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.

The rhodopsin genes of Drosophila melanogaster are expressed in nonoverlapping subsets of photoreceptor cells within the insect visual system. Two of these genes, Rh3 and Rh4, are known to display complementary expression patterns in the UV-sensitive R7 photoreceptor cell population of the compound eye. In addition, we find that Rh3 is expressed in a small group of paired R7 and R8 photoreceptor cells at the dorsal eye margin that are apparently specialized for the detection of polarized light. In this paper we present a detailed characterization of the cis-acting requirements of both Rh3 and Rh4. Promoter deletion series demonstrate that small regulatory regions (less than 300 bp) of both R7 opsin genes contain DNA sequences sufficient to generate their respective expression patterns. Individual cis-acting elements were further identified by oligonucleotide-directed mutagenesis guided by interspecific sequence comparisons. Our results suggest that the Drosophila rhodopsin genes share a simple bipartite promoter structure, whereby the proximal region constitutes a functionally equivalent promoter "core" and the distal region determines cell-type specificity. The expression patterns of several hybrid rhodopsin promoters, in which all or part of the putative core regions have been replaced with the analogous regions of different rhodopsin promoters, provide additional evidence in support of this model.

We have constructed a series of strains to facilitate the generation and analysis of clones of genetically distinct cells in developing and adult tissues of Drosophila. Each of these strains carries an FRT element, the target for the yeast FLP recombinase, near the base of a major chromosome arm, as well as a gratuitous cell-autonomous marker. Novel markers that carry epitope tags and that are localized to either the cell nucleus or cell membrane have been generated. As a demonstration of how these strains can be used to study a particular gene, we have analyzed the developmental role of the Drosophila EGF receptor homolog. Moreover, we have shown that these strains can be utilized to identify new mutations in mosaic animals in an efficient and unbiased way, thereby providing an unprecedented opportunity to perform systematic genetic screens for mutations affecting many biological processes.

We have made a P-element derivative called Pc[ry], which carries the selectable marker gene rosy, but which acts like a nondefective, intact P element. It transposes autonomously into the germline chromosomes of an M-strain Drosophila embryo and it mobilizes in trans the defective P elements of the singed-weak allele. Frameshift mutations introduced into any of the four major open reading frames of the P sequence were each sufficient to eliminate the transposase activity, but none affected signals required in cis for transposition of the element. Complementation tests between pairs of mutant elements suggest that a single polypeptide comprises the transposase. We have examined transcripts of P elements both from natural P strains and from lines containing only nondefective Pc[ry] elements, and have identified two RNA species that appear to be specific for autonomous elements.

Many animals maintain an internal representation of their heading as they move through their surroundings. Such a compass representation was recently discovered in a neural population in the Drosophila melanogaster central complex, a brain region implicated in spatial navigation. Here, we use two-photon calcium imaging and electrophysiology in head-fixed walking flies to identify a different neural population that conjunctively encodes heading and angular velocity, and is excited selectively by turns in either the clockwise or counterclockwise direction. We show how these mirror-symmetric turn responses combine with the neurons' connectivity to the compass neurons to create an elegant mechanism for updating the fly's heading representation when the animal turns in darkness. This mechanism, which employs recurrent loops with an angular shift, bears a resemblance to those proposed in theoretical models for rodent head direction cells. Our results provide a striking example of structure matching function for a broadly relevant computation.

To compare appetitive and aversive visual memories of the fruit fly Drosophila melanogaster, we developed a new paradigm for classical conditioning. Adult flies are trained en masse to differentially associate one of two visual conditioned stimuli (CS) (blue and green light as CS) with an appetitive or aversive chemical substance (unconditioned stimulus or US). In a test phase, flies are given a choice between the paired and the unpaired visual stimuli. Associative memory is measured based on altered visual preference in the test. If a group of flies has, for example, received a sugar reward with green light in the training, they show a significantly higher preference for the green stimulus during the test than another group of flies having received the same reward with blue light. We demonstrate critical parameters for the formation of visual appetitive memory, such as training repetition, order of reinforcement, starvation, and individual conditioning. Furthermore, we show that formic acid can act as an aversive chemical reinforcer, yielding weak, yet significant, aversive memory. These results provide a basis for future investigations into the cellular and molecular mechanisms underlying visual memory and perception in Drosophila.

We tested whether Drosophila larvae can associate odours with a mechanosensory disturbance as a punishment, using substrate vibration conveyed by a loudspeaker (buzz:). One odour (A) was presented with the buzz, while another odour (B) was presented without the buzz (A/B training). Then, animals were offered the choice between A and B. After reciprocal training (A/B), a second experimental group was tested in the same way. We found that larvae show conditioned escape from the previously punished odour. We further report an increase of associative performance scores with the number of punishments, and an increase according to the number of training cycles. Within the range tested (between 50 and 200 Hz), however, the pitch of the buzz does not apparently impact associative success. Last, but not least, we characterized odour-buzz memories with regard to the conditions under which they are behaviourally expressed--or not. In accordance with what has previously been found for associative learning between odours and bad taste (such as high concentration salt or quinine), we report that conditioned escape after odour-buzz learning is disabled if escape is not warranted, i.e. if no punishment to escape from is present during testing. Together with the already established paradigms for the association of odour and bad taste, the present assay offers the prospect of analysing how a relatively simple brain orchestrates memory and behaviour with regard to different kinds of 'bad' events.

The behavioral state of an animal can dynamically modulate visual processing. In flies, the behavioral state is known to alter the temporal tuning of neurons that carry visual motion information into the central brain. However, where this modulation occurs and how it tunes the properties of this neural circuit are not well understood. Here, we show that the behavioral state alters the baseline activity levels and the temporal tuning of the first directionally selective neuron in the ON motion pathway (T4) as well as its primary input neurons (Mi1, Tm3, Mi4, Mi9). These effects are especially prominent in the inhibitory neuron Mi4, and we show that central octopaminergic neurons provide input to Mi4 and increase its excitability. We further show that octopamine neurons are required for sustained behavioral responses to fast-moving, but not slow-moving, visual stimuli in walking flies. These results indicate that behavioral-state modulation acts directly on the inputs to the directionally selective neurons and supports efficient neural coding of motion stimuli.

Analyzing Drosophila melanogaster neural expression patterns in thousands of three-dimensional image stacks of individual brains requires registering them into a canonical framework based on a fiducial reference of neuropil morphology. Given a target brain labeled with predefined landmarks, the BrainAligner program automatically finds the corresponding landmarks in a subject brain and maps it to the coordinate system of the target brain via a deformable warp. Using a neuropil marker (the antibody nc82) as a reference of the brain morphology and a target brain that is itself a statistical average of data for 295 brains, we achieved a registration accuracy of 2 μm on average, permitting assessment of stereotypy, potential connectivity and functional mapping of the adult fruit fly brain. We used BrainAligner to generate an image pattern atlas of 2954 registered brains containing 470 different expression patterns that cover all the major compartments of the fly brain.