Antagonistic roles of Wnt5 and the Drl receptor in patterning the Drosophila antennal lobe

Numerous studies have shown that ingrowing olfactory axons exert powerful inductive influences on olfactory map development. From an overexpression screen, we have identified wnt5 as a potent organizer of the olfactory map in Drosophila melanogaster. Loss of wnt5 resulted in severe derangement of the glomerular pattern, whereas overexpression of wnt5 resulted in the formation of ectopic midline glomeruli. Cell type-specific cDNA rescue and mosaic experiments showed that wnt5 functions in olfactory neurons. Mutation of the derailed (drl) gene, encoding a receptor for Wnt5, resulted in derangement of the glomerular map, ectopic midline glomeruli and the accumulation of Wnt5 at the midline. We show here that drl functions in glial cells, where it acts upstream of wnt5 to modulate its function in glomerular patterning. Our findings establish wnt5 as an anterograde signal that is expressed by olfactory axons and demonstrate a previously unappreciated, yet powerful, role for glia in patterning the Drosophila olfactory map.     

Convergent projections of Drosophila olfactory neurons to specific glomeruli in the antennal lobe

Candidate Drosophila olfactory receptors (ORs) provide molecular tools to investigate how the organization of the Drosophila olfactory system determines the coding of olfactory stimuli. Neurons in the third antennal segment and maxillary palp appear to express different ORs. Individual olfactory neurons send axonal projections to glomeruli in the antennal lobe. Using transgenic flies, we provide evidence that the neurons expressing a given OR gene, which have cell bodies distributed among neurons expressing other ORs, converge in their projections to topographically fixed glomeruli in the antennal lobe. This convergence allows for the formation of an odotopic map in the antennal lobe whose organization could provide a basis for olfactory discrimination in Drosophila.     

Sensory processing in the Drosophila antennal lobe increases reliability and separability of ensemble odor representations

Here we describe several fundamental principles of olfactory processing in the Drosophila melanogaster antennal lobe (the analog of the vertebrate olfactory bulb), through the systematic analysis of input and output spike trains of seven identified glomeruli. Repeated presentations of the same odor elicit more reproducible responses in second-order projection neurons (PNs) than in their presynaptic olfactory receptor neurons (ORNs). PN responses rise and accommodate rapidly, emphasizing odor onset. Furthermore, weak ORN inputs are amplified in the PN layer but strong inputs are not. This nonlinear transformation broadens PN tuning and produces more uniform distances between odor representations in PN coding space. In addition, portions of the odor response profile of a PN are not systematically related to their direct ORN inputs, which probably indicates the presence of lateral connections between glomeruli. Finally, we show that a linear discriminator classifies odors more accurately using PN spike trains than using an equivalent number of ORN spike trains.     

Dendritic excitability and synaptic plasticity

Most synaptic inputs are made onto the dendritic tree. Recent work has shown that dendrites play an active role in transforming synaptic input into neuronal output and in defining the relationships between active synapses. In this review, we discuss how these dendritic properties influence the rules governing the induction of synaptic plasticity. We argue that the location of synapses in the dendritic tree, and the type of dendritic excitability associated with each synapse, play decisive roles in determining the plastic properties of that synapse. Furthermore, since the electrical properties of the dendritic tree are not static, but can be altered by neuromodulators and by synaptic activity itself, we discuss how learning rules may be dynamically shaped by tuning dendritic function. We conclude by describing how this reciprocal relationship between plasticity of dendritic excitability and synaptic plasticity has changed our view of information processing and memory storage in neuronal networks.     

Static magnetic field modulates rhythmic activities of a cluster of large local interneurons in Drosophila antennal lobe

With the development of superconducting magnets, the chances of exposure to intense static magnetic fields (SMFs) have increased. Therefore, safety concerns related to magnetic field exposure need to be studied, especially the effects of magnetic field exposure on the central nervous system. Only a limited number of studies prove a direct connection between magnetic fields and electrophysiological signal processing. Here we described a cluster of large local interneurons (LNs) located laterally to each antennal lobe of Drosophila melanogaster, which exhibit extensive arborizations throughout the whole antennal lobe. Dual recordings showed that these large LNs demonstrated rhythmic spontaneous activities that correlated with other LNs and projection neurons (PNs) in the olfactory circuit. The results suggest that 3.0-T SMF can interfere with the properties of the action potential, rhythmic spontaneous activities of large LNs, and correlated activity in pairs of ipsilateral large LN/LN in the olfactory circuit. This indicates that Drosophila can be an ideal intact neural circuit model and that the activities of the olfactory circuit can be used to evaluate the effects of magnetic field stimulations.     

