Tetrodotoxin

APP-CTM1, APP-CT11, PS1_NT, Flotilin-2 and APP_NTH-452 homemade rabbit polyclonal antibodies were generously provided by Dr. Gopal Thinakaran (University of Chicago, Chicago, IL) as described previously (Deyts et al., 2012 (link); Vetrivel et al., 2009 (link)). Monoclonal anti-MAP2, GAP-43 (clone 7B10) and GAPDH were purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal DCC (clone G97-449) antibody was purchased from BD Biosciences (San Diego, CA). Polyclonal phospho-(Ser/Thr) PKA substrate antibody and phospho-CREB (Ser133) antibodies were purchased from Cell Signaling Technology (Danvers, MA) and EMD Millipore (Billerica, MA), respectively. Monoclonal Alexa-647 and Alexa-555, and polyclonal Alexa-555 secondary antibodies were purchased from Invitrogen (Carlsbad, CA). IRDye 680 and IRDye 800CW-conjugated secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE). γ-secretase inhibitor Compound E was generously provided by Dr. Todd E. Golde (University of Florida, Gainesville, FL) (Seiffert et al., 2000 (link)). Cis-N-(2-phenylcyclopentyl) azacyclotridec-1-en-2-amine (MDL,12-330A) was obtained from Enzo Life Science (Farmingdale, NY). Tetrodotoxin was purchased from Tocris Bioscience (distributed by Fisher Scientific, Pittsurgh, PA). Unless indicated, all other reagents were purchased from Sigma-Aldrich.

These were freshly prepared prior to use. Carbachol, atropine, L-NAME (N_ω-nitro-L-arginine methyl ester hydrochloride), propranolol, phentolamine (each from Sigma, UK) and tetrodotoxin (Tocris, UK) were each dissolved in distilled water (dH₂O). Asimadoline (generous gift from Tioga Inc, USA), ICI204448 and nor-binaltorphimine (both Tocris, UK) were dissolved to 10 mM in DMSO.

Rat Na_V1.2 (NM_012647.1) was handled in pcDNA1, as previously described (Plant et al., 2006 (link)). Human SUMO1₁₀₁ (BCOO5899) was amplified from a brain cDNA library (Clontech, Mountain View, CA) and inserted into pMAX as described before (Rajan et al., 2005 (link)). Sequences encoding mCherry or CFP were inserted as described (Plant et al., 2010 (link)) at the N-terminus of SUMO1 or Na_V1.2 respectively. Mutations were introduced with Pfu Quikchange PCR (Agilent, Santa Clara, CA). Purified SUMO1₉₇ and SENP1 were purchased from Boston Biochem (Cambridge, MA). SUMO1₉₇-T95K (introducing a diagnostic trypsin cleavage site) was produced as a His-tagged protein fused to the TEV cleavage domain in pET28a using the bacterial strain BL21 (DE3) and isolated by routine procedures cleaving the His-tag with TEV Protease before dialysis against 140 mm KCl, 0.5 mm CaCl₂, 5 mm HEPES, pH 7.4. Protein concentration was determined by BCA assay (Thermo Fisher, Waltham, MA). The sodium channel blockers, tetrodotoxin, 4–9-anhydrotetrodotoxin and µ-conotoxin-TIIIA were purchased from Tocris (Bristol, U.K.), Focus Biomolecules (Plymouth Meeting, PA) and CS Bio (Menlo Park, CA), respectively, and handled in buffers with 0.1% BSA.

Atropine sulfate, guanethidine monosulphate, N^ω-nitro-L-argininemethylester, 6-hydroxy dopamine, and ascorbic acid were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Tetrodotoxin, Substance P, N-acetyl-L-tryptophan 3,5-bis (trifluoromethyl) benzyl ester, GR159897, and SB218795 were purchased from Tocris (Bristol, UK). Mouse anti-TH antibody (1:2000) was purchased from Chemicon International (Temecula, CA, USA). Biotinylated anti-mouse IgG antibody (1:1000) and nickel-intensified 3,3′-diaminobenzidine tetrahydrochloride (DAB Substrate Kit for peroxidase) were purchased from Vector Laboratories (Burlingame, CA, USA). Neutral-buffered formaldehyde (NBF) and xylene were purchased from Carlo Erba (Milan, Italy).

Mouse pups were euthanized by deep anesthesia by isofluorane followed by decapitation. Organotypic hippocampal slice cultures were prepared as described previously [49 (link)] from p4-p6 wild-type mice and were cultured for 10–12 days before transfection. A biolostic particle delivery system (Helios^® Gene Gun System, Bio-Rad) was used to introduce fluorescent GFP labels to obtain sparse transfection of neurons. Two to six days after transfection, neurons in sparsely GFP-labeled CA1 hippocampal regions were chosen for imaging. Individual spines in the striatum radiatum on secondary apical dendrites were chosen for observation. MNI-caged L-glutamate (4-methoxy-7-nitroindolinyl-caged L-glutamate, Tocris) was uncaged with a train of 820-nm laser pulses (3.5–4 mW under the objective, 30 times at 1 Hz) near a spine of interest. Pulse duration was varied 4-8ms based on depth of the spine in tissue, allowing for reliable uncaging without excess light exposure. Experiments were performed at room temperature in ACSF solution containing (in mM): 127 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 4 CaCl2, 25 glucose, 0.001 tetrodotoxin (Tocris) and 4 MNI-caged L-glutamate, bubbled with 95% O2 and 5% CO2.

Chem-LTP was performed on mouse cortical neurons at days in vitro (DIV) 15–17 as previously described, with slight modifications (Hussain et al., 2014 (link)). Briefly, neurons were incubated for 1 h in artificial cerebrospinal fluid (ACSF; 125 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl₂, 25 mM HEPES, pH 7.4, 33 mM glucose, 1 mM MgCl₂, 500 nM tetrodotoxin (Tocris), 20 μM bicuculline (Tocris), 1 μM strychnine (Sigma)) prior to 10-min incubation with 200 μM glycine in magnesium-free ACSF to induce chem-LTP (Figure 1A). For the 20 and 40 min time points, neurons were recovered in ACSF for another 10 or 30 min prior to lysis. To block NMDAR activity, 50 μM D-APV (Tocris) was added to the ACSF 10 min prior to the induction of LTP, and remained present throughout the treatment.