nmda receptor antagonist dl ap5  (Tocris)

Journal: ACS Chemical Neuroscience

Article Title: Reactive Astrocytes with Reduced Function of Glutamate Transporters in the App NL‑G‑F Knock-in Mice

doi: 10.1021/acschemneuro.4c00714

Figure Lengend Snippet: Passive membrane properties of astrocytes are altered in App NL‑G‑F mice compared to WT mice. (a) Resting membrane potential (RMP) of astrocytes in App NL‑G‑F mice ( n = 9) is not significantly different compared to WT ( n = 9, p = 0.16, unpaired t test). (b) App NL‑G‑F ( n = 9) astrocytes have significantly higher input resistance than WT ( n = 9, * p = 0.02, unpaired t test), (c) I – V plot showing a shift in astrocyte voltage response to current injection in WT hippocampal slices ( n = 9, red) compared to App NL‑G‑F ( n = 9, green). (d) Scheme illustrating the setup for recording synaptically activated glutamate transporter current (STC). Schaffer collaterals (SC) were stimulated using a bipolar electrode leading to glutamate release. Representative trace showing astrocyte current response (red) to single pulse SC stimulation in the presence of glutamate receptor blocker (NBQX, AP5) in (e) WT and (h) App NL‑G‑F mice. Representative trace showing astrocyte current response to single-pulse SC stimulation (red) superimposed over the astrocytic current response in the presence of glutamate transporter blocker (TFB-TBOA, blue) in the same astrocyte in (f) WT and (i) App NL‑G‑F mice. STC peak amplitude and decay time constant were obtained after best fitting to a single exponential function (black line) in (g) WT and in (j) App NL‑G‑F mice. (k) STC decay time constant in WT ( n = 5) and App NL‑G‑F mice ( n = 4, p = 0.22, n.s.= non significant, unpaired t test) (l) STC peak amplitude significantly decreased in App NL‑G‑F mice ( n = 4) compared to WT ( n = 5, * p = 0.003, unpaired t test). Bars show mean ± SEM.

Article Snippet: GABA A receptor antagonist: PTX (Picrotoxin, 50 μM), AMPA receptor antagonist NBQX (1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo­[f]­quinoxaline-7-sulfonamide hydrate; 10 μM), NMDA receptor antagonist DL-AP5 (DL-2-Amino-5-phosphonopentanoic acid; 50 μM), EAAT blocker TFB-TBOA ((3S)-3-3-4-(Trifluoromethyl) benzoyl amino phenyl methoxy- l -aspartic acid, 1 μM) and Kainic acid (KA), were obtained from Tocris Bioscience (Bristol, U.K.).

Techniques: Membrane, Injection

Journal: ACS Chemical Neuroscience

Article Title: Reactive Astrocytes with Reduced Function of Glutamate Transporters in the App NL‑G‑F Knock-in Mice

doi: 10.1021/acschemneuro.4c00714

Figure Lengend Snippet: Astrocytes in App NL‑G‑F mice exhibit a reduced rate of glutamate clearance compared to WT mice. Representative superimposed traces showing the astrocytic current recording after 9 (black) and 10 pulses (red) of 50 Hz SC stimulation in the presence of NBQX and AP5 in (a) WT and (e) App NL‑G‑F mice. The resultant trace showing the astrocytic response to the 10th pulse was obtained by subtracting the response of 9 pulses from 10 pulses in (b) WT and (f) App NL‑G‑F mice. Representative traces showing the response of TFB-TBOA (blue) in the same astrocyte superimposed over the astrocytic current obtained due to the 10th pulse in (c) WT and (g) App NL‑G‑F mice. STC was isolated by subtracting the response of TFB-TBOA from the 10th pulse. This isolated STC was best fitted by a single exponential function (black line) to obtain the decay time constant (d) WT (h) App NL‑G‑F mice and peak amplitude of STC. (i) Decay time constant of the STC by the 10th pulse, which indicates the rate of glutamate clearance, was significantly increased in App NL ‑ G‑F mice ( n = 6) compared to WT ( n = 5, unpaired t test, * p = 0.0339) whereas (j) peak amplitude of STC by was significantly lower in App NL‑G‑F mice ( n = 6) vs WT ( n = 5, unpaired t test, * p = 0.003). Bars show mean ± SEM.

