Maturation of synapses and their integration into neural systems during development

We aim: (i) to describe the sequence of events that takes place while synapses mature; (ii) to study the number and spacing of identifyable synapses during development. For these aims, we have selected well characterised synapses from two central neural systems of the locust: (i) Output synapses of large ocellar interneurons that form part of a pathway for flight stabilisation, and (ii) input synapses onto the lobula giant movement detector (LGMD) that is part of a neural circuit for avoidance of collisions. In adult locusts, the physiological properties and morphological characteristics of all of these synapses have been well studied and the synapses are readily localised. They include both excitatory and inhibitory connections, and some function in a phasic and some in a tonic way, so that a range of different synapses will be covered. We shall use histological methods and anterograde labelling methods of neuronal tracts to identify the neurons and their processes in nymphs and embryos. Electron microscopy and molecular markers for the synaptic vesicle proteins synaptotagmin and synapsin will then help us localise the synases and study their development. Using these methods, we shall focus on specific questions: (i) which sequence of events leads to the formation of mature, central synapses at the ultrastructural level. (ii) when certain proteins of the synaptic release machinery assemble while synapses mature and (iii) how the synaptic pattern characteristic of the adult is established during development. I (Dr. Gerd Leitinger) will be able to use my experience in most of the appropriate methods and carry out the experiments in the Department of Histology and Embryology in Graz. The equipment necessary is already present at that department, and the theme of research will fit well into the neurobiological emphasis of the group headed by Univ. Prof. Dr. Maria Anna Pabst (head of the department). This project will also be carried out in collaboration with two experienced scientists from the Department of Neuroscience, University of Newcastle upon Tyne. Dr. Peter J. Simmons and Dr. Claire F. Rind are experts in the fields of neurophysiology and neuroanatomy and have recently performed many detailed studies on the L-neuron and LGMD systems. Collaboration between the Newcastle and Graz group will consist of the exchange of ideas and the discussion of data gained, and I shall be able to use an electron microscope with tilting stage function in Newcastle.

The different visual systems of the locust - compound eyes as well as single eyes - are particularly well suited for studying developing sites of contact between neurons. Large interneurons (neurons which are connected to photoreceptors) in the single eye system, for example, are readily identified with microscopical techniques because of the size and location of their processes, and their sites of contact (synapses) to other neurons can already be identified after hatching. However, these synapses are rearranged during later developmental stages, which consist of several larval instars. In the compound eye visual system, processes of the lobula giant movement detector neuron are readily identified because of their characteristic arrangement, and this arrangement can be found in early instars. In order to detect proteins which are part of the synaptic structure in both these neurons, we applied specific labelling methods to tissue taken from larvae and adult locusts. In collaboration with scientists of the University of Newcastle upon Tyne, UK (Dr. Peter Simmons, Dr. Claire Rind), we used both light microscopy and electron microscopy for these studies. We thus found the synaptic protein synapsin in interneurons, but not in photoreceptors of the single and compound eye; this difference may be due to differences in the mode of operation of the synapses of these neurons. Moreover, we found that the synaptic proteins synapsin and synaptotagmin accumulate soon after hatching within the interneurons in the single eye system, which indicates that synapses are soon formed. However, we found that these synaptic locations are reorganised during development: whereas they are evenly distributed between the periphery and the centre of the brain in young instars, these synaptic locations are later only found in central areas. The giant movement detecting neurons of the compound eye also develop early. They exhibit a fan of finger-like processes that are each met by two processes of input neurons. This characteristic arrangement of input processes is crucial for the functioning of the giant movement detectors, and we have already detected this in young instars. However, further experiments will be necessary to find out whether these young processes exhibit functioning synapses. Another major part of the project concerned cell culture of neurons of the snail, Helix. Pairs of snail neurons were isolated in Turin in cooperation with Dr. Ferdinando Fiumara of Turin University (Italy). After cultivating the cell pairs under stimulating conditions, they developed synaptic contact sites whose function was studied in Turin. In Graz, we were able to demonstrate using electron microscopy that these sites showed all the features of synaptic contacts.