Analyzing Synaptic Modulation of <em>Drosophila melanogaster</em> Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

German Center for Neurodegenerative Diseases (DZNE)

Asymmetric cell divisions generate cellular diversity. In Drosophila, embryonic neuroblasts target cell fate determinants basally, rotate their spindles by 90 degrees to align with the apical-basal axis, and divide asymmetrically in a stem cell-like fashion. In this process, apically localized Bazooka recruits Inscuteable and other proteins to form an apical complex, which then specifies spindle orientation and basal localization of the cell fate determinants and their adapter proteins such as Miranda. Here we report that Miranda localization requires the unconventional myosin VI Jaguar (Jar). In jar null mutant embryos, Miranda is delocalized and the spindle is misoriented, but the Inscuteable crescent remains apical. Miranda directly binds to Jar, raising the possibility that Miranda and its associated proteins are translocated basally by this actin-based motor. Our studies demonstrate that a class VI myosin is necessary for basal protein targeting and spindle orientation in neuroblasts.

The Drosophila myosin VI Jaguar is required for basal protein targeting and correct spindle orientation in mitotic neuroblasts.

A series of unconventional microtubule organizing centers play a fundamental role during egg chamber development in Drosophila. To gain a better understanding of their molecular nature, we have studied the centrosomal component gamma-tubulin during Drosophila oogenesis. We find that although single mutations in either of the two gamma-tubulin genes identified in Drosophila do not affect oogenesis progression the simultaneous depletion of both protein products has severe consequences. The combination of loss-of-function mutant alleles for the two gamma-tubulin genes leads to mitotic defects in female germ cells, resulting in agametic ovaries. A combination of weaker mutant alleles instead allows female germ-cell development to proceed, although the resulting egg chambers display pleiotropic abnormalities, most frequently affecting the number of nurse cells and oocytes per egg chamber. Thus, gamma-tubulin is required for several processes at different stages of germ-cell development and oogenesis, including oocyte determination and differentiation. Our data provide a functional link between a component of the peri-centriolar material, such as gamma-tubulin, and microtubule organization during Drosophila oogenesis. In addition, our results show that gamma-tubulin is required for female germ-cell proliferation and that the two gamma-tubulins present in Drosophila are functionally equivalent during female germ-cell development and oogenesis.

gamma-Tubulin function during female germ-cell development and oogenesis in Drosophila.

Space-filling neurons extensively sample their receptive fields with fine dendritic branches. In this study we show that a member of the conserved Robo receptor family, Robo, and its ligand Slit regulate the dendritic differentiation of space-filling neurons. Loss of Robo or Slit function leads to faster elongating and less branched dendrites of the complex and space-filling class IV multi-dendritic dendrite-arborization (md-da) neurons in the Drosophila embryonic peripheral nervous system, but not of the simpler class I neurons. The total dendrite length of Class IV neurons is not modified in robo or slit mutant embryos. Robo mediates this process cell-autonomously. Upon Robo over-expression in md-da neurons the dendritic tree is simplified and time-lapse analysis during larval stages indicates that this is due to reduction in the number of newly formed branches. We propose that Slit, through Robo, provides an extrinsic signal to coordinate the growth rate and the branching level of space-filling neurons, thus allowing them to appropriately cover their target field.

Slit and Robo regulate dendrite branching and elongation of space-filling neurons in Drosophila.

Dendritic spines are a characteristic feature of a number of neurons in the vertebrate nervous system and have been implicated in processes that include learning and memory. In spite of this, there has been no comprehensive analysis of the presence of spines in a classical genetic system, such as Drosophila, so far. Here, we demonstrate that a subset of processes along the dendrites of visual system interneurons in the adult fly central nervous system, called LPTCs, closely resemble vertebrate spines, based on a number of criteria. First, the morphology, size, and density of these processes are very similar to those of vertebrate spines. Second, they are enriched in actin and devoid of tubulin. Third, they are sites of synaptic connections based on confocal and electron microscopy. Importantly, they represent a preferential site of localization of an acetylcholine receptor subunit, suggesting that they are sites of excitatory synaptic input. Finally, their number is modulated by the level of the small GTPase dRac1. Our results provide a basis to dissect the genetics of dendritic spine formation and maintenance and the functional role of spines.

Characterization of dendritic spines in the Drosophila central nervous system.

