Learning allows animals to adapt their behaviour to changes in the environment. In humans and other mammals, memories are stored in the hippocampus and cerebellum, whereas in insects, they are stored inside the mushroom bodies (MB). Here, MB-intrinsic Kenyon cells (KCs) form plastic synapses to MB output neurons (MBONs) that are modulated by the reinforcing action of dopaminergic neurons (DANs). Despite decades of research on the MB, the main neurotransmitter underlying the plastic KC → MBON synapse has remained a mystery. Here, I show that this synapse is cholinergic in the fruit fly Drosophila melanogaster. MBONs show fast excitatory responses to direct acetylcholine (ACh) application. KCs synthesise ACh-related proteins ChAT and VAChT. MBONs express and require nicotinic ACh receptors (nAChRs) to become fully activated by odour presentation. Lastly, artificial activation of KCs leads to MBON calcium responses that are blocked by nicotinic antagonists and genetic reduction of VAChT in KCs. Short neuropeptide F (sNPF) may play a role as a modulatory co-transmitter that can either excite or inhibit specific MBONs and DANs. The retrieval of memories is state-dependent and known to potentially change the original memory. Fruit flies need to be hungry to express appetitive memories. Hunger state depends on insulin signalling that activates the GABAergic MBON MVP2, while appetitive memory retrieval depends on decreased activity in M4/6 MBONs. Here, I show that optogenetic MVP2 activation acutely inhibits M4/6 odour responses, rendering MVP2 an inhibitory MBON interneuron. I also show that other MBONs are functionally connected to DANs, thus linking memory reinforcement and retrieval pathways in a way that enables the updating of the original memory. These findings show that associative memories in Drosophila are initially formed at cholinergic-MBON synapses, and can be retrieved and modified through an intricate KC-MBON-DAN network.

Gamma-aminobutyric acid (GABA) and short neuropeptide F (sNPF) are widespread signalling molecules in the brain of insects. In order to understand more about the signalling and to some extent start to unravel the functional roles of these two substances, this study has examined the locations of the transmitters and their receptors in the brain of the fruit fly Drosophila melanogaster using immunocytochemistry in combination with Gal4/UAS technique. The main focus is GABA and sNPF in feeding circuits and in the olfactory system. We found both GABA receptor types in neurons in many important areas of the Drosophila brain including the antennal lobe, mushroom body and the central body complex. The metabotropic GABAB receptor (GABABR) is expressed in a pattern similar to the ionotropic GABAAR, but some distribution differences can be distinguished (paper I). The insulin producing cells contain only GABABR, whereas the GABAAR is localized on neighbouring neurons. We found that GABA regulates the production and release of insulin-like peptides via GABABRs (paper II). The roles of sNPFs in feeding and growth have previously been established, but the mechanisms behind this are unclear. We mapped the distribution of sNPF with antisera to the sNPF precursor and found the peptide in a large variety of interneurons, including the Kenyon cells of the mushroom bodies, as well as in olfactory sensory neurons that send axons to the antennal lobe (paper III). We also mapped the distribution of the sNPF receptor in larval tissues and found localization in six median neurosecretory cells that are not insulin-producing cells, in neuronal branches in the larval antennal lobe and in processes innervating the mushroom bodies (paper IV). In summary, we have studied two different signal substances in the Drosophila brain (GABA and sNPF) in some detail. We found that these substances and their receptors are widespread, that both sNPF and GABA act in very diverse systems and that they presumably play roles in feeding, metabolism and olfaction. / At the time of doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.