Dcg 4

Carbachol (CCh), CGP55845, NBQX, DCG-IV, D-APV, picrotoxin, atropine, mecamylamine, nitrocaramiphen, DAU5884, AM251, GDP-β-S were purchased from Tocris (UK). GSK-5 was synthesised in-house at Eli Lilly and Co. Stock solutions of these compounds were made by dissolving in water. The selective muscarinic M₁ and M₄ receptor agonist Compound 1 was synthesised in-house at Sosei Heptares and dissolved in DMSO for stock solution. The purity of the final compounds was determined by HPLC or LC/MS analysis to be >95%. Additional experimental details relating to the synthesis of Compound 1 and associated structures are described in detail in WO2015/118342 which relates to the invention of agonists of the muscarinic M₁ receptor and/or M₄ receptor and which are useful in the treatment of muscarinic M₁/M₄ receptor-mediated diseases.

Recordings were done in a submersion-type chamber perfused with ACSF (29.5°C–30.5°C, 2 ml/min). Synaptic responses were evoked through glass bipolar stimulating electrodes placed in the dentate granule cell layer to activate MFs with half-maximal stimulation intensity (0.2 ms pulse duration at 0.067 Hz), and recorded extracellularly in the stratum lucidum of CA3. Paired-pulse facilitation (PPF) was measured at 25, 50, 100, 200, 400, 1000, and 2000 ms interstimulus intervals (ISIs). To induce mfLTP, three trains of 100 Hz (1 sec) stimuli were given at 20 sec intervals. All experiments were done in the presence of 100 μM D,L-2-amino-5-phosphonovaleric acid (D,L-APV) (Sigma-Aldrich) to isolate the presynaptic NMDAR-independent mossy fiber long-term potentiation (mfLTP) [20] (link). At the end of each experiment, 1 μM (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl) glycine (DCG-IV) (Tocris Bioscience) was added, and blockade ≥80% were taken to be MF inputs. Field potential slopes were measured, and data are expressed as mean ± standard error of mean.

Stock solutions for all drugs applied by i.h. injection were diluted in physiological saline (0.9%). ACET, d-(−)-2-amino-5-phosphonopentanoic acid (D-AP5), DCG-IV, LY367385, MCPG, and MPEP were all sourced from Tocris, Untied Kingdom; sodium pentobarbitone and urethane were obtained from Sigma-Aldrich, United Kingdom; UBP161 was synthesized in-house as reported previously (Irvine et al., 2012 (link)). The compounds, ACET (0.1 nmol), d-AP5 (1 nmol), UBP161 (1 nmol), LY367385 (0.3 and 1 nmol), MPEP (0.3 and 1 nmol), MCPG (1, 5 and 50 nmol), and interleaved vehicle controls, were injected in a 2 μL volume i.h. over a 10 min period. DCG-IV (0.01 nmol) was injected in a 1 μL volume i.h. over a 10 min period, 90 min after tetanization. The concentrations of mGlu agonist or antagonists were similar to those used by others (Manahan-Vaughan and Reymann, 1997 (link); Manahan-Vaughan et al., 1998 (link); Naie and Manahan-Vaughan, ,).

MK-801, NBQX, CGP-55845, nimodipine, DCG-IV, and GYKI 53655 were obtained from Tocris-Cookson. LY 303070 was obtained from ABX advanced biochemical compounds. BoTX was obtained from List Biological. All other chemicals and drugs were purchased from Sigma-Aldrich.

Picrotoxin (GABA_AR antagonist; 100 μM), NBQX (AMPAR antagonist; 5 μM), QX-314 (Na⁺-channel blocker; 5 mM), and CGP55845 (GABA_BR antagonist; 2 μM) were obtained from Abcam (Cambridge, UK). R-CPP (NMDAR antagonist; 5–10 μM), LY341495 (mGluR2 antagonist; 0.5 μM), DCG-IV (mGluR2 agonist; 1 μM), and DL-TBOA (EAAT antagonist; 25–50 μM) were purchased from Tocris Bioscience (Minneapolis, MN). Strychnine (GlyR antagonist; 1 μM) and all other drugs and chemicals were obtained from Sigma Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).

L-glutamate was purchased from Sigma. DCG-IV, L-AP4, MNI137 and LY487379 were from Tocris Bioscience. LSP4-2022 was a provided by Dr. F. Acher (Paris, France). Glutamate-pyruvate transaminase (GPT) was purchased from Roche. Lipofectamine 2000 and Fluo-4-AM were from Life Technologies. SNAP-Green was from NEN Biolabs.