Insect mushroom bodies are critical for olfactory associative learning. We have carried out an extensive quantitative description of the synaptic organization of the calyx of adult Drosophila melanogaster, the main olfactory input region of the mushroom body. By using high-resolution confocal microscopy, electron microscopy-based three-dimensional reconstructions, and genetic labeling of the neuronal populations contributing to the calyx, we resolved the precise connections between large cholinergic boutons of antennal lobe projection neurons and the dendrites of Kenyon cells, the mushroom body intrinsic neurons. Throughout the calyx, these elements constitute synaptic complexes called microglomeruli. By single-cell labeling, we show that each Kenyon cell's claw-like dendritic specialization is highly enriched in filamentous actin, suggesting that this might be a site of plastic reorganization. In fact, Lim kinase (LimK) overexpression in the Kenyon cells modifies the shape of the microglomeruli. Confocal and electron microscopy indicate that each Kenyon cell claw enwraps a single bouton of a projection neuron. Each bouton is contacted by a number of such claw-like specializations as well as profiles of gamma-aminobutyric acid-positive neurons. The dendrites of distinct populations of Kenyon cells involved in different types of memory are partially segregated within the calyx and contribute to different subsets of microglomeruli. Our analysis suggests, though, that projection neuron boutons can contact more than one type of Kenyon cell. These findings represent an important basis for the functional analysis of the olfactory pathway, including the formation of associative olfactory memories.

Synaptic organization in the adult Drosophila mushroom body calyx.

How does the sensory environment shape circuit organization in higher brain centers? Here we have addressed the dependence on activity of a defined circuit within the mushroom body of adult Drosophila. This is a brain region receiving olfactory information and involved in long-term associative memory formation. The main mushroom body input region, named the calyx, undergoes volumetric changes correlated with alterations of experience. However, the underlying modifications at the cellular level remained unclear. Within the calyx, the clawed dendritic endings of mushroom body Kenyon cells form microglomeruli, distinct synaptic complexes with the presynaptic boutons of olfactory projection neurons. We developed tools for high-resolution imaging of pre- and postsynaptic compartments of defined calycal microglomeruli. Here we show that preventing firing of action potentials or synaptic transmission in a small, identified fraction of projection neurons causes alterations in the size, number, and active zone density of the microglomeruli formed by these neurons. These data provide clear evidence for activity-dependent organization of a circuit within the adult brain of the fly.

Structural long-term changes at mushroom body input synapses.

Plastic changes at the presynaptic sites of the mushroom body (MB) principal neurons called Kenyon cells (KCs) are considered to represent a neuronal substrate underlying olfactory learning and memory. It is generally believed that presynaptic and postsynaptic sites of KCs are spatially segregated. In the MB calyx, KCs receive olfactory input from projection neurons (PNs) on their dendrites. Their presynaptic sites, however, are thought to be restricted to the axonal projections within the MB lobes. Here, we show that KCs also form presynapses along their calycal dendrites, by using novel transgenic tools for visualizing presynaptic active zones and postsynaptic densities. At these presynapses, vesicle release following stimulation could be observed. They reside at a distance from the PN input into the KC dendrites, suggesting that regions of presynaptic and postsynaptic differentiation are segregated along individual KC dendrites. KC presynapses are present in γ-type KCs that support short- and long-term memory in adult flies and larvae. They can also be observed in α/β-type KCs, which are involved in memory retrieval, but not in α'/β'-type KCs, which are implicated in memory acquisition and consolidation. We hypothesize that, as in mammals, recurrent activity loops might operate for memory retrieval in the fly olfactory system. The newly identified KC-derived presynapses in the calyx are, inter alia, candidate sites for the formation of memory traces during olfactory learning.

Presynapses in Kenyon cell dendrites in the mushroom body calyx of Drosophila.

Dendrites represent the compartment of neurons primarily devoted to collecting and computating input. Far from being static structures, dendrites are highly dynamic during development and appear to be capable of plastic changes during the adult life of animals. During development, it is a combination of intrinsic programs and external signals that shapes dendrite morphology; input activity is a conserved extrinsic factor involved in this process. In adult life, dendrites respond with more modest modifications of their structure to various types of extrinsic information, including alterations of input activity. Here, the author reviews classical and recent evidence of dendrite plasticity in invertebrates and vertebrates and current progress in the understanding of the molecular mechanisms that underlie this plasticity. Importantly, some fundamental questions such as the functional role of dendrite remodeling and the causal link between structural modifications of neurons and plastic processes, including learning, are still open.

Dendritic structural plasticity.

