Developmentally programmed remodeling of the Drosophila olfactory circuit

Neural circuits are often remodeled after initial connections are established. The mechanisms by which remodeling occurs, in particular whether and how synaptically connected neurons coordinate their reorganization, are poorly understood. In Drosophila, olfactory projection neurons (PNs) receive input by synapsing with olfactory receptor neurons in the antennal lobe and relay information to the mushroom body (MB) calyx and lateral horn. Here we show that embryonic-born PNs participate in both the larval and adult olfactory circuits. In the larva, these neurons generally innervate a single glomerulus in the antennal lobe and one or two glomerulus-like substructures in the MB calyx. They persist in the adult olfactory circuit and are prespecified by birth order to innervate a subset of glomeruli distinct from larval-born PNs. Developmental studies indicate that these neurons undergo stereotyped pruning of their dendrites and axon terminal branches locally during early metamorphosis. Electron microscopy analysis reveals that these PNs synapse with MB gamma neurons in the larval calyx and that these synaptic profiles are engulfed by glia during early metamorphosis. As with MB gamma neurons, PN pruning requires cell-autonomous reception of the nuclear hormone ecdysone. Thus, these synaptic partners are independently programmed to prune their dendrites and axons.     

The Drosophila larval visual system: high-resolution analysis of a simple visual neuropil

The task of the visual system is to translate light into neuronal encoded information. This translation of photons into neuronal signals is achieved by photoreceptor neurons (PRs), specialized sensory neurons, located in the eye. Upon perception of light the PRs will send a signal to target neurons, which represent a first station of visual processing. Increasing complexity of visual processing stems from the number of distinct PR subtypes and their various types of target neurons that are contacted. The visual system of the fruit fly larva represents a simple visual system (larval optic neuropil, LON) that consists of 12 PRs falling into two classes: blue-senstive PRs expressing Rhodopsin 5 (Rh5) and green-sensitive PRs expressing Rhodopsin 6 (Rh6). These afferents contact a small number of target neurons, including optic lobe pioneers (OLPs) and lateral clock neurons (LNs). We combine the use of genetic markers to label both PR subtypes and the distinct, identifiable sets of target neurons with a serial EM reconstruction to generate a high-resolution map of the larval optic neuropil. We find that the larval optic neuropil shows a clear bipartite organization consisting of one domain innervated by PRs and one devoid of PR axons. The topology of PR projections, in particular the relationship between Rh5 and Rh6 afferents, is maintained from the nerve entering the brain to the axon terminals. The target neurons can be subdivided according to neurotransmitter or neuropeptide they use as well as the location within the brain. We further track the larval optic neuropil through development from first larval instar to its location in the adult brain as the accessory medulla.     

Postembryonic development of serotonin-immunoreactive neurons in the central nervous system of the blowfly,Calliphora erythrocephala

Summary The postembryonic development of serotonin-immunoreactive (5-HTi) neurons was studied in the optic lobe of the blowfly. In the adult fly there are 24 5-HTi neurons invading each optic lobe. The perikarya of two of these neurons are situated in the dorso-caudal part of the protocerebrum (LBO-5HT neurons; large bilateral optic lobe 5-HTi neurons). The cell bodies of the remaining 22 neurons are located anteriorly at the medial base of the medulla (2 innervating the lobula, LO-5HT neurons; and 20 neurons innervating the medulla, ME-5HT neurons). The two central neurons (LBO-5HT neurons) are derived from metamorphosing larval neurons, while the ME- and LO-5HT neurons are imaginai optic lobe neurons differentiating during pupal development.The 5-HTi neurons of the optic lobe seem to have different ancestors. The LBO-5HT neurons are probably derived from segmental protocerebral neuroblasts, whereas the ME-and LO-5HT neurons are most likely derived from the inner optic anlage. The first 5-HTi fibers to reach the imaginal optic lobes are seen in the late third instar larva and are derived from the LBO-5HT neurons. The first ME- and LO-5HT neurons become immunoreactive at 24 h (10%) pupal development. At about 96 h (40%) of pupal development all the 5-HTi neurons of the optic lobes have differentiated and attained their basic adult morphology. The further development mainly entails increase in volume of arborizations and number of finer processes. The differentiation and outgrowth of 5-HTi processes follows that of, e.g., columnar neurons in the optic lobe neuropils. Hence, 5-HTi processes invade neuropil relatively late in the differentiation of the optic lobe.     

ARIA is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction

ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses.     

Fate mapping of Drosophila embryonic mitotic domain 20 reveals that the larval visual system is derived from a subdomain of a few cells.

In an attempt to study the fates of cells in the dorsal head region of Drosophila embryos at gastrulation, we used the photoactivated gene expression system to mark small numbers of cells in selected mitotic domains. We found that mitotic domain 20, which is a cluster of approximately 30 cells on the dorsal posterior surface, gives rise to various ectodermal cell types in the head, including dorsal pouch epithelium, the optic lobe, and head sensory organs, including Bolwig's organ, the larval photoreceptor organ. We found that the optic lobe and larval photoreceptors share the same origin of a few adjacent cells near the center of mitotic domain 20, suggesting that within the mitotic domain, there is a subdomain from which the larval visual system is specified. In addition to the components of the larval visual system, this central region of mitotic domain 20 also generates a part of the eye-antennal disc placode; cells that gives rise to the adult visual system. We also observed that a significant amount of cell death occurred within this domain and used cell ablation experiments to determine the ability of the embryo to compensate for cell loss.     