Article Snippet: GABA A receptor antagonist: PTX (Picrotoxin, 50 μM), AMPA receptor antagonist NBQX (1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo­[f]­quinoxaline-7-sulfonamide hydrate; 10 μM), NMDA receptor antagonist DL-AP5 (DL-2-Amino-5-phosphonopentanoic acid; 50 μM), EAAT blocker TFB-TBOA ((3S)-3-3-4-(Trifluoromethyl) benzoyl amino phenyl methoxy- l -aspartic acid, 1 μM) and Kainic acid (KA), were obtained from Tocris Bioscience (Bristol, U.K.).

Techniques: Isolation

Journal: Nature Neuroscience

Article Title: Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding

doi: 10.1038/s41593-024-01615-5

Figure Lengend Snippet: a , The implementation of TurboID in primary cortical neurons. b , Doxycycline (Dox) induction of TurboID-PSD95 (Flag) and its biotinylation pattern (streptavidin) in the presence and absence of biotin shown by western blots. c , IF to detect TurboID expression and biotinylation in primary cortical neurons transduced with TurboID-PSD95 after 30 min of biotin incubation. DAPI (blue) marker for nuclei, Flag (red) stain for TurboID and streptavidin (cyan) to demarcate the biotinylated proteins. Magnification, ×40. d , Diagram of neuronal activation workflow with TurboID labeling: neurons are first silenced with TTX (sodium channel blocker) and DL-AP5 (NMDA receptor antagonist) to standardize activity levels in culture; then, activated with KCl (dep) for 1 h or by DGPH for 10 min; and, finally, incubated with the same silencers and biotin to induce biotinylation and allow for recovery (30 min for KCl and 20 min for DHPG). Colors: salmon (resting); burgundy (depolarized by KCl); cyan (DHPG). e , Distribution of streptavidin signal (cyan) in Pan-TurboID-transduced or TurboID-PSD95-transduced activated neurons. DAPI (blue) marker for nuclei. Magnification, ×20. f , Fura-2 AM staining (left) and quantification (right) in resting and KCl-depolarized neurons. Each circle represents information from one field (data are mean ± s.d., 15 fields total from three biological replicates). Significance was derived from the biological replicates and calculated using the two-tailed, unpaired Student’s t -test. Scale bars, 50 μm.

Article Snippet: Before KCl depolarization on the 12th day, neurons were silenced with 1 µM sodium channel blocker tetrodotoxin (TTX) (Abcam, ab120054) and 100 µM NMDA receptor antagonist DL-2-amino-5-phosphopentanoic acid (DL-AP5) (Abcam, b120004) for 2 h at 37 °C and 5% CO 2 .

Techniques: Western Blot, Expressing, Transduction, Incubation, Marker, Staining, Activation Assay, Labeling, Activity Assay, Derivative Assay, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Cerebrospinal Fluid-Contacting Neurons Sense pH Changes and Motion in the Hypothalamus

doi: 10.1523/JNEUROSCI.3359-17.2018

Figure Lengend Snippet: Electrophysiological properties of hypothalamic CSF-c neurons. A, Whole-cell current-clamp recording of a hypothalamic CSF-c neuron showing its firing pattern. A brief current injection (20 pA, 20 ms; left) elicited a single action potential, whereas longer current injections (20 pA, 500 ms; right) generated repetitive firing. B, CSF-c neuron showing spontaneous GABA- and glutamate-mediated postsynaptic potentials (top trace) that were successively blocked by gabazine (20 μm), CNQX (40 μm), and AP5 (50 μm; bottom traces), respectively. C, Voltage responses to 12 consecutive hyperpolarizing and depolarizing current injections. The red and green traces illustrate a single action potential and spike frequency adaptation evoked by depolarizing steps, respectively. D, I–V curve showing a linear current–voltage relationship.

Article Snippet: The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2, an extract of the sea anemone, Anthopleura elegantissima toxin-2, (1 μ m ; catalog #500527; Calbiochem), the connexin hemichannel blocker lanthanum chloride (100 μ m ; catalog #298182; Sigma-Aldrich), the GABA A receptor antagonist gabazine (20 μ m ; catalog #1262; Tocris Bioscience), the NMDA receptor antagonist AP5 (50 μ m ; catalog #0105; Tocris Bioscience), the AMPA receptor antagonist CNQX (40 μ m ; catalog #1045; Tocris Bioscience), and tetrodotoxin (TTX, 1.5 μ m ; catalog #T8024; Sigma-Aldrich).