Modulation of olfactory information processing in the antennal lobe of Manduca sexta by serotonin.

The nervous system copes with variability in the external and internal environment by using neuromodulators to adjust the efficacy of neural circuits. The role of serotonin (5HT) as a neuromodulator of olfactory information processing in the antennal lobe (AL) of Manduca sexta was examined using multichannel extracellular electrodes to record the responses of ensembles of AL neurons to olfactory stimuli. In one experiment, the effects of 5HT on the concentration-response functions for two essential plant oils across a range of stimulus intensities were examined. In a second experiment, the effect of 5HT on the ability of ensembles to discriminate odorants from different chemical classes was examined. Bath application of 5HT enhanced AL unit responses by increasing response duration and firing rate, which in turn increased the amount of spike time cross-correlation and -covariance between pairs of units. 5HT had the greatest effect on overall ensemble activation at higher odorant concentrations, resulting in an increase in the gain of the dose-response function of individual units. Additionally, response thresholds shifted to lower odorant concentrations for some units, suggesting that 5HT increased their sensitivity. Serotonin enhanced ensemble discrimination of different concentrations of individual odorants as well as discrimination of structurally dissimilar odors at the same concentration. Given the known circadian fluctuations of 5HT in the AL of this species, these findings support the hypothesis that 5HT periodically enhances sensitivity and responsiveness in the AL of Manduca to maximize efficiency when the requirement for olfactory acuity is the greatest.     

Dendritic and synaptic pathology in experimental autoimmune encephalomyelitis

Evidence has shown that excitotoxicity may contribute to the loss of central nervous system axons and oligodendrocytes in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Because dendrites and synapses are vulnerable to excitotoxicity, we examined these structures in acute and chronic models of EAE. Immunostaining for microtubule-associated protein-2 showed that extensive dendritic beading occurred in the white matter of the lumbosacral spinal cord (LSSC) during acute EAE episodes and EAE relapses. Retrograde labeling confirmed that most motoneuron dendrites were beaded in the white matter of the LSSC in acute EAE. In contrast, only mild swelling was observed in the gray matter of the LSSC. Dendritic beading showed marked recovery during EAE remission and after EAE recovery. In addition, synaptophysin, synapsin I, and PSD-95 immunoreactivities were significantly reduced in both the gray and white matter of the LSSC during acute EAE episodes and EAE relapses, but showed partial recovery during EAE remission and after EAE recovery. Pathologically, both dendritic beading and the reduction in synaptic protein immunoreactivity were well correlated with inflammatory cell infiltration in the LSSC at different EAE stages. We propose that dendritic and synaptic damage in the spinal cord may contribute to the neurological deficits in EAE.     

Central processing of pulsed pheromone signals by antennal lobe neurons in the male moth Agrotis segetum

Male moths use female-produced pheromones as orientation cues during the mate-finding process. In addition to the needs of evaluating the quality and quantity of the pheromone signal, the male moth also needs to resolve the filamentous structure of the pheromone plume to proceed toward the releasing point successfully. To understand how a discontinuous olfactory signal is processed at the central level, we used intracellular recording methods to characterize the response patterns of antennal lobe (AL) neurons to pulsatile stimulation with the full female-produced pheromone blend and its single components in male turnip moths, Agrotis segetum. Air puffs delivered at frequencies of 1, 3, 5, 7, or 10 Hz were used to carry the stimulus. Two types of AL neurons were characterized according to their capabilities to resolve stimulus pulses. The most common type could resolve at least 1-Hz pulses, thus termed fast neurons; another type could not resolve any pulses, thus termed slow neurons. When fast neurons were excited by stimuli, they always displayed biphasic response patterns, a depolarization phase followed by a hyperpolarization phase. This pattern could be evoked by stimulation with both the single pheromone components and the blend. The pulse-resolving capability of the fast neurons correlated significantly with the size of the hyperpolarization phase. When the amplitude was higher and the fall time of the hyperpolarization faster, the neuron could follow more pulses per second. Moreover, interactions between different pheromone components eliciting different response patterns did not improve the pulse-resolving capability of fast neurons.     

Cytoarchitecture and the patterning of fushi tarazu expression in the Drosophila blastoderm

In the Drosophila embryo at the blastoderm stage, the segmentation gene fushi tarazu (ftz) is expressed in a seven-banded pattern. The generation of this pattern, like many other segmentation gene expression patterns, coincides with the formation of cell membranes around the blastoderm nuclei. To test the role of cellularization in resolving the banded ftz pattern, we used cytoskeletal inhibitors (colcemid and cytochalasin B) to block cellularization. We found that banded ftz RNA and protein patterns can form without cellular structure. We also tested the importance of rapid degradation of the ftz RNA, using cycloheximide to block degradation. RNA degradation is essential to maintain the banded ftz pattern in a syncytium, but is not required to maintain the pattern in a cellularized embryo. A latticework of cytoskeletal microtubules that forms during cellularization appears to be a key component in localizing the ftz mRNA. We conclude that RNA degradation and cellular structure normally work together to localize ftz RNA to its sites of synthesis.     

Distributed representation of social odors indicates parallel processing in the antennal lobe of ants

In colonies of eusocial Hymenoptera cooperation is organized through social odors, and particularly ants rely on a sophisticated odor communication system. Neuronal information about odors is represented in spatial activity patterns in the primary olfactory neuropile of the insect brain, the antennal lobe (AL), which is analog to the vertebrate olfactory bulb. The olfactory system is characterized by neuroanatomical compartmentalization, yet the functional significance of this organization is unclear. Using two-photon calcium imaging, we investigated the neuronal representation of multicomponent colony odors, which the ants assess to discriminate friends (nestmates) from foes (nonnestmates). In the carpenter ant Camponotus floridanus, colony odors elicited spatial activity patterns distributed across different AL compartments. Activity patterns in response to nestmate and nonnestmate colony odors were overlapping. This was expected since both consist of the same components at differing ratios. Colony odors change over time and the nervous system has to constantly adjust for this (template reformation). Measured activity patterns were variable, and variability was higher in response to repeated nestmate than to repeated nonnestmate colony odor stimulation. Variable activity patterns may indicate neuronal plasticity within the olfactory system, which is necessary for template reformation. Our results indicate that information about colony odors is processed in parallel in different neuroanatomical compartments, using the computational power of the whole AL network. Parallel processing might be advantageous, allowing reliable discrimination of highly complex social odors.     

A glimpse into dorso-ventral patterning of the Drosophila eye

During organogenesis in all multi-cellular organisms, axial patterning is required to transform a single layer organ primordium into a three-dimensional organ. The Drosophila eye model serves as an excellent model to study axial patterning. Dorso-ventral (DV) axis determination is the first lineage restriction event during axial patterning of the Drosophila eye. The early Drosophila eye primordium has a default ventral fate, and the dorsal eye fate is established by onset of dorsal selector gene pannier (pnr) expression in a group of cells on the dorsal eye margin. The boundary between dorsal and ventral compartments called the equator is the site for Notch (N) activation, which triggers cell proliferation and differentiation. This review will focus on (1) chronology of events during DV axis determination; (2) how early division of eye into dorsal and ventral compartments contributes towards the growth and patterning of the fly retina, and (3) functions of DV patterning genes.     

Syndapin I is the phosphorylation-regulated dynamin I partner in synaptic vesicle endocytosis

Dynamin I is dephosphorylated at Ser-774 and Ser-778 during synaptic vesicle endocytosis (SVE) in nerve terminals. Phosphorylation was proposed to regulate the assembly of an endocytic protein complex with amphiphysin or endophilin. Instead, we found it recruits syndapin I for SVE and does not control amphiphysin or endophilin binding in rat synaptosomes. After depolarization, syndapin showed a calcineurin-mediated interaction with dynamin. A peptide mimicking the phosphorylation sites disrupted the dynamin-syndapin complex, not the dynamin-endophilin complex, arrested SVE and produced glutamate release fatigue after repetitive stimulation. Pseudophosphorylation of Ser-774 or Ser-778 inhibited syndapin binding without affecting amphiphysin recruitment. Site mutagenesis to alanine arrested SVE in cultured neurons. The effects of the sites were additive for syndapin I binding and SVE. Thus syndapin I is a central component of the endocytic protein complex for SVE via stimulus-dependent recruitment to dynamin I and has a key role in synaptic transmission.     

