The formation of a stable olfactory memory requires activity within several brain regions. The honeybee provides a valuable model to map complex olfactory learning tasks onto certain brain areas. To this end, we used injections of the local anaesthetic procaine to reversibly block spike activity in a specific brain region, the mushroom body (MB). We first investigated the physiological effects of procaine on cultured MB neurons from adult honeybee brains. Using the whole-cell configuration of the patch-clamp technique, we show that procaine blocks voltage-gated Na+ and K+ currents in a dose-dependent manner between 0.1 and 10 mm. The effects are reversible within a few minutes of wash. Lidocaine acts similarly, but is less effective at the tested concentrations. We then studied the role of the MBs during reversal learning by blocking the neural activity within these structures by injecting procaine. During reversal learning bees learn to revert their responses to two odorants, one rewarded (A+) and one unrewarded (B-), if their contingencies are changed (A- vs B+). Injecting procaine into the MBs impaired reversal learning. Procaine treatment during acquisition left the later retention of the initial learning (A+ vs B-) intact. Similarly, a differential conditioning task involving novel odorants (C+ vs D-) was intact under procaine treatment. Our experiments show that local injections of procaine can be used to map learning tasks onto specific regions of the insect brain. We conclude that intact MB activity is required for the acquisition of reversal learning, but not for simple differential learning tasks.

Stonins are a small family of evolutionarily conserved clathrin adaptor complex AP-2μ-related factors that may act as cargo-specific sorting adaptors in endocytosis and perhaps beyond. Whereas little is known about the localization and function of stonin 1, recent work suggests that stonin 2 serves as a linker between the endocytic proteins AP-2 and Eps15 and the calciumsensing synaptic vesicle (SV) protein synaptotagmin 1. The molecular determinants involved in the recognition of SV cargo by the μ-homology domain of stonin 2 are evolutionarily conserved from worm to man, thereby identifying stonin 2 and its invertebrate homologs uncoordinated (UNC)-41 and stoned B as endocytic adaptors dedicated to the retrieval of surface-stranded SV proteins, most notably synaptotagmin. In this review, we summarize the current state of knowledge about mammalian stonins with a special focus on the role of stonin 2 in SV recycling at presynaptic nerve terminals.

Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery.