Strychnine

Miniature inhibitory postsynaptic currents (mIPSCs) were recorded in voltage-clamp mode (Vhold: −60 mV) from labeled NTS neurons in slices prepared from normoxic or CIH rats. Currents were low-pass-filtered at 2 kHz and sampled at 10 kHz. Each recording was performed in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM, Tocris, Minneapolis, MN) to block AMPA receptors and tetrodotoxin (TTX, 1 µM, Tocris) to block Na⁺ channel-mediated action potentials, thereby eliminating excitatory glutamatergic inputs. mIPSCs were collected over a 4 min baseline period and continually recorded for another 5 min after addition of bath SR95531 (25 µM, Tocris) to abolish GABA_A receptor (GABA_AR)-mediated currents and isolate GlyR-mediated mIPSCs. In some experiments, strychnine (3 µM Tocris) was used to block glycine currents. Our recording conditions (heightened intracellular chloride concentration of 72 mM, ECl⁻ = approximately −15 mV) made glycinergic and GABAergic currents appear inward at our holding potential of −60 mV.

Recombinant rat CCL2 was obtained from R&D Systems (Minneapolis, MN, USA). Minocycline was purchased from Sigma (St Louis, MO, USA). BMS CCR2 22, picrotoxin, strychnine, tetrodotoxin, 2-amino-5-phosphonovalerate (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were obtained from Tocris Bioscience (Bristol, UK). WP9QY was purchased from Santa Cruz (Dallas, TX, USA).

Borosilicate glass microelectrodes were filled with intracellular solution containing (in mm): 120 Cs-methanesulfonate, 5 TEA-Cl, 10 HEPES, 10 BAPTA, 3 NaCl, 2 QX 314-Cl, 4 ATP-Mg, 0.4 GTP-Na2, and 10 phosphocreatine-Tris2 (pH 7.3, 280 mOsm), and a red fluorescent dye (Sulforhodamine 101; Sigma-Aldrich). Voltage-clamp recordings were performed at the reversal potential for chloride and cations, respectively, −67 and +15 mV. Membrane potential recordings were obtained in current clamp mode (I = 0 nA). Cs-based solution suppressed potassium channel activity to improve voltage-clamp recordings. While Cs likely also altered the resting potential and amplitude of the recorded membrane voltage response, this was not expected to substantially alter the timing of the stimulus evoked response. Post hoc assessment of dendritic morphology from dye fills was used to confirm α-type identity of all electrophysiologically recorded ganglion cells. To test for inhibitory circuit contributions to phase advancing in ganglion cells we used a loss-of-function approach. Glycine receptors were blocked with strychnine (1 μm; Tocris); GABA_a receptors were blocked with SR95531 (gabazine, 50 μm; Tocris); GABA_a-ρ (former GABA_c) receptors were blocked with the selective, competitive antagonist TPMPA (50 μm; Tocris).

DNQX was obtained from Sigma-Aldrich. D-2-amino-5-phosphonopentanoic acid (APV), bicuculline, [D-Ala², N-Me-Phe⁴, Gly-ol⁵]-enkephalin (DAMGO), forskolin, and naloxone were obtained from Tocris. Strychnine was obtained from Tocris or Abcam.

Tetrahydropyridin-4-yl methylphosphinic acid (TPMPA), strychnine, DNQX and D-AP5, GF 109203X were purchased from Tocris Bioscience (Park Ellisville, MO, USA). LPS, minocycline, TTX, glutamine, GABA and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma (St. Louis, MO, USA). Recombinant human IL-1β and IL-1ra proteins were purchased from R&D Systems (Minneapolis, MN, USA). PKCI 19–31 was obtained from EMD Biosciences (San Diego, CA, USA). All pharmacological agents were applied by perfusion into the recording chamber unless indicated.

Under baseline conditions, neurons were incubated in HEPES-buffered saline (HBS) containing (in mM) the following: 119 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 30 Glucose, 10 HEPES, pH 7.4. Pharmacological induction of LTP in cultured hippocampal neurons was achieved via brief (5 min) exposure to a Mg²⁺ −free HBS solution supplemented with (in mM): 0.4 Glycine (Fisher, Waltham, MA), 0.02 Bicuculline (Tocris), and 0.003 Strychnine (Tocris, Bristol, UK) Neurons were immediately washed with warm HBS after Glycine stimulation and imaged.