We designed a novel imaging technique based on frustrated total internal reflection (FTIR) to obtain high resolution and high contrast movies. This FTIR-based Imaging Method (FIM) is suitable for a wide range of biological applications and a wide range of organisms. It operates at all wavelengths permitting the in vivo detection of fluorescent proteins. To demonstrate the benefits of FIM, we analyzed large groups of crawling Drosophila larvae. The number of analyzable locomotion tracks was increased by implementing a new software module capable of preserving larval identity during most collision events. This module is integrated in our new tracking program named FIMTrack which subsequently extracts a number of features required for the analysis of complex locomotion phenotypes. FIM enables high throughput screening for even subtle behavioral phenotypes. We tested this newly developed setup by analyzing locomotion deficits caused by the glial knockdown of several genes. Suppression of kinesin heavy chain (khc) or rab30 function led to contraction pattern or head sweeping defects, which escaped in previous analysis. Thus, FIM permits forward genetic screens aimed to unravel the neural basis of behavior.

Ensheathment of axons by glial membranes is a key feature of complex nervous systems ensuring the separation of single axons or axonal fascicles. Nevertheless, the molecules that mediate the recognition and specific adhesion of glial and axonal membranes are largely unknown. We use the Drosophila midline of the embryonic central nervous system as a model to investigate these neuron glia interactions. During development, the midline glial cells acquire close contact to commissural axons and eventually extend processes into the commissures to wrap individual axon fascicles. Here, we show that this wrapping of axons depends on the interaction of the neuronal transmembrane protein Neurexin IV with the glial Ig-domain protein Wrapper. Although Neurexin IV has been previously described to be an essential component of epithelial septate junctions (SJ), we show that its function in mediating glial wrapping at the CNS midline is independent of SJ formation. Moreover, differential splicing generates two different Neurexin IV isoforms. One mRNA is enriched in septate junction-forming tissues, whereas the other mRNA is expressed by neurons and recruited to the midline by Wrapper. Although both Neurexin IV isoforms are able to bind Wrapper, the neuronal isoform has a higher affinity for Wrapper. We conclude that Neurexin IV can mediate different adhesive cell-cell contacts depending on the isoforms expressed and the context of its interaction partners.

Kinesin heavy chain (Khc) is crucially required for axonal transport and khc mutants show axonal swellings and paralysis. Here, we demonstrate that in Drosophila khc is equally important in glial cells. Glial-specific downregulation of khc by RNA interference suppresses neuronal excitability and results in spastic flies. The specificity of the phenotype was verified by interspecies rescue experiments and further mutant analyses. Khc is mostly required in the subperineurial glia forming the blood-brain barrier. Following glial-specific knockdown, peripheral nerves are swollen with maldistributed mitochondria. To better understand khc function, we determined Khcdependent Rab proteins in glia and present evidence that Neurexin IV, a well known blood-brain barrier constituent, is one of the relevant cargo proteins. Our work shows that the role of Khc for neuronal excitability must be considered in the light of its necessity for directed transport in glia.

Specialized glial subtypes provide support to developing and functioning neural networks. Astrocytes modulate information processing by neurotransmitter recycling and release of neuromodulatory substances, whereas ensheathing glial cells have not been associated with neuromodulatory functions yet. To decipher a possible role of ensheathing glia in neuronal information processing, we screened for glial genes required in the Drosophila central nervous system for normal locomotor behavior. Shopper encodes a mitochondrial sulfite oxidase that is specifically required in ensheathing glia to regulate head bending and peristalsis. shopper mutants show elevated sulfite levels affecting the glutamate homeostasis which then act on neuronal network function. Interestingly, human patients lacking the Shopper homolog SUOX develop neurological symptoms, including seizures. Given an enhanced expression of SUOX by oligodendrocytes, our findings might indicate that in both invertebrates and vertebrates more than one glial cell type may be involved in modulating neuronal activity.