The branched morphology of dendrites represents a functional hallmark of distinct neuronal types. Nonetheless, how diverse neuronal class-specific dendrite branches are generated is not understood. We investigated specific classes of sensory neurons of Drosophila larvae to address the fundamental mechanisms underlying the formation of distinct branch types. We addressed the function of fascin, a conserved actin-bundling protein involved in filopodium formation, in class III and class IV sensory neurons. We found that the terminal branchlets of different classes of neurons have distinctive dynamics and are formed on the basis of molecularly separable mechanisms; in particular, class III neurons require fascin for terminal branching whereas class IV neurons do not. In class III neurons, fascin controls the formation and dynamics of terminal branchlets. Previous studies have shown that transcription factor combinations define dendrite patterns; we find that fascin represents a downstream component of such programs, as it is a major effector of the transcription factor Cut in defining class III-specific dendrite morphology. Furthermore, fascin defines the morphological distinction between class III and class IV neurons. In fact, loss of fascin function leads to a partial conversion of class III neurons to class IV characteristics, while the reverse effect is obtained by fascin overexpression in class IV neurons. We propose that dedicated molecular mechanisms underlie the formation and dynamics of distinct dendrite branch types to elicit the accurate establishment of neuronal circuits.

Fascin controls neuronal class-specific dendrite arbor morphology.

A hallmark of the central nervous system is its spatial and functional organization in synaptic layers. During neuronal development, axons form transient contacts with potential post-synaptic elements and establish synapses with appropriate partners at specific layers. These processes are regulated by synaptic cell-adhesion molecules. In the Drosophila visual system, R7 and R8 photoreceptor subtypes target distinct layers and form en passant pre-synaptic terminals at stereotypic loci of the axonal shaft. A leucine-rich repeat transmembrane protein, Capricious (Caps), is known to be selectively expressed in R8 axons and their recipient layer, which led to the attractive hypothesis that Caps mediates R8 synaptic specificity by homophilic adhesion. Contradicting this assumption, our results indicate that Caps does not have a prominent role in synaptic-layer targeting and synapse formation in Drosophila photoreceptors, and that the specific recognition of the R8 target layer does not involve Caps homophilic axon-target interactions. We generated flies that express a tagged synaptic marker to evaluate the presence and localization of synapses in R7 and R8 photoreceptors. These genetic tools were used to assess how the synaptic profile is affected when axons are forced to target abnormal layers by expressing axon guidance molecules. When R7 axons were mistargeted to the R8-recipient layer, R7s either maintained an R7-like synaptic profile or acquired a similar profile to r8s depending on the overexpressed protein. When R7 axons were redirected to a more superficial medulla layer, the number of presynaptic terminals was reduced. These results indicate that cell-surface molecules are able to dictate synapse loci by changing the axon terminal identity in a partially cell-autonomous manner, but that presynapse formation at specific sites also requires complex interactions between pre- and post-synaptic elements.

Assessing the role of cell-surface molecules in central synaptogenesis in the Drosophila visual system.

Neural activity contributes to the regulation of the properties of synapses in sensory systems, allowing for adjustment to a changing environment. Little is known about how synaptic molecular components are regulated to achieve activity-dependent plasticity at central synapses. Here, we found that after prolonged exposure to natural ambient light the presynaptic active zone in Drosophila photoreceptors undergoes reversible remodeling, including loss of Bruchpilot, DLiprin-α, and DRBP, but not of DSyd-1 or Cacophony. The level of depolarization of the postsynaptic neurons is critical for the light-induced changes in active zone composition in the photoreceptors, indicating the existence of a feedback signal. In search of this signal, we have identified a crucial role of microtubule meshwork organization downstream of the divergent canonical Wnt pathway, potentially via Kinesin-3 Imac. These data reveal that active zone composition can be regulated in vivo and identify the underlying molecular machinery.

Molecular Remodeling of the Presynaptic Active Zone of Drosophila Photoreceptors via Activity-Dependent Feedback.

Dynamic rearrangements of the actin cytoskeleton are crucial for cell shape and migration. In this issue, Squarr et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201508081) show that the cadherin superfamily protein Fat2 regulates actin-rich protrusions driving collective cell migration during Drosophila melanogaster egg morphogenesis through its interaction with the WAVE regulatory complex.

Gaia Tavosanis

Dendrite Differentiation

German Center for Neurodegenerative Diseases (DZNE)

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Gaia Tavosanis

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Dendrite Differentiation

German Center for Neurodegenerative Diseases (DZNE)

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Dendrite Differentiation