. Annals of neurology, 80(2), 233-246.

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Transcriptomic Profiling of Hindbrain Neurons

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Ex vivo cultured E12.5 hindbrain neurons were treated with 2 μM trichostatin A (TSA) (MBJ. JM-1606-1) at the culture Day 1 and incubated for 16 hours. As for the KCl treatment, cultured hindbrain neurons were treated with a cocktail of neuronal activity blockers (TDN cocktail = 1 μM tetrodotoxin (TTX)(TOCRIS, 1069) + 100 μM D-AP5 (Sigma, A8054) + 20 μM NBQX (TOCRIS, 0373) at Day 1 for an over-night incubation, and 55 mM KCl containing medium was treated at Day 2 in the presence or absence of 35 μM GSK-J4 (Sigma, SML0701), 50 μM A-485 (TOCRIS, 6387) or 10 μM Flavopiridol (Sigma, F3055) after a rinse. As for Drg11^tdTomato/+ cultured hindbrain neuorns, tdTomato⁺ neurons were sorted immediately after the KCl treatment. Processing of these cells was adapted for further analyses (mRNA-seq, ATAC-seq and ChIP-seq, see below and Supplementary Methods).

Kitazawa T., Machlab D., Joshi O., Maiorano N., Kohler H., Ducret S., Kessler S., Gezelius H., Soneson C., Papasaikas P., López-Bendito G., Stadler M.B, & Rijli F.M. (2021). A Unique Bipartite Polycomb Signature Regulates Stimulus-Response Transcription during Development. Nature genetics, 53(3), 379-391.

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Preparation of Mouse Cerebellar Slices

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Slices were prepared from P17–P90 male CD1 EAAT4-GFP or EAAT4-GlyT2-GFP mice. This strain was obtained by crossing EAAT4-GFP mice with a line expressing GlyT2-GFP (gift from H.U. Zeilhofer) (Zeilhofer et al., 2005 (link)), in which some GoCs express GFP. Mice were anesthetized by inhalation of isoflurane, and then killed by decapitation. Transverse slices were prepared as previously described (Valera et al., 2012 (link)). The cerebellum was dissected out and placed in cold artificial cerebrospinal fluid (ACSF) bubbled with carbogen (95% O₂, 5% CO₂), containing (in mM): NaCl 120, KCl 3, NaHCO₃ 26, NaH₂PO₄ 1.25, CaCl₂ 2.5, MgCl₂ 2, glucose 10 and minocycline 0.00005 (Sigma-Aldrich, USA). Then 300 µm-thick transverse slices were prepared (Microm HM 650V, Microm, Germany) in potassium-based medium, containing (in mM): K-gluconate 130, KCl 14.6, EGTA 2, HEPES 20, glucose 25, minocycline 0.00005 and D-AP5 0.05 (Sigma-Aldrich). After cutting, slices were soaked in a sucrose-based medium at 34°C, containing (in mM): sucrose 230, KCl 2.5, NaHCO₃ 26, NaH₂PO₄ 1.25, glucose 25, CaCl₂ 0.8, MgCl₂ 8, and minocyclin 0.00005 (Sigma-Aldrich) and maintained in a water bath at 34°C in bubbled ACSF.

Valera A.M., Binda F., Pawlowski S.A., Dupont J.L., Casella J.F., Rothstein J.D., Poulain B, & Isope P. (2016). Stereotyped spatial patterns of functional synaptic connectivity in the cerebellar cortex. eLife, 5, e09862.

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Methamphetamine Addiction Pathway Study

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Methamphetamine was purchased from the National Institute for Food and Drug Control (Beijing, China). The selective DR1 antagonist SCH23390, DR2 antagonist raclopride, the mitogen-activated protein kinase inhibitor U0126 and the PI3K inhibitor wortmannin were purchased from Tocris Cookson Ltd. (Bristol, United Kingdom). The Akt/PKB inhibitor triciribine PKA inhibitor H89, NMDAR antagonist D-AP5 and the kainate receptor agonist kainate (KA) were obtained from Sigma-Aldrich (United Kingdom).
Stock solutions, at thousand times the final concentration, were made up in water or in DMSO, and stored in individual aliquots at −20°C. Final solutions were prepared freshly on the day of the experiment.

