Erich Buchner

Bruchpilot Promotes Active Zone Assembly, Ca2+ Channel Clustering, and Vesicle Release

The molecular organization of presynaptic active zones during calcium influx–triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) were entirely lost, Ca2+ channels were reduced in density, evoked vesicle release was depressed, and short-term plasticity was altered. BRP-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity.

Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae

During classical conditioning, a positive or negative value is assigned to a previously neutral stimulus, thereby changing its significance for behavior. If an odor is associated with a negative stimulus, it can become repulsive. Conversely, an odor associated with a reward can become attractive. By using Drosophila larvae as a model system with minimal brain complexity, we address the question of which neurons attribute these values to odor stimuli. In insects, dopaminergic neurons are required for aversive learning, whereas octopaminergic neurons are necessary and sufficient for appetitive learning. However, it remains unclear whether two independent neuronal populations are sufficient to mediate such antagonistic values. We report the use of transgenically expressed channelrhodopsin-2, a light-activated cation channel, as a tool for optophysiological stimulation of genetically defined neuronal populations in Drosophila larvae. We demonstrate that distinct neuronal populations can be activated simply by illuminating the animals with blue light. Light-induced activation of dopaminergic neurons paired with an odor stimulus induces aversive memory formation, whereas activation of octopaminergic/tyraminergic neurons induces appetitive memory formation. These findings demonstrate that antagonistic modulatory subsystems are sufficient to substitute for aversive and appetitive reinforcement during classical conditioning.

Elementary movement detectors in an insect visual system

In the theoretical part of the present work the input-output relation for a multi-input system is developed into a functional power series. This is formally equivalent to a decomposition of the system into a sum of all possible combinations of 1-, 2-, 3-... input subsystems. The average response of the system to a uniformly moving patern is known to be a Fourier series with respect to spatial frequency. The coefficients of the series are linear combinations of the “weights” by which different subsystems contribute to the total reaction. If a system can be shown to have essential nonlinearities of no higher than second order it is possible to calculate, from a Fourier analysis of the average movement response, the “weight” by which the nonlinear interaction between any two input elements contributes to the total reaction. This interaction is termed “elementary movement detector”. By the analysis presented here the arrangement of the elementary movement detectors may be determined for a two-dimensional array of input elements and the strength of their contributions to the total movement reaction may be calculated. Special experimental methods have been developed which allow one to apply this analysis to the visual system of the fruitfly Drosophila. The preliminary data presented show that the direction sensitive optomotor response of Drosophila can be attributed predominantly to the contributions from two “elementary movement detectors” which interconnect neighbouring visual elements. The detectors are oriented in the hexagonal array of the compound eye at +30° and at-30° with respect to the horizontal line of symmetry. A weak contribution from a detector between neighbouring elements along the horizontal line of symmetry is suggested by the present data. In the course of the analysis the contrast transfer properties of the compound eye are characterized.

The rôle of retinula cell types in visual behavior ofDrosophila melanogaster

SummaryWe propose that inDrosophila melanogaster the optomotor response to both horizontal and vertical movement is mediated predominantly by the 6 large retinula cells (R1–6) in each facet of the compound eye. Evidence is presented which indicates that this may also be true for most of the other visual responses which at present can be quantitatively studied. These responses include visually controlled landing, pattern-induced orientation of flying and walking animals, the abnormal jump reflex of the mutant Hk1 (Kaplan, 1976) and probably also phototaxis. The only function for which the small retinula cells R7 and/or R8 seem to be required so far is spectral wavelength discrimination in phototaxis at high light intensity. Our hypothesis is based on studies of the receptor deficient mutantssevenless, outer rhabdomeres absent andreceptor degeneration B as well as on results of bleaching experiments by which the retinula cells R1–6 of the eye color mutantwhite can be reversibly blocked.Visual performance of wild typeDrosophila in the optomotor response reflects receptor properties (visual acuity, spectral sensitivity and polarization sensitivity) expected for the R1–6 receptor subsystem. The notion of a ‘high sensitivity’ and a ‘high acuity’ state which was proposed earlier on the basis of experiments on various visual mutants is in agreement with the present results but their interpretation as reflecting properties of different receptor subsystems must be abandoned. Experimental data on wild type also suggest the existence of such an adaptational mechanism; this, however, remains to be demonstrated more conclusively.

