Vagal afferent activation of NTS neurons is mediated by 5-HT3/7 receptors (Ramage & Villalón, 2008). Using fast cyclic voltammetry it was shown that extracellular 5-HT in the NTS increases in response to vagal afferent stimulation (Millar et al., 2009). Experiments were carried out to investigate the characteristics of this response. Sprague-Dawley rats (200-275g) were anesthetised with α-chloralose (120 mg kg-1, i.v), neuromuscular blocked (α-bungarotoxin 30 μg, i.v.) and artificially ventilated. Depth of anaesthesia was assessed by the stability of BP and HR following a noxious stimulus and additional anaesthetic was given when necessary. The left vagus nerve was exposed and tied distally to the stimulating site. A carbon fibre microelectrode (tip Ø 7 x 100 μm; Millar & Pelling, 2001) was lowered into the NTS until the evoked action potentials from vagal stimulation (1Hz, 100μA, 1ms) were maximal. Recording sites were confirmed histologically. A modified form of fast differential voltammetry was used for electrochemical detection of 5-HT. Electrochemical signals were quantified by differential recording of the peak oxidation and re-reduction currents. All values (n=3-6) are means ± S.E.M. Comparisons between means were made with Student’s unpaired t-test. P<0.05 was considered to be significant. At threshold (40-100µA) for electrically-evoked activity over the frequency range of 5 to 50Hz no 5-HT could be detected. At 5x threshold there was a frequency dependent increase in the 5-HT concentration, ranging from 6±2 to 22±8 nM. At 10 & 30x threshold this relationship was not observed, although 50Hz still caused a maximum increase of 17±4 (10x) and 26±10nM (30x). Stimulation of cardiopulmonary C-fibres with phenylbiguanide (50 μg kg-1, i.a.) caused an increased in 5-HT of 7 ± 1 nM. Topical application of CdCl2 (10mM, 30μl) attenuated the electrically evoked (20Hz, 10x threshold) increase in 5-HT (3 ± 0.4 c.f. 7 ± 0.6nM). Changes in HR and BP associated with vagal stimulation were attenuated. Blockade of the serotonin transporter (SERT) with fluoxetine (1mg kg-1; i.v.) failed to increase release. However, the OCT3 (uptake2) and plasma membrane monoamine transporter (PMAT) blocker decynium-22 (D-22, 1 mg kg-1, i.v., Hayer-Zillgen et al, 2002) caused a significant increase in electrically evoked 5-HT (107 ± 52 %) c.f. DMSO (25 µl) control. This was associated with a significant increase (176 ± 84%) in the evoked bradycardia. Both vagal stimulation and D-22 caused hypotension, however chlorisondamine (1 mg kg-1, i.v.) had no effect on the increase in 5-HT despite blocking the evoked hypotension and causing a fall in BP. It is concluded that the 5-HT release in the NTS evoked by vagal afferent stimulation is removed by OCT3/PMAT not by SERT.