Effects of transgenic expression of botulinum toxins in Drosophila.

Clostridial neurotoxins (botulinum toxins and tetanus toxin) disrupt neurotransmitter release by cleaving neuronal SNARE proteins. We generated transgenic flies allowing for conditional expression of different botulinum toxins and evaluated their potential as tools for the analysis of synaptic and neuronal network function in Drosophila melanogaster by applying biochemical assays and behavioral analysis. On the biochemical level, cleavage assays in cultured Drosophila S2 cells were performed and the cleavage efficiency was assessed via western blot analysis. We found that each botulinum toxin cleaves its Drosophila SNARE substrate but with variable efficiency. To investigate the cleavage efficiency in vivo, we examined lethality, larval peristaltic movements and vision dependent motion behavior of adult Drosophila after tissue-specific conditional botulinum toxin expression. Our results show that botulinum toxin type B and botulinum toxin type C represent effective alternatives to established transgenic effectors, i.e. tetanus toxin, interfering with neuronal and non-neuronal cell function in Drosophila and constitute valuable tools for the analysis of synaptic and network function.

Aging Drosophila melanogaster display altered pre- and postsynaptic ultrastructure at adult neuromuscular junctions.

Although age-related changes in synaptic plasticity are an important focus within neuroscience, little is known about ultrastructural changes of synaptic morphology during aging. Here we report how aging affects synaptic ultrastructure by using fluorescence and electron microscopy at the adult Drosophila neuromuscular junction (NMJ) of ventral abdominal muscles. Mainly four striking morphological changes of aging NMJs were revealed. 1) Bouton size increases with proportionally rising number of active zones (AZs). 2) Synaptic vesicle density at AZs is increased in old flies. 3) Late endosomes, cisternae, and multivesicular bodies accumulate in the presynaptic terminal, and vesicles accumulate between membranes of the terminal bouton and the subsynaptic reticulum. 4) The electron-dense pre- and postsynaptic apposition is expanded in aging NMJs, which is accompanied by an expansion of the postsynaptic glutamate receptor fields. These findings suggest that aging is possibly accompanied by impaired synaptic vesicle release and recycling and a potentially compensatory expansion of AZs and postsynaptic densities.

Behavioral consequences of dopamine deficiency in the Drosophila central nervous system.

The neuromodulatory function of dopamine (DA) is an inherent feature of nervous systems of all animals. To learn more about the function of neural DA in Drosophila, we generated mutant flies that lack tyrosine hydroxylase, and thus DA biosynthesis, selectively in the nervous system. We found that DA is absent or below detection limits in the adult brain of these flies. Despite this, they have a lifespan similar to WT flies. These mutants show reduced activity, extended sleep time, locomotor deficits that increase with age, and they are hypophagic. Whereas odor and electrical shock avoidance are not affected, aversive olfactory learning is abolished. Instead, DA-deficient flies have an apparently "masochistic" tendency to prefer the shock-associated odor 2 h after conditioning. Similarly, sugar preference is absent, whereas sugar stimulation of foreleg taste neurons induces normal proboscis extension. Feeding the DA precursor L-DOPA to adults substantially rescues the learning deficit as well as other impaired behaviors that were tested. DA-deficient flies are also defective in positive phototaxis, without alteration in visual perception and optomotor response. Surprisingly, visual tracking is largely maintained, and these mutants still possess an efficient spatial orientation memory. Our findings show that flies can perform complex brain functions in the absence of neural DA, whereas specific behaviors involving, in particular, arousal and choice require normal levels of this neuromodulator.

Visual targeting of motor actions in climbing Drosophila.

Drosophila melanogaster flies cross surmountable gaps in their walkway of widths exceeding their body length with an astounding maneuver but avoid attempts at insurmountable gaps by visual width estimation. Different mutant lines affect specific aspects of this maneuver, indicating a high complexity and modularity of the underlying motor control. Here we report on two mutants, ocelliless(1) and tay bridge(1), that, although making a correct decision to climb, fail dramatically in aiming at the right direction. Both mutants show structural defects in the protocerebral bridge, a central complex neuropil formed like a handlebar spanning the brain hemispheres. The bridge has been implicated in step-length control in walking flies and celestial E-vector orientation in locusts. In rescue experiments using tay bridge(1) flies, the integrity of the bridge was reestablished, concomitantly leading to a significant improvement of their orientation at the gap. Although producing directional scatter, their attempts were clearly aimed at the landing site. However, this partial rescue was lost in these flies at a reduced-visibility landing site. We therefore conclude that the protocerebral bridge is an essential part of a visual targeting network that transmits directional clues to the motor output via a known projection system.

