Dihydro β erythroidine hydrobromide dhβe

In the gramicidin perforation experiments, GABA_AR responses were isolated by using a cocktail of drugs containing 0.2 µM tetrodotoxin (TTX, Latoxan Laboratory, France), 4 mM kynurenic acid (Millipore Sigma), 10 µM (+)-tubocurarine (Millipore Sigma), 5 µM dihydro-β-erythroidine hydrobromide (DHΒE, Bio-Techne, Lille, France), and 3 µM strychnine (Millipore Sigma), which blocked voltage-dependent Na⁺ action potentials and glutamate, cholinergic, and glycinergic inputs to MNs, respectively. VU0240551 (Bio-Techne, France) (10 µM) was applied to specifically block KCC2. Serotonin (5-HT, 10 µM) was purchased from Millipore Sigma. Methysergide maleate (10 µM) and ketanserin tartrate (10 µM) (Bio-techne) were used to evaluate the involvement of 5-HTRs in the effect of 5-HT. Combined, these antagonists are known to efficiently block 5-HT_2A-CR [31 (link),32 (link)].

In gramicidin perforated experiments and in experiments performed for assessing the KCC2 efficacy, GABA_AR responses were isolated by using a cocktail of drugs containing 0.2 μM tetrodotoxin (TTX, Latoxan Laboratory, France), 4 mM kynurenic acid (Millipore Sigma), 10 μM (+)-tubocurarine (Millipore Sigma), 5 μM Dihydro-β-erythroidine hydrobromide (DHβE, Bio-techne, France), and 3 μm strychnine (Millipore Sigma) that respectively blocked voltage-dependent Na⁺ action potentials, and glutamate, cholinergic, and glycinergic input to MNs. In CsCl experiments, IPSCs were isolated pharmacologically using DL-AP5 40 μM ((2R)-amino-5-phosphonovaleric acid, Bio-techne, France) and CNQX 20 μM (6-cyano-7-nitroquinoxaline-2,3-dione, Bio-techne, France). mIPSCs were isolated in the presence of 0.2 μM TTX (Latoxan, France). GABA and glycine mIPSCs were isolated by adding 3 μM strychnine or 3 μM GABAzine (SR 95531 hydrobromide, Bio-techne, France), respectively. These blockers were added to the cocktail containing 0.2 µM TTX, 4 mM kynurenic acid, 10 μM (+)-tubocurarine and 5 μM DHβE. In experiments assessing the KCC2 efficacy, 10 µM bumetanide (Millipore Sigma) was applied to block NKCC1 and 10 µM VU0240551 (Bio-techne, France) (10 µM) to block KCC2. Serotonin (5-HT, 10 µM), dopamine (DA, 100 µM) and N-Methyl-D-aspartic acid (NMDA, 10 µM) were from Millipore Sigma.

DFP and all test pharmaceuticals were obtained from the same sources for the in vitro and in vivo studies. DFP was purchased from Sigma (St. Louis, MO, USA) and stored at −80 °C. The DFP’s purity of ≥90% was confirmed via nuclear magnetic resonance as previously described [28 (link)]. Atropine sulfate (AS), 2-pralidoxime (2-PAM), and paraoxon were purchased from Sigma and stored at room temperature or −20 °C. Manufacturer certificates of analysis indicated their purities to be as follows: AS (lot #BCBM6966V), >97%; 2-PAM (lot #MKCG3184), >99%; and paraoxon (#MFCD00007316), 99.9%. Nicotine was purchased from Fisher Scientific (Fisher Scientific; Fair Lawn, NJ, USA). Manufacturer certificates of analysis indicated that the purity of the nicotine (#AC181420050) was >99%. Mecamylamine hydrochloride was purchased from AK Scientific (Union City, CA, USA). Dihydro-β-erythroidine hydrobromide (DHβE) and methyllycaconitine citrate (MLA) were purchased from Tocris (Bristol, UK). Manufacturer certificates of analysis indicated their purities to be as follows: MEC (lot #TC23203), >98%; DHβE (batch #11A/249912), >98%; and MLA (batch #23A/251394), >98%.