Techniques: Injection, Generated

Journal: The Journal of Neuroscience

Article Title: Cerebrospinal Fluid-Contacting Neurons Sense pH Changes and Motion in the Hypothalamus

doi: 10.1523/JNEUROSCI.3359-17.2018

Figure Lengend Snippet: Hypothalamic CSF-c neurons are activated by both acidic and alkaline pH. A, Change to acidic (pH 6.5) as well as to alkaline (pH 8.0) extracellular pH (gray area) depolarized the membrane potential (10–12 mV) and triggered action potentials. Upon return to pH 7.4, firing ceased and the membrane potential repolarized back to the control value (−63 mV). In this and all subsequent recordings, gabazine (20 μm), AP5 (50 μm), and CNQX (40 μm) were applied to exclude any indirect, synaptically mediated effects. B, In the presence of TTX (1.5 μm), both acidic and alkaline pH resulted in depolarization of the membrane potential that recovered after return to pH 7.4. C, Photomicrographs of the CSF-c neuron recorded in A after intracellular labeling with Neurobiotin (green, arrow), showing that the cell expressed somatostatin (magenta). Scale bars, 20 μm. D, Mean membrane potential changes in hypothalamic CSF-c neurons for each pH condition [mean ± SD; n = 15; Student's paired t test: ***p < 0.001 significant difference vs pH 7.4 at 6.5 (p = 1.43 × 10−19, t14 = 22.7), at 6.8 (p = 5.56 × 10−18, t14 = 19.76), at 7.1 (p = 1.97 × 10−14, t14 = 14.35), at 7.7 (p = 2.20 × 10−13, t14 = 13.0), at 8.0 (p = 2.24 × 10−20, t14 = 24.33), and at 8.3 (p = 1.38 × 10−21, t14 = 26.99)]. E, In voltage-clamp mode, frequent inward current deflections appeared at pH 6.5 and 8.0 with a maximal amplitude of ∼10 pA. F, Mean frequency increase of events (5–15 pA) in response to acidic and alkaline pH [mean ± SEM; n = 7; Student's paired t test: ***p < 0.001 significant difference vs pH 7.4 at pH 6.5 (p = 2.34 × 10−7; t6 = 25.59) and at 8 (p = 4.48 × 10−8; t6 = 33.79)]. G, Unitary current deflections recorded at acidic and alkaline pH. TTX (1.5 μm) was present in B, E–G.

Article Snippet: The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2, an extract of the sea anemone, Anthopleura elegantissima toxin-2, (1 μ m ; catalog #500527; Calbiochem), the connexin hemichannel blocker lanthanum chloride (100 μ m ; catalog #298182; Sigma-Aldrich), the GABA A receptor antagonist gabazine (20 μ m ; catalog #1262; Tocris Bioscience), the NMDA receptor antagonist AP5 (50 μ m ; catalog #0105; Tocris Bioscience), the AMPA receptor antagonist CNQX (40 μ m ; catalog #1045; Tocris Bioscience), and tetrodotoxin (TTX, 1.5 μ m ; catalog #T8024; Sigma-Aldrich).

Techniques: Membrane, Control, Labeling

Journal: The Journal of Neuroscience

Article Title: Cerebrospinal Fluid-Contacting Neurons Sense pH Changes and Motion in the Hypothalamus

