Picrotoxin

Reagents for ACSF and internal solutions, biocytin, NBQX, BaCl₂, and picrotoxin were obtained from Sigma-Aldrich. CNQX, AP-5, DHK, DL-TBOA, TTX, ouabain, and D-serine were obtained from Tocris. L-glutamic acid from BioTrend and SR-101 from Invitrogen. NBQX, CNQX, and DL-TBOA were dissolved in DMSO. picrotoxin was dissolved in EtOH. AP-5, D-serine, TTX, ouabain, and DHK were dissolved in ddH₂O.
In both patch-clamp and two-photon imaging experiments, following baseline recordings, drugs were applied in the external solution for at least 15 min prior to recordings. For the double pharmacology imaging experiments (Fig. 9) following baseline recordings, DHK (300 μM) was first applied and recordings were acquired 20 min later. Subsequently, DHK (300 μM) and DL-TBOA (68 μM) were applied for 20 min before recording. In a number of recordings in which we applied much higher doses of DL-TBOA (300 µM), we observed a change in baseline iGluSnFr fluorescence (similar to ref. ^{18 (link)}), a reduced amplitude of the evoked responses and cellular swelling often accompanied by a lateral or Z drift. These experiments had to be excluded, which explains the low n value for this set of experiments (Supplementary Fig. 9).

Secretin (30 nM–1 μM; rat, Tocris); Secretin antagonist [3 μM; Secretin 5–27; TFTSELSRLQDSARLQRLLQGLV (Williams et al., 2012 (link))], GABA_A-R blocker picrotoxin [100 μM, Sigma; (Seidl et al., 2014 (link); Keshavarzi et al., 2015 (link))].

Chemicals including lithium chloride (LiCl), picrotoxin, strychnine hydrochloride (strychnine), and chemicals for ACSF were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetrodotoxin citrate (TTX), DL-AP5 (AP5), and CNQX disodium salt (CNQX) were purchased from Tocris Bioscience (Avonmouth, Bristol, UK). Stocks were diluted (usually by 1000 times) to a working concentration in ACSF before bath application.

Stock solutions of memantine (Sigma, St. Louis, MO) were dissolved in standard aCSF (see above) at 1, 3, or 10 mM, stored at −80°C until the day of experiments, and diluted 1 : 1000 to a final concentration of 1, 3, or 10 μM on the day of experiments. For the two lowest concentrations of picrotoxin (Sigma) used here (0.1 and 1 μM), 10x stock solutions were prepared (1 and 10 μM, resp.) and diluted to the appropriate concentration on the day of the experiment. Given that picrotoxin does not dissolve in water at high concentrations, 10 and 100 μM were dissolved in aCSF on the day of the experiment. Bacterial expression and purification of rPrP were performed as described previously [29 (link)], and the protein concentration was determined by measuring absorbance at 276 nm using the appropriate extinction coefficients.

Neuronal cultures were incubated for 40 min with either aaMMP-9 or iaMMP-9 in MM (400 ng/ml). The cells were then treated for 30 min with GM6001 (25 μM) dissolved in DMSO or treated with DMSO alone as a control. For cLTP in neuronal cultures, 50 μM forskolin, 50 μM picrotoxin, and 0.1 μM rolipram (Sigma-Aldrich; all dissolved in DMSO) were bath-applied (described previously). cLTP was applied to neurons in the presence or absence of iaMMP-9 (400 ng/ml) for 40 min. For the control experiments, hippocampal cultures were treated with DMSO.

The MOR agonist DAMGO (H-2535, Bachem), MOR antagonist CTAP (H-3698, Bachem), delta opioid receptor agonist [D-Pen²,D-Pen⁵]-enkephalin (DPDPE; H-2905m, Bachem), α7 nicotinic acetylcholine receptor antagonist methyllycaconitine citrate (MLA; 1029, Tocris), AMPA antagonist NBQX (0373, Tocris), tetrodotoxin citrate (ARCD-0640, ARC Inc.), EtOH (E7148, Sigma-Aldrich), and GABA_A receptor antagonist picrotoxin (P1675, Sigma-Aldrich) were used.