Esp 32

˙OH production was estimated by measuring the extracellular levels of 2,3-dihydroxybenzoic acid (2,3-DHBA) formed through the nonenzymatic hydroxylation of salicylic acid^{42 (link)} according to the protocol of Teismann and Ferger^{43 (link)} with modifications^{44 (link)}. A microdialysis probe with a cellulose membrane (3 mm in length and 0.22 mm in diameter) with or without a thin fused-silica needle (0.15 mm in diameter) for drug administration (MI-A-I-8-03 or A-I-8-03, respectively; Eicom) was inserted into the striatum through the guide cannula and perfused with modified Ringer’s solution (147 mM NaCl, 3 mM KCl, 1.3 mM CaCl₂, and 1 mM MgCl₂) containing 5 mM sodium salicylate (Nacali Tesque; Kyoto, Japan) at a flow rate of 2 μL/min using a microsyringe pump (ESP-32; Eicom). The dialysate was collected in an autoinjector (EAS-20; Eicom) in 40-μL fractions every 20 min and injected into an inert HPLC system (Eicom) that was equipped with an electrochemical detector (ECD-300; Eicom) consisting of a graphite working electrode at +500 mV vs. an Ag/AgCl reference electrode. Separation was carried out on an Eicompac SC-5ODS column (2.1 × 150 mm) at 25 °C with a mobile phase that consisted of 100 mM sodium phosphate buffer (pH 6.0) containing 13.4 μM EDTA and 2% (v/v) methanol at a flow rate of 230 μL/min.

A linear dialysis probe (OP-100-10, Eicom, Kyoto, Japan) was implanted in the submandibular glands of rats under inhalation anesthesia with nitrous oxide, oxygen, and isoflurane (2%). The semipermeable membrane region (10 mm) of the probe was implanted in the left submandibular gland along the long axis (Figure 1). The probes were perfused with Ringer’s solution at a speed of 2 µL/min by a micro-infusion pump (ESP-32, Eicom, Kyoto, Japan). The Ringer’s solution consisted of 147 mM NaCl, 2.2 mM CaCl₂, 4.02 mM KCl, and the cholinesterase inhibitor Eserine (physostigmine; 100 µM). To elicit the release of acetylcholine from the nerve endings, the microdialysis probe was perfused with Ringer’s solution containing a higher than usual amount of KCI (147 mM NaCl, 2.2 mM CaCl₂, 100 mM KCl, 100 µM Eserine) (high-K⁺ Ringer’s solution). The perfused solution was switched to high-K⁺ Ringer’s solution and back to regular Ringer’s solution by using an SI-60 liquid switch (Eicom, Kyoto, Japan).
Each sampling period lasted 15 min (sample volume = 30 µL), which allowed for the sufficient collection of effluent for a quantitative determination of acetylcholine level. Each 15 min sample was collected in a chilled microtube containing 3 µL of 3 nM IPHC as an internal standard.