A genetic analysis of visual system development in Drosophilia melanogaster.

The eyes and optic lobes of adult Drosophila melanogaster comprise a highly organized system of interconnected neurons. The eye and optic lobe primordia are physically separate during the embryonic and larval stages of development, and these tissues do not come into contact until the third larval instar, as a consequence of axons growing from the receptor cells of the developing eyes to the primordial optic lobes. After this contact, the axons of the eyes arrange themselves into their complex and orderly adult pattern. Simultaneously, the optic lobe cells begin elaborating axons which organize into their precise adult array. One question posed by this system is: Does cellular pattern formation in either the eyes or optic lobes depend on eye-brain interactions, or do the two tissues organize autonomously? To answer this question, mutations were found which cause abnormal ommatidial array in the eyes and which also perturb the normal adult axon array in the optic lobes. By means of X ray-induced somatic recombination and by genetically controlled mitotic chromosome loss (gynandromorph formation), flies mosaic for genotypically mutant and normal tissue were constructed. Analysis of the neuronal array in mosaic flies in which eye and optic lobe tissue differed genotypically showed that the axon array phenotype of the optic lobe depends on the genotype of the eye tissue innervating that lobe, while the eye phenotype does not depend on optic lobe genotype. Thus, the axonal organization of the D. melanogaster optic lobe has been shown to depend on the transmission of information from the eyes to the optic lobes.     

The fine structure of the lateral ocellus of the dobsonfly larva

The lateral ocelli of the dobsonfly (Protohermes grandis, Neuroptera) larva have been examined with light and electron microscopy. The larva has six ocelli on both sides of the head, each containing a single corneal lens. A conical crystalline body, of some 10–20 cells is situated immediately posterior to the lens. From 100 to 300 elongated retinular cells are arranged perpendicular to the crystalline body except at the innermost surface of the lens, where they are absent. The distal process of each retinular cell is enclosed by a tube-like rhabdom formed by the close association of microvilli from the same and adjacent distal processes. The distal process contains many mitochondria, multivesicular bodies, microtubles and pigment granules. In the dark-adapted ocellus the pigment granules are concentrated near the nucleus which lies under the rhabdomic layer. The granules diffuse toward the rhabdomic microvilli during light adaptation. Each retinular cell has a single axon, which extends from the ocellus as an ocellar nerve fiber into the optic lobe, where it frequently synapses upon second order neurons. In addition to these afferent synapses, there are two other synaptic combinations: (1) a feedback synapse from a second order neuron to a retinular axon, and (2) a synapse between second order neurons. These results suggest that photic signals reach the more proximal part of the brain via second order neurons after some degree of integration in the optic lobe.     

Connectivity of identified central synapses in the cricket is normal following regeneration and blockade of presynaptic activity

Cercal sensory neurons in the cricket innervate interneurons in the central nervous system (CNS) and provide a model system for studying the formation of central synapses. When axons of the sensory neurons were transected during larval development, the cell bodies and the soma-bearing portion of axons, which are located within the cercus, survived but lost their excitability for 9-10 days. During this period, the sensory neurons grew new axons and reinnervated the terminal abdominal ganglion. Physiological recordings showed that sensory neurons of known identity reestablished monosynaptic contacts with their normal postsynaptic interneuron. Moreover, each synapse exhibited a characteristic strength indistinguishable from the intact synapse in an unoperated cricket. Since this selective connectivity was apparent immediately after the excitability of the axotomized sensory neurons was restored, action potentials in the sensory neurons appear to be unnecessary for normal synaptic regeneration to occur. Consistent with this, the reinnervation process was unaffected even when action potentials in the sensory neurons were blocked by tetrodotoxin (TTX) immediately following axotomy until just before testing. During the normal course of development, the characteristic strength of individual synapses changes systematically, resulting in the developmental rearrangement of these synapses (Chiba et al., 1988). This synaptic rearrangement was also unaffected when action potentials in the sensory neurons were blocked by TTX for the last 30% of larval development. Therefore, in the cricket cercal sensory system, both regeneration of the central synapses following axotomy of the presynaptic sensory neurons and the normal rearrangement of connectivity during larval development appear not to require axonal action potentials.     