Analysis of a spatial orientation memory in Drosophila.

Flexible goal-driven orientation requires that the position of a target be stored, especially in case the target moves out of sight. The capability to retain, recall and integrate such positional information into guiding behaviour has been summarized under the term spatial working memory. This kind of memory contains specific details of the presence that are not necessarily part of a long-term memory. Neurophysiological studies in primates indicate that sustained activity of neurons encodes the sensory information even though the object is no longer present. Furthermore they suggest that dopamine transmits the respective input to the prefrontal cortex, and simultaneous suppression by GABA spatially restricts this neuronal activity. Here we show that Drosophila melanogaster possesses a similar spatial memory during locomotion. Using a new detour setup, we show that flies can remember the position of an object for several seconds after it has been removed from their environment. In this setup, flies are temporarily lured away from the direction towards their hidden target, yet they are thereafter able to aim for their former target. Furthermore, we find that the GABAergic (stainable with antibodies against GABA) ring neurons of the ellipsoid body in the central brain are necessary and their plasticity is sufficient for a functional spatial orientation memory in flies. We also find that the protein kinase S6KII (ignorant) is required in a distinct subset of ring neurons to display this memory. Conditional expression of S6KII in these neurons only in adults can restore the loss of the orientation memory of the ignorant mutant. The S6KII signalling pathway therefore seems to be acutely required in the ring neurons for spatial orientation memory in flies.

Locomotor control by the central complex in Drosophila-An analysis of the tay bridge mutant.

Several aspects of locomotor control have been ascribed to the central complex of the insect brain; however, the role of distinct substructures of this complex is not well known. The tay bridge1 (tay1) mutant of Drosophila melanogaster was originally isolated on the basis of reduced walking speed and activity. In addition, tay1 is defective in the compensation of rotatory stimuli during walking and histologically, tay1 causes a mid-sagittal constriction of the protocerebral bridge, a constituent of the central complex. Cloning of the tay gene revealed that it encodes a novel protein with no significant homology to any known protein. To associate the behavioral phenotypes with the anatomical defect in the protocerebral bridge, we used different driver lines to express the tay cDNA in various neuronal subpopulations of the central brain in tay1-mutant flies. These experiments showed an association of the aberrant walking speed and activity with the structural defect in the protocerebral bridge. In contrast, the compensation of rotatory stimuli during walking was rescued without a restoration of the protocerebral bridge. The results of our differential rescue approach are supported by neuronal silencing experiments using conditional tetanus toxin expression in the same subset of neurons. These findings show for the first time that the walking speed and activity is controlled by different substructures of the central brain than the compensatory locomotion for rotatory stimuli.

The carrot, not the stick: appetitive rather than aversive gustatory stimuli support associative olfactory learning in individually assayed Drosophila larvae.

The ability to learn is universal among animals; we investigate associative learning between odors and "tastants" in larval Drosophila melanogaster. As biologically important gustatory stimuli, like sugars, salts, or bitter substances have many behavioral functions, we investigate not only their reinforcing function, but also their response-modulating and response-releasing function. Concerning the response-releasing function, larvae are attracted by fructose and repelled by sodium chloride and quinine; also, fructose increases, but salt and quinine suppress feeding. However, none of these stimuli has a nonassociative, modulatory effect on olfactory choice behavior. Finally, only fructose but neither salt nor quinine has a reinforcing effect in associative olfactory learning. This implies that the response-releasing, response-modulating and reinforcing functions of these tastants are dissociated on the behavioral level. These results open the door to analyze how this dissociation is brought about on the cellular and molecular level; this should be facilitated by the cellular simplicity and genetic accessibility of the Drosophila larva.