Altered synaptic transmission in Drosophila hyperkinetic mutants

Synaptic transmission in Drosophila can be altered by mutations in specific genes. For example, mutations in the Shaker (Sh) gene, which encodes the rapidly inactivating A-type potassium channel, cause repetitive nerve firing and prolonged transmitter release at the neuromuscular junction. Here we show that mutations in the Hyperkinetic (Hk) gene also affect the properties of synaptic transmission at the neuromuscular junction. In particular, we find that whereas single or low frequency nerve stimulation evokes a wild type postsynaptic response, at higher frequencies of nerve stimulation, each stimulus results in repetitive nerve firing and increased postsynaptic response, which is similar to that observed in Sh mutants. Various experiments suggest that this increased postsynaptic response results from prolonged depolarization of the nerve terminal, leading to increased transmitter release at the neuromuscular junction. The similarity in phenotypes between Sh and Hk mutants, along with the observation that Sh is epistatic to Hk in its effects on synaptic transmission, suggest that Hk acts on synaptic transmission by an effect on A-type potassium channels.     

Neuromuscular synaptic patterning requires the function of skeletal muscle dihydropyridine receptors

Developing skeletal myofibers in vertebrates are intrinsically 'pre-patterned' for motor nerve innervation. However, the intrinsic factors that regulate muscle pre-patterning remain unknown. We found that a functional skeletal muscle dihydropyridine receptor (DHPR, the L-type Ca(2+) channel in muscle) was required for muscle pre-patterning during the development of the neuromuscular junction (NMJ). Targeted deletion of the β1 subunit of DHPR (Cacnb1) in mice led to muscle pre-patterning defects, aberrant innervation and precocious maturation of the NMJ. Reintroducing Cacnb1 into Cacnb1(-/-) muscles reversed the pre-patterning defects and restored normal development of the NMJ. The mechanism by which DHPRs govern muscle pre-patterning is independent of their role in excitation-contraction coupling, but requires Ca(2+) influx through the L-type Ca(2+) channel. Our findings indicate that the skeletal muscle DHPR retrogradely regulates the patterning and formation of the NMJ.     

Delayed synaptic transmission in Drosophila cacophonynull embryos

Ca(2+) influx through the Drosophila N-type Ca(2+) channel, encoded by cacophony (cac), triggers fast synaptic transmission. We now ask whether the cac Ca(2+) channel is the Ca(2+) channel solely dedicated for fast synaptic transmission. Because the cac(null) mutation is lethal, we used cac(null) embryos to address this question. At the neuromuscular junction in HL3 solution, no fast synchronous synaptic transmission was detected on nerve stimulation. When the wild-type cac gene was introduced in the cac(null) background, fast synaptic transmission recovered. However, even in cac(null) embryos, nerve stimulation infrequently induced delayed synaptic events in the minority of cells in 1.5 mM [Ca(2+)](e) and in the majority of cells in 5 mM [Ca(2+)](e). The number of delayed quantal events per stimulus was greater in 5 mM [Ca(2+)](e) than in 1.5 mM. Thus the delayed release is [Ca(2+)](e) dependent. Plectreurys toxin II (PLTXII) (10 nM; a spider toxin analog) depressed the frequency of delayed events, suggesting that voltage-gated Ca(2+) channels, other than cac Ca(2+) channels, are contributing to them. However, delayed events were not affected by 50 microM La(3+). The frequency of miniature synaptic currents in cac(null) embryos was approximately 1/2 of control, whereas in high K(+) solutions, it was approximately 1/135. The hypertonicity response was approximately 1/10 of control. These findings indicate that the number of release-ready vesicles is smaller in cac(null) embryos. Taken together, the cac Ca(2+) channel is indispensable for fast synaptic transmission in normal conditions, and another type of Ca(2+) channel, the non-cac, PLTXII-sensitive Ca(2+) channel, is contributing to delayed release in cac(null) embryos.     

