Electrical stimulations were delivered via a bipolar stimulation electrode (0.1–1 mA, 100 μs; FHC Inc.). To study the effect of the mGlu7 receptor at thalamic presynaptic sites, the stimulation electrode was placed either in the VPM to evoke excitatory postsynaptic currents (eEPSCs) in TRN neurons, held at -60 mV (inward currents), or in the TRN to evoke inhibitory postsynaptic currents (eIPSCs) within adjacent TRN neurons or VPM neurons, held at 0 mV (outward currents). The group III mGluRs agonist D ,L -AP4 (1.2 mM, Tocris Bioscience) and the mGlu7 receptor NAM ADX71743 (10 μM) were applied and the effects compared between the two genotypes.
Dl ap4
Manufactured by Bio-Techne
Sourced in United Kingdom
DL-AP4 is a laboratory equipment product from Bio-Techne. It is designed to perform a specific function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolating or interpreting the intended use.
Automatically generated - may contain errors
Lab products found in correlation
Qx 314 bromide, by Bio-Techne (1 mentions)
Matlab, by MathWorks (1 mentions)
Multiclamp 700b amplifier, by Molecular Devices (1 mentions)
D ap5, by Abcam (1 mentions)
Igor pro, by Wavemetrics (1 mentions)
Ubp 310, by Abcam (1 mentions)
Picrotoxin, by Bio-Techne (1 mentions)
Dl ap5, by Bio-Techne (1 mentions)
6 7 dinitroquinoxaline 2 3 dione dnqx, by Bio-Techne (1 mentions)
1 2 5 6 tetrahydropyridin 4 yl methylphosphinic acid tpmpa, by Bio-Techne (1 mentions)
Glutamate, by Bio-Techne (1 mentions)
L ap4, by Bio-Techne (1 mentions)
Mmpip, by Bio-Techne (1 mentions)
Bicuculline, by Merck Group (1 mentions)
Kynurenic acid, by Merck Group (1 mentions)
Bacl2, by Merck Group (1 mentions)
Ames medium, by Merck Group (1 mentions)
Digidata 1440, by Molecular Devices (1 mentions)
7 protocols using dl ap4
1
Presynaptic mGlu7 Receptor Modulation
2
Synaptic Blockade via Glutamatergic Antagonists
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The synaptic blockade was achieved using a combination of two glutamatergic antagonists: 80 μM 6,7-dinitroquinoxaline-2,3-dione (DNQX, Tocris Bioscience) and 130 μM (DL-AP4, Tocris Bioscience) in aCSF.
3
Electrophysiological Characterization of DSGCs
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DSGCs were identified using 2-photon imaging in mouse lines with fluorescent labeling or were identified by their extracellular spiking responses in wild type mice. Electrodes were pulled from borosilicate glass capillaries to a resistance 3–6 MΩ. For extracellular recordings, electrodes were filled with Ringer’s solution. For voltage clamp recordings, electrodes contained the following in mM: 112.5 CH3CsO3S, 7.75 CsCl, 1 MgSO4, 10 EGTA, 10 HEPES, 5 QX-314-bromide (Tocris). For Ca2+ imaging current clamp recordings, electrodes were filled with the following in mM: 115 K-Gluconate, 7.7 KCl, 10 HEPES, 1 MgCl2, 2 ATP-Na2, 1 GTP-Na, five phosphocreatine, 2 QX-314, 0.2 Oregon Green Bapta-1, and 0.05 Sulphorhodamine 101. For some recordings, QX-314 was omitted and 1 µM TTX was included in the bath solution instead. Signals were sampled at 10 kHz and filtered at 2 kHz in a MultiClamp 700B amplifier (Molecular Devices). Analysis was performed using custom routines in MATLAB (Mathworks) and Igor Pro (Wavemetrics). The following pharmacological agents were added directly to the superfusion solution: DL-AP4 (50 µM; Tocris Bioscience), D-AP5 (50 µM; Abcam Biochemicals), UBP-310 (10 µM; Abcam Biochemicals), TTX (0.5–1 µM; Abcam Biochemicals), CNQX (20 µM; Tocris Bioscience).
4
Synaptic Blockade in Retina Explant
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Blocking of various synapses was achieved by bath applying various pharmacological substances dissolved in aCSF, which superfused the explanted retina as described. Gap junctions were blocked with 50 µM MFA (Sigma Aldrich) that was applied for 30 min before the data were recorded. Glutamatergic signaling was blocked using a combination of three compounds: 100 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX; Tocris Bioscience, Bristol, UK) was used to block the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamatergic synapses, 100 µM DL-AP5 (Tocris Bioscience) was used to block the N-methyl-D-aspartate (NMDA) glutamatergic synapses, and 150 µM DL-AP4 (Tocris Bioscience) was used to block the metabotropic (mGluR6) synapses. As an mGluR6 agonist, DL-AP4 did not allow the investigation of synapses downstream of the transfected ON bipolar cells. Gamma-aminobutyric acid (GABA)a and GABAc receptors were antagonized with 20 µM picrotoxin and 50 µM 1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), respectively (Tocris Bioscience).
5
Synthesis of Glutamate Receptor Ligands
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L-AP4, DL-AP4, MMPIP, and glutamate were purchased from Tocris. LSP4-2022 and ADX71743 were synthesized in house using methods reported in the study by Klar et al. (18 (link)). VU06010608 and VU6010953 were synthesized in house using methods reported in the study by Reed et al. (26 (link), 28 (link)).
6
Retinal Ganglion Cell Recordings
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Methods for path-clamp recordings from ipRGCs were similar to methods previously described73 (link). Following at least one hour of dark adaptation, mice were euthanized by cervical dislocation after isoflurane anesthesia. Eyes were enucleated under dim red light, and the cornea was slit to remove the lens, followed by retina extraction into oxygenated, bicarbonate-buffered Ames media (US Biologicals), 3 mM kynurenic acid (Sigma), 50 µM DL-AP4 (Tocris), and 50 µM (+)-bicuculline (Sigma). The retinas were then cut into quarters, mounted on a nitrocellulose filter with a hole providing access to the retinal ganglion cell layer, which were stored in the same solution in the dark at room temperature. Retinas could be maintained for more than 4 hours under these conditions before recording.
7
Ex-vivo Transretinal ERG Recording
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Mice were dark adapted overnight prior to the day of experiment and were euthanized by CO2 incubation. Eyes were enucleated under dim red light immediately after euthanasia followed by dissection under infrared illumination. The retinas were gently detached from posterior eye cups and were stored in dark in a dish containing oxygenated Ames medium at room temperature until recording. Recordings were conducted using previously described methods61 . The retinas were mounted photoreceptors facing up in a closed chamber and were continuously superfused with oxygenated Ames medium (Sigma) at a flow rate of 3–5 mL/min. For isolating the a-wave of ERG, 50 μM DL-AP4 (Tocris) and 100 μM BaCl2 (Sigma) were included in the Ames medium. The recording chamber was maintained at 35–36 °C and retinas were allowed to adapt to the chamber temperature for at least 15 min before experiments. Ex-vivo transretinal ERG recordings were made in scotopic conditions by presenting light flashes produced by LEDs (Thor Labs). The ERG signals were amplified using a differential amplifier (Warner Instruments), low-pass filtered at 300 Hz (Krohn Hite Corp.), digitized using digidata 1440 (Molecular Devices), and were recorded at a sampling frequency of 10 kHz using pClamp 10 software.
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