Li Y., Xie X., Xing H., Yuan X., Wang Y., Jin Y., Wang J., Vreugdenhil M., Zhao Y., Zhang R, & Lu C. (2019). The Modulation of Gamma Oscillations by Methamphetamine in Rat Hippocampal Slices. Frontiers in Cellular Neuroscience, 13, 277.

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Signaling Pathways Activation in Neurodegenerative Diseases

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VEGF₁₆₅ (VEGF), SU1498, UBP310, rabbit monoclonal anti‐GluA2, goat polyclonal anti‐Flk1, and anti‐mouse IgG‐Cy5 were purchased from Abcam (Cambridge, UK). Rabbit polyclonal anti‐phospho‐PKCα (Thr638, p‐PKCα), rabbit monoclonal anti‐PKCα, and rabbit polyclonal anti‐GluA1 were purchased from Abways Technology (Shanghai, China). Rabbit polyclonal anti‐pan‐cadherin and rabbit polyclonal anti‐Flk1 were purchased from Cell Signaling Technology (Beverly, MA). Mouse monoclonal anti‐phosphotyrosine (PY20), rabbit IgG, and mouse polyclonal anti‐GFAP were purchased from Merck Millipore (Billerica, MA). Anti‐rabbit IRDye 680 and anti‐mouse IRDye 800CW were purchased from LI‐COR bioscience (Lincoln, NC). Rabbit polyclonal anti‐Flt1, rabbit polyclonal anti‐Flk1, and horseradish peroxidase (HRP)‐conjugated anti‐rabbit/mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Glutamate, CNQX, and D‐AP5 were purchased from Sigma‐Aldrich (St. Louis, MO). Fluo‐4AM, lipofectamine 2000, anti‐goat IgG‐Alexa Fluor 488, anti‐rabbit IgG‐Alexa Fluor 594, and rabbit monoclonal anti‐phosphoserine (pSer) were purchased from Thermo Fisher Scientific (Waltham, MA). Calphostin C was purchased from Tocris Bioscience (Bristol, UK).

Kou Z., Mo J., Wu K., Qiu M., Huang Y., Tao F., Lei Y., Lv L, & Sun F. (2019). Vascular endothelial growth factor increases the function of calcium‐impermeable AMPA receptor GluA2 subunit in astrocytes via activation of protein kinase C signaling pathway. Glia, 67(7), 1344-1358.

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Reagent Preparation for Nucleosome Stimulation

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NaHS, L-Glu, D-AP5, and potamine sky blue were purchased from Sigma-Aldrich (St. Louis, MO, United States). Concentrations of 0.02 mol/L were prepared for L-Glu, NAHS, AOAA, and D-AP5 solutions, and 2% Potamine sky blue solution was prepared for the identification of nucleosome stimulation sites. All reagents were prepared in 0.9% NaCl solution.

Sun H., Li C., Shi Y., Wang Y., Li J., Fan L., Yu Y., Ji X., Gao X., Hou K, & Li Y. (2024). Investigating the L-Glu-NMDA receptor-H2S-NMDA receptor pathway that regulates gastric function in rats’ nucleus ambiguus. Frontiers in Pharmacology, 15, 1389873.

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Pharmacological Manipulation of Synaptic Transmission

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All drugs, including d‐2‐amino‐5‐phosphonopentanoate (D‐AP5; 50 μm), D,L‐AP5 (100 μm), dizocilpine (MK‐801; 20 μm), memantine (10 μm; Sigma‐Aldrich UK), the glutamate transporter inhibitor d,l‐threo‐benzyloxyaspartic acid (TBOA; 30 μm), the Group II mGluR antagonist LY 341495 (200 nm) and tetrodotoxin (TTX; 100 nm; all Tocris UK) were added to the perfusion solution.

Wild A.R., Bollands M., Morris P.G, & Jones S. (2015). Mechanisms regulating spill‐over of synaptic glutamate to extrasynaptic NMDA receptors in mouse substantia nigra dopaminergic neurons. The European Journal of Neuroscience, 42(9), 2633-2643.