A Drosophila kinesin required for synaptic bouton formation and synaptic vesicle transport

The morphological transition of growth cones to synaptic boutons characterizes synaptogenesis. Here we have isolated mutations in immaculate connections (imac; CG8566), a previously uncharacterized Drosophila gene encoding a member of the Kinesin-3 family. Whereas earlier studies in Drosophila implicated Kinesin-1 in transporting synaptic vesicle precursors, we find that Imac is essential for this transport. An unexpected feature of imac mutants is the failure of synaptic boutons to form. Motor neurons lacking imac properly target to muscles but remain within target fields as thin processes, a structure that is distinct from either growth cones or mature terminals. Few active zones form at these endings. We show that the arrest of synaptogenesis is not a secondary consequence of the absence of transmission. Our data thus indicate that Imac transports components required for synaptic maturation and provide insight into presynaptic maturation as a process that can be differentiated from axon outgrowth and targeting.     

Revisiting synaptic vesicle pool localization in the Drosophila neuromuscular junction

The synaptic vesicles are organized in distinct populations or 'pools': the readily releasable pool (the first vesicles released upon stimulation), the recycling pool (which maintains release under moderate stimulation) and the reserve pool (which is called into action only upon strong, often unphysiological stimulation). A major question in the field is whether the pools consist of biochemically different vesicles or whether the pool tag is a spatial one (with the recycling vesicles found next to the release sites, and the reserve ones farther away). A strong and stable spatial segregation has been proposed in the last decade in the Drosophila larval neuromuscular junction – albeit based solely on light microscopy experiments. We have tested here this hypothesis using electron microscopy (EM) photoconversion. We found the recycling and reserve pools to be thoroughly intermixed at the EM level, indicating that spatial location is irrelevant for the functional properties of the vesicle.     

Biphasic modulation of synaptic transmission by hypertonicity at the embryonic Drosophila neuromuscular junction

Puff-application of hypertonic saline (sucrose added to external saline) causes a transient increase in the frequency of spontaneous miniature synaptic currents (mSCs) at the neuromuscular junctions of Drosophila embryos. The frequency gradually returns to pre-application levels. External Ca²⁺ is not needed for this response, but it may modify it. At 50 m m added sucrose, for example, enhanced spontaneous release was observed only in the presence of external Ca²⁺, suggesting that Ca²⁺ augments the response. In a high-K⁺ solution, in which the basal mSC frequency was elevated, higher sucrose concentrations produced an increase in mSC frequency that was followed (during and after the hypertonic exposure) by depression, with the magnitude of both effects increasing with hypertonicity between 100 and 500 m m. Evoked release by nerve stimulation showed only depression in response to hypertonicity. We do not believe that the depression of spontaneous or evoked release can be explained by the depletion of releasable quanta, however, since the frequency of quantal release did not reach levels compatible with this explanation and the enhancement and depression could be obtained independent of one another. In a mutant lacking neuronal synaptobrevin, only the depression of mSC frequency was induced by hypertonicity. Conversely, only the enhancing effect was observed in wild-type embryos when the mSC frequency was elevated with forskolin in Ca²⁺-free saline. In cultured embryonic Drosophila neurons, Ca²⁺ signals that were induced by high K⁺ and detected by Fura-2, were reduced by hypertonicity, suggesting that the depressing response is due to a direct effect of hypertonicity on Ca²⁺ influx.     

Genetic and molecular analysis of synaptic vesicle recycling in Drosophila

Following exocytosis, one of the major presynaptic events is replenishing synaptic vesicles (SVs) to ensure the possibility of continuous synaptic transmission. The nerve terminal is thought to recycle SVs through clathrin-mediated endocytosis and by a clathrin-independent pathway called "kiss and run". This review highlights the use of the genetic model organism, the fruit fly (Drosophila melanogaster), in dissecting the molecular mechanisms of clathrin-mediated endocytosis in recycling SVs at neuromuscular junctions (NMJs). Analyses of endocytotic mutants in Drosophila indicate that clathrin-mediated endocytosis may be essential for SV recycling, including a putative fast recycling mechanism uncovered recently. Further, a rather complex picture begins to emerge suggesting that clathrin-mediated endocytosis involves several sequential steps mediated by a large number of proteins. Finally, these studies also reveal that SV proteins may be selectively retrieved into nascent SVs by clathrin accessory proteins and defects in protein retrieval have significant impacts on synaptic transmission. Following the completion of the Drosophila Genome Project and the development of gene targeting and RNAi approaches, genetic studies in Drosophila have become increasingly efficient. Hence, Drosophila is expected to continue to serve as an important model organism for studies of SV recycling.