A Cysteine-String Protein is Expressed in Retina and Brain of Drosophila

Antibodies can be used to identify tissue- and stage-specifically expressed genes. A monoclonal antibody MAB ab49 from a hybridoma library screened for immunohistochemical staining in the adult nervous system of Drosophila melanogaster was found to selectively bind to all neuropil regions and to synaptic boutons of motor neurons. In Western blots of homogenized brains the antibody recognizes two proteins of 32 and 34 kD. Using this antibody we have isolated seven cDNA clones that derive from two polyadenylated mRNA splice variants of a gene located at 79E1-2 on polytene chromosomes. The two mRNAs code for two inferred proteins of 249 and 223 amino acids, respectively, which are identical except for their C-terminals and a central deletion of 21 amino acids in the second protein. Both contain a contiguous string of 11 cysteine residues. In situ hybridization to frozen head sections detects expression of this gene in retina and neuronal perikarya. The 32 and 34 kD brain proteins that presumably are localized predominantly in synaptic terminals of photoreceptors and most if not all neurons may correspond to two variant cysteine-string proteins as they are of similar molecular weight and share an antigenic binding site for MAB ab49.

Genetically Expressed Cameleon in Drosophila melanogaster Is Used to Visualize Olfactory Information in Projection Neurons

Complex external stimuli such as odorants are believed to be internally represented in the brain by spatiotemporal activity patterns of extensive neuronal ensembles. These activity patterns can be recorded by optical imaging techniques. However, optical imaging with conventional fluorescence dyes usually does not allow for resolving the activity of biologically defined groups of neurons. Therefore, specifically targeting reporter molecules to neuron populations of common genetic identity is an important goal. We report the use of the genetically encoded calcium-sensitive fluorescence protein cameleon 2.1 in the Drosophila brain. We visualized odorant-evoked intracellular calcium concentration changes in selectively labeled olfactory projection neurons both postsynaptically in the antennal lobe, the primary olfactory neuropil, and presynaptically in the mushroom body calyx, a structure involved in olfactory learning and memory. As a technical achievement, we show that calcium imaging with a genetically encoded fluorescence probe is feasible in a brain in vivo. This will allow one to combine Drosophilas advanced genetic tools with the physiological analysis of brain function. Moreover, we report for the first time optical imaging recordings in synaptic regions of the Drosophila mushroom body calyx and antennal lobe. This provides an important step for the use of Drosophila as a model system in olfaction.

Presynaptic Dysfunction in Drosophila csp Mutants

Cysteine string proteins are synapse-specific proteins. In Drosophila, csp deletion mutants exhibit temperature-sensitive paralysis and early death. Here, we report that neuromuscular transmission is impaired presynaptically in these csp mutant larvae. At 22 degrees C, evoked transmitter release is depressed relative to wild type and rescued controls, and high frequency stimulation of the nerve leads to sporadic failures. At 30 degrees C, stimulus-evoked responses decline gradually before failing completely. When the temperature is returned to 22 degrees C, evoked responses recover. Spontaneous release events persist at both 22 degrees C and 30 degrees C. Since nerve conduction and postsynaptic sensitivity are unaffected, these data indicate that csp mutations disrupt depolarization-secretion coupling. This disruption explains the cellular basis of the temperature-sensitive paralysis of these organisms.

Behavioural Analysis of Spatial Vision in Insects

Two aspects of spatial visual orientation in insects constitute the central theme of this chapter: The detection of movement and the evaluation of the position of contrast elements in the visual world. In the first section the visual stimulus situation of an insect moving freely in its natural surround is described. The received “flow field” can be decomposed into three components which result from rotatory and translatory self-movement of the animal and from moving objects (e.g. birds). The next section on basic behavioural phenomena outlines the techniques of open- and closed-loop experiments and describes a few simple experiments on visual movement and position detection in flies. On the basis of these experiments an expression is derived which describes the rotatory component of a fly’s flight path through space. The equation is equivalent to the phenomenological equation of Reichardt and Poggio (1976). The third section investigates the basic principles underlying movement and position detection. Comparison of two schemes for movement detection, the gradient scheme and the correlation scheme, with measured behavioural responses demonstrates that the visual system of flies utilises a correlation-based mechanism for the detection of large- field movement. For position detection again two schemes are discussed which are based on flicker detection and movement detection. A final decision on which of the two schemes might be more relevant for fixation and tracking of moving objects by flies seems not yet possible. In the fourth section interactions between elementary movement and position detectors are deduced from behavioural experiments. In the discussion various more sophisticated aspects of visual spatial orientation behavior of insects are reviewed with emphasis on recent literature (1979 & later).

Flies lacking all synapsins are unexpectedly healthy but are impaired in complex behaviour

Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine‐regulate neurotransmitter release by phosphorylation‐dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock‐out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock‐out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified ‘model’ synapse of the larval nerve muscle preparation revealed no difference between wild‐type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild‐type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.

Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

SummaryUsing a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.