Glucagon-like peptide-1 (GLP-1) acts in the brain to affect homeostatic processes, such as thermoregulation, cardiovascular control and food intake. GLP-1 is produced in the lower brainstem by preproglucagon (PPG) neurons in the nucleus of the solitary tract (NTS). Whilst these neurons are morphologically well described in several species, little is known about their physiology. Most evidence for their role in energy homeostasis comes from studies using cFos as a marker of neuronal activity. Additionally, patch-clamp recordings in mouse brain slices have revealed electrical responses to satiety hormones such as leptin and cholecystokinin (CCK). Here, we characterise functional properties of the PPG neurons using a genetically-encoded calcium indicator, GCaMP3. PPG-Cre/ROSA26-GCaMP3 mice express GCaMP3 from the ROSA26 locus in a Cre recombinase-dependent manner. As Cre recombinase is expressed under the control of the glucagon promoter, GCaMP3 fluorescence only occurs in pancreatic α-cells, intestinal L-cells and PPG neurons. Experiments were performed on 200 μm thick in vitro brainstem slices from PPG-Cre/ROSA26-GCaMP3 mice cut on a vibratome. Intracellular Ca2+ concentration [Ca]i was recorded optically (excitation 488 nm; emission 515±10 nm) from NTS PPG neurons under various experimental conditions. PPG neurons receive strong glutamatergic input, and indeed 100 μM glutamate added to acute brain slices induced [Ca2+]i rises in 89% of imaged neurons (n=143). Similarly, stimulation with CCK (100 nM; n=12) increased [Ca2+]i in 42% of cells, whereas 58% of cells were unresponsive, confirming patch-clamp data. 1mM ATP, an important glial transmitter in the NTS, also increased [Ca2+]i in 89% of PPG cells analysed (n=28) and induced rapid, transient changes in [Ca2+]i in fibres surrounding the cell bodies (n=22). These Ca2+ spikes were reduced by 92±4% upon inhibition of synaptic transmission with 0.5 μM TTX, 20 μM DNQX, and 25 μM APV. 20 μM 5-HT increased [Ca2+]i in fibres surrounding cell bodies by 143±9% (n=61). Unlike ATP, the 5-HT response was not blocked by inhibition of synaptic transmission suggesting that the PPG neurons express functional 5-HT receptors. However, stimulation with the 5-HT3-receptor agonist phenylbiguanide (PBG; 1μM or 10μM) had no effect on [Ca2+]i (n=27) and similarly, 5-HT-induced increases in [Ca2+]i were unaffected by the 5-HT2A receptor antagonist ketanserine (1μM; n=21). These results demonstrate that GCaMP3 expressed selectively in PPG neurons has similar sensitivity to electrophysiology for assessing the response to signals that modulate food intake. Thus, it is a valuable tool to interrogate in vitro the regulation of PPG neurons, with potentially better understanding of population responses than patch-clamp electrophysiology.