One of the two main central clusters of pro-opiomelanocortin (POMC) containing neurones is in the nucleus of the solitary tract (NTS), which receives vagal afferent inputs. POMC can be cleaved into the endogenous opioid β-endorphin. Thus, β-endorphin release by POMC neurones may modulate vagal afferent processing within the NTS. To map the anatomical organisation and projection pattern of NTS POMC neurones we used an adeno-associated viral vector (AAV) to drive channelrhodopsin2-mCherry expression in mice selectively expressing cre-recombinase/GFP in POMC neurones (1). We examined the cardiorespiratory effects of NTS POMC neuronal activation in the working heart-brainstem preparation (WHBP, 2). POMC-Cre mice (n=3) received NTS microinjections of AAV-EF1α-DIO-hChR2-mCherry under anaesthesia (ketamine 70mg.kg-1/medetomidine 0.45mg.kg-1 i.p.). Three weeks later mice were anaesthetised with 5% halothane (until decerebration) for set-up of the WHBP. NTS POMC neurones were activated using a laser and cardiorespiratory responses measured. Naloxone (1-5µM) was used to block opioid receptors and effects compared with responses to DAMGO (2mM), a μ-opioid agonist, microinjection into the NTS and vagal preganglionic cell groups (n=2). Brains were post-fixed and histology performed to determine the location and projection pattern of NTS POMC neurones, based on mCherry fluorescence. Immunohistochemistry was performed for choline acetyltransferase, to label vagal preganglionic neurones, and GFP, to label POMC neurones and imaged using fluorescence microscopy. Immuno- for POMC neurones (GFP) and β-endorphin was also performed in the NTS/hypothalamus. GFP positive cells were seen in the hypothalamic arcuate nucleus and NTS (200-300 somata in the caudal NTS). β-endorphin positive POMC neurones were found in the arcuate but not NTS. β-endorphin labelled fibres were found close to NTS POMC neurones. The AAV efficiently and selectively transduced NTS POMC neurones (~180); mCherry was distributed over a 1.4mm rostrocaudal distance. Most mCherry-labelled fibres were seen within the NTS but fibres also projected ventrally and ventrolaterally to the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (NA) (n=2). Opto-activation of transduced NTS POMC neurones caused a reproducible, frequency dependent bradycardia, transient apnoea and exaggerated respiratory sinus arrhythmia. These cardiorespiratory responses were reversibly abolished by naloxone (n=1), but not mimicked by DAMGO microinjection into the NTS (n=2). This subgroup of POMC neurones forms an output path from the NTS to other brainstem autonomic centres, including the DMV and NA. Our results suggest that NTS POMC neurones can play a role in modulating vagal afferent processing via opioid release and may mediate cardiorespiratory responses.