Dl 2 amino 5 phosphonopentanoic acid ap5

Carbamazepine (Sigma Aldrich, Saint Louis, MO, USA) was added to perfused mock CSF at 10, 100 and 1,000 μM. Gabapentin (Sigma Aldrich) was added to perfused mock CSF at 1.0, 10 and 100 μM. NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid (AP5, Sigma Aldrich) was added to perfused mock CSF at 30 μM. These concentrations were determined from preliminary experiments and previous studies [16 (link), 22 (link)]. Optical records using electrical stimulation were taken 20 min after the start of superfusion with control mock CSF and were taken 20 min after switching to drug-containing mock CSF. To induce activation of the NMDA receptor, we used low Mg²⁺ concentration solution (in mM): NaCl, 126; KCl, 5; CaCl₂, 2.6; MgSO₄, 0.8; NaH₂PO₄, 1.25; NaHCO₃, 26 and glucose, 30. In these series of experiments, optical records using electrical stimulation were taken 20 min after the start of superfusion with control mock CSF and were taken 20 min after switching to low Mg²⁺ concentration solution, and then, taken 20 min after switching to low Mg²⁺ solution containing 30 μM AP5, 1,000 μM Carbamazepine or 100 μM Gabapentin.

Neuronal activity was modified using previously described cLTP or chemical LTD protocols1 (link)2 (link)12 (link)26 (link). Briefly, maintenance media was replaced with an extracellular recording solution containing the following: 125 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 0 mM MgCl₂, 5 mM HEPES, 33 mM D-glucose and supplemented with 0.5 μM tetrodotoxin, 20 μM Bicuculline pH 7.3, 290 mOsm l⁻¹, for 10–15 min. For cLTP, this media was supplemented with 200 μM glycine or 200 μM glycine plus 50 μM DL-2-Amino-5-phosphonopentanoic acid (AP5) (Sigma) for 3 min. For cLTD, the media was supplemented with 20 μM NMDA (Sigma) plus 10 μM glycine for 3 min. The solution was then replaced with fresh extracellular recording solution (containing 0 mM MgCl₂ for LTP or 2 mM MgCl₂ for LTD) for the indicated times before experimentation. Cells were continually maintained at 37 °C for the duration of activity stimulation.

To isolate mGluR1a-mediated excitatory postsynaptic currents (mGluR1a-EPSCs), antagonists of non-NMDA (6-cyano-7-nitroquinoxaline-2,3-dione; CNQX, 20 μM), NMDA (DL-2-amino-5-phosphonopentanoic acid; AP5, 50 μM), and GABA-A (gabazine, 5 μM) receptors (all from Sigma, Oakville, ON, Canada), as well as the glutamate transporter blocker DL-threo-b-benzyloxyaspartic acid (TBOA, 30 μM) (Tocris, Ellisville, MO,USA) were bath-applied. In some experiments, the mGluR1a receptor antagonist (S)(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385, 100 μM), the TRP channel antagonist 1–2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)proproxy]ethyl-1H-imidazole (SKF96365, 30 μM) (both drugs from Tocris), or the phospholipase C inhibitor 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122, 10 μM) (Calbiochem, Gibbstown, NJ, USA), were added to the external solution.