doi: 10.1523/JNEUROSCI.3359-17.2018

Figure Lengend Snippet: Response to acidic and alkaline pH is mediated by different channels. A, Whole-cell current clamp in the presence of gabazine (20 μm), AP5 (50 μm), and CNQX (40 μm). Application of APETx2 (1 μm) abolished the response to acidic pH but not to alkaline pH in the same cell. B, Recordings from a hypothalamic CSF-c neuron in voltage-clamp mode. No current events were seen at pH 7.4 (black traces) in the presence of gabazine (20 μm), AP5 (50 μm), CNQX (40 μm), and TTX (1.5 μm). Inward current deflections appeared after a decrease or increase in the extracellular pH. The current events recorded in acidic pH (6.5) were completely blocked in the presence of APETx2 (1 μm; red trace), whereas those recorded in alkaline pH (8.0; blue trace) remained. C, Mean frequency increase of events (5–15 pA) in response to acidic and alkaline pH in the presence of APETx2 [mean ± SEM; n = 3; Student's paired t test: no significant difference vs pH 7.4 at 6.5 (p = 1, t2 = 0), but a significant difference vs pH 7.4 at 8 (**p < 0.01, p = 0.007, t2 = 11.55)]. D, Hypothalamic somatostatin-immunopositive CSF-c neurons (green) do not express the PKD2L1 channel (magenta). Scale bar, 20 μm. E, Spinal somatostatin-CSF-c neurons (green) coexpress PKD2L1 (arrows; magenta). Scale bar, 20 μm. cc, Central canal. F, Whole-cell current clamp in the presence of gabazine (20 μm), AP5 (50 μm), and CNQX (40 μm) showing the response to both acidic (pH 6.5; red trace) and alkaline (pH 8.0; blue trace) pH. The connexin hemichannel blocker lanthanum (100 μm) abolished the alkaline response but not the acidic response. G, Mean frequency increase of action potential firing in response to acidic and alkaline pH before and after application of lanthanum chloride (100 and 70 μm) [before application (control): means ± SEM; n = 5; Student's paired t test: ***p < 0.001 significant difference vs pH 7.4 at 6.5 (p = 1.31 × 10−4, t4 = 14.51) and at 8 (p = 9.43 × 10−5, t4 = 15.77)]. In the presence of lanthanum, a complete (100 μm) or partial (70 μm) blockade of the spiking response to alkaline pH was seen [means ± SEM; n = 5; Student's paired t test: ***p < 0.001 significant difference at pH 8 vs control in the presence of lanthanum at 100 μm (p = 9.4 × 10−5, t4 = 15.77; as well as at 70 μm (p = 9.3 × 10−4, t4 = 8.76)]. A tendency for a small, nonsignificant (n.s.) frequency reduction was also observed at acidic pH (6.5) versus control in the presence of lanthanum at 100 μm (p = 0.06, t4 = 2.58). H, The recorded CSF-c neuron in F intracellularly labeled with Neurobiotin expressed somatostatin. Scale bars, 10 μm. I, Whole-cell current clamp of a hypothalamic CSF-c neuron in control condition and in the presence of gabazine (20 μm), AP5 (50 μm), and CNQX (40 μm) showing that this cell did not respond to either acidic (pH 6.5; red trace) or alkaline (pH 8.0; blue trace) pH. J, The recorded CSF-c neuron in I intracellularly labeled with Neurobiotin did not express somatostatin. Scale bars, 10 μm.

Article Snippet: The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2, an extract of the sea anemone, Anthopleura elegantissima toxin-2, (1 μ m ; catalog #500527; Calbiochem), the connexin hemichannel blocker lanthanum chloride (100 μ m ; catalog #298182; Sigma-Aldrich), the GABA A receptor antagonist gabazine (20 μ m ; catalog #1262; Tocris Bioscience), the NMDA receptor antagonist AP5 (50 μ m ; catalog #0105; Tocris Bioscience), the AMPA receptor antagonist CNQX (40 μ m ; catalog #1045; Tocris Bioscience), and tetrodotoxin (TTX, 1.5 μ m ; catalog #T8024; Sigma-Aldrich).

Techniques: Control, Labeling

Journal: The Journal of Neuroscience

Article Title: Cerebrospinal Fluid-Contacting Neurons Sense pH Changes and Motion in the Hypothalamus

doi: 10.1523/JNEUROSCI.3359-17.2018

Figure Lengend Snippet: The same hypothalamic CSF-c neuron is sensitive to both fluid movement and pH changes. A, In vitro preparation of the hypothalamus with CSF-c neurons protruding into the third ventricle. An aCSF-filled pressure pipette was placed close to a bulb-like ending of a recorded hypothalamic CSF-c neuron. Scale bar, 20 μm. B, A short fluid-pulse (80 ms) elicited receptor potential responses and action potentials while holding the membrane potential at −65 mV and −55 mV, respectively, in the presence of gabazine (20 μm), CNQX (40 μm), and AP5 (50 μm). C, Receptor potential elicited by fluid pulse stimulation (20 psi, 80 ms) was blocked by application of the ASIC3 blocker APETx2 (1 μm). D, Complete blockade of responses after application of APETx2 (n = 4). E, In the same hypothalamic CSF-c neuron as in C, exposure to acidic (pH 6.5) as well as alkaline (pH 8.0) pH depolarized the membrane potential (10–12 mV) and triggered action potentials (with GABA and glutamate receptor antagonists present).

Article Snippet: The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2, an extract of the sea anemone, Anthopleura elegantissima toxin-2, (1 μ m ; catalog #500527; Calbiochem), the connexin hemichannel blocker lanthanum chloride (100 μ m ; catalog #298182; Sigma-Aldrich), the GABA A receptor antagonist gabazine (20 μ m ; catalog #1262; Tocris Bioscience), the NMDA receptor antagonist AP5 (50 μ m ; catalog #0105; Tocris Bioscience), the AMPA receptor antagonist CNQX (40 μ m ; catalog #1045; Tocris Bioscience), and tetrodotoxin (TTX, 1.5 μ m ; catalog #T8024; Sigma-Aldrich).

Techniques: In Vitro, Transferring, Membrane