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Neuronal mEPSC Recordings in Hippocampus

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Coverslips with hippocampal 9–11 DIV neurons were continuously perfused with artificial CSF (aCSF) containing (in mM): 130 NaCl, 5.4 KCl, 2 CaCl₂, 1.2 MgCl₂, 20 HEPES, 15 glucose; pH 7.4. Miniature excitatory postsynaptic currents (mEPSC) were recorded in the whole-cell configuration from pyramidal shaped neurons voltage clamped at −70 mV using an Axopatch 200B amplifier (Molecular Devices). The intracellular solution contained (in mM): 120 Cs-gluconate, 17.5 CsCl, 10 Na-HEPES, 4 Mg-ATP, 0.4 Na-GTP, 10 Na₂-creatine phosphate, 0.2 Na-EGTA, pH 7.4, 300 mOsm. To pharmacologically isolate mEPSCs, the aCSF contained 0.5 μM TTX, 50 μM D-AP5 and 50 μM picrotoxin (Sigma). Whole-cell currents were digitized at 10 kHz and filtered at 2 kHz. Cells with series resistance >20 MΩ or that changed by ≥20% during the recording are excluded. mEPSCs were detected and analyzed using the MiniAnalysis program (Synaptosoft).

Xu X., Garcia J., Ewalt R., Nason S, & Pozzo-Miller L. (2017). The BDNF val-66-met Polymorphism Affects Neuronal Morphology and Synaptic Transmission in Cultured Hippocampal Neurons from Rett Syndrome Mice. Frontiers in Cellular Neuroscience, 11, 203.

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Acute Brain Slice Preparation and Pharmacological Modulation

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300 μm thick acute coronal brain slices (≈1.7 mm posterior to bregma) were made after the deep anesthesia of the mice with halothane (Sigma). Isolated brain was sliced by Vibratome (VT-1200, Leica, Nussloch, Germany) in a cold, high-sucrose, artificial cerebrospinal fluid (ACSF) solution containing (in mM): 240 Sucrose, 26 NaHCO₃, 2.5 KCl, 1.0 CaCl₂, 4 MgCl₂, 1.25 NaH₂PO₄, and 10 D(+)-glucose, pH 7.4, by gassing with 95% O₂ / 5% CO₂. Slices were then placed in an interface chamber filled with ACSF oxygenated with 95% O₂ / 5% CO₂.Slices were incubated at 36°C for at least 1 hour. During the experiment, a slice was placed on the microscope stage chamber with continuous perfusion of ACSF at 1–2 ml/min. The temperature of ACSF on the recording chamber was adjusted to 33 ± 0.5 °C by a temperature controller (TC-344B, Warner Instruments, CT) throughout the experiment. When required, the following drugs were dissolved in perfused ACSF for perfusion: 50 μM D-AP5, 40 μM CNQX, 40 μM bicuculline and 1 μM tetrodotoxin (TTX) (all from Sigma). A minimum of 10 minutes of drug perfusion was done to ensure proper delivery.

Nakajima R, & Baker B.J. (2018). Mapping of excitatory and inhibitory postsynaptic potentials of neuronal populations in hippocampal slices using the GEVI, ArcLight. Journal of physics D: Applied physics, 51(50), 504003.

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Quantification of Hydrogen Sulfide Signaling

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NaHS (2, 4, and 8 nmol), PDTC (2 nmol), D-AP5 (2 nmol), and pontamine sky blue were all purchased from Sigma-Aldrich (St. Louis, MO, United States). NaHS was dissolved in 0.9% normal saline, while the other chemicals were dissolved in dimethyl sulfoxide. Goat serum, anti-CBS rabbit pAb, FITC-conjugated goat anti-rabbit IgG, Cy3 conjugated Goat anti-mouse IgG, and anti-c-Fos mouse pAb were purchased from Servicebio.

Li C., Sun H., Shi Y., Yu Y., Ji X., Li E., Zhou X., Liu X., Xue X, & Sun H. (2022). Effects of Exogenous Hydrogen Sulfide in the Hypothalamic Paraventricular Nucleus on Gastric Function in Rats. Frontiers in Pharmacology, 12, 806012.

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