Expression of two different isoforms of fasciclin II during postembryonic central nervous system remodeling in <Emphasis Type="Italic">Manduca sexta</Emphasis>

Insect metamorphosis serves as a useful model to investigate postembryonic development in the central nervous system, because the transformation between larval and adult life is accompanied by a remodeling of neural circuitry. Most changes are controlled by ecdysteroids, but activity-dependent mechanisms and cell surface signals also play a role. This immunocytochemical study investigates the expression patterns of two isoforms of the neural cell adhesion molecule, fasciclin II (FasII), during postembryonic ventral nerve cord remodeling in the moth, Manduca sexta. Both the expression of the glycosyl-phosphatidylinositol (GPI)-linked isoform and the transmembrane isoform of Manduca FasII (TM-MFasII) are regulated in a stereotyped spatio-temporal pattern. TM-MFasII is expressed in a stage-specific manner in a subset of neurons. Subsets of central axons express high levels during outgrowth supporting a functional role for TM-FasII during pathfinding. Dendritic localization is not found at any stage of metamorphosis, suggesting no homophilic interactions of TM-MFasII during central synapse development. GPI-MFasII is expressed in a stage-specific manner, most likely only in glial cells. The larval and adult stages show almost no GPI-MFasII expression, whereas during pupal life, positive GPI-MFasII labeling is present around synaptotagmin-negative tracts or commissures, so that either homophilic stabilization of glial boundaries or heterophilic neuron-glial interactions possibly stabilize the axons within their tracts. GPI-MFasII expression is not co-localized with synaptotagmin-positive central terminals, rendering a role for synapse development unlikely. Neither isoform is expressed in all neurons of a specific class at any developmental stage, indicating that MFasII functions are restricted to specific subsets of neurons or to individual neurons. The support of the German Science Foundation (Du 331/4–1) and of Arizona State University to C.D. is greatly appreciated.     

Genetic dissection of trophic interactions in the larval optic neuropil of Drosophila melanogaster

The larval visual system of Drosophila melanogaster consists of two bilateral clusters of 12 photoreceptors, which express Rhodopsin 5 and 6 (Rh5 and Rh6) in a non-overlapping manner. These neurons send their axons in a fascicle, the larval optic nerve (LON), which terminates in the larval optic neuropil. The LON is required for the development of a serotonergic arborization originating in the central brain and for the development of the dendritic tree of the circadian pacemakers, the small ventral lateral neurons (LNv) [Malpel, S., Klarsfeld, A., Rouyer, F., 2002. Larval optic nerve and adult extra-retinal photoreceptors sequentially associate with clock neurons during Drosophila brain development. Development 129, 1443-1453; Mukhopadhyay, M., Campos, A.R., 1995. The larval optic nerve is required for the development of an identified serotonergic arborization in Drosophila melanogaster. Dev. Biol., 169, 629-643]. Here, we show that both Rh5- and Rh6-expressing fibers overlap equally with the 5-HT arborization and that it, in turn, also contacts the dendritic tree of the LNv. The experiments described here aimed at determining whether Rh5- or Rh6-expressing fibers, as well as the LNv, influence the development of this serotonergic arborization. We conclude that Rh6-expressing fibers play a unique role in providing a signal required for the outgrowth and branching of the serotonergic arborization. Moreover, the innervation of the larval optic neuropil by the 5-HT arborization depends on intact Rac function. A possible role for these serotonergic processes in modulating the larval circadian rhythmicity and photoreceptor function is discussed.     

Development of neural lineages derived from the sine oculis positive eye field of Drosophila

The Anlage of the Drosophila visual system, called eye field, comprises a domain in the dorso-medial neurectoderm of the embryonic head and is defined by the expression of the early eye gene sine oculis (so). Beside the eye and optic lobe, the eye field gives rise to several neuroblasts that contribute their lineages to the central brain. Since so expression is only very short lived, the later development of these neuroblasts has so far been elusive. Using the P-element replacement technique [Genetics, 151 (1999) 1093] we generated a so-Gal4 line driving the reporter gene LacZ that perdures in the eye field derived cells throughout embryogenesis and into the larval period. This allowed us to reconstruct the morphogenetic movements of the eye field derived lineages, as well as the projection pattern of their neurons. The eye field produces a dorsal (Pc1/2) and a ventral (Pp3) group of three to four neuroblasts each. In addition, the target neurons of the larval eye, the optic lobe pioneers (OLPs) are derived from the eye field. The embryonically born (primary) neurons of the Pp3 lineages spread out at the inner surface of the optic lobe. Together with the OLPs, their axons project to the dorsal neuropile of the protocerebrum. Pp3 neuroblasts reassume expression of so-Gal4 in the larval period and produce secondary neurons whose axonal projection coincides with the pattern formed by the primary Pp3 neurons. Several other small clusters of neurons that originate from outside the eye field, but have axonal connections to the dorsal protocerebrum, also express so and are labeled by so-Gal4 driven LacZ. We discuss the dynamic pattern of the so-positive lineages as a tool to reconstruct the morphogenesis of the larval brain.     

Mechanisms underlying olfactory neuronal connectivity in Drosophila-the atonal lineage organizes the periphery while sensory neurons and glia pattern the olfactory lobe

Patterning of the antennal lobe of adult Drosophila occurs through a complex interaction between sensory neurons, glia, and central neurons of larval and adult origin. Neurons from the olfactory sense organs are organized into distinct fascicles lined by glial cells. The glia originate from one of the three types of sensory lineages-specified by the proneural gene atonal. Gain-of-function as well as loss-of-function analysis validates a role for cells of the Atonal lineage in the ordered fasciculation of sensory neurons. Upon entry of the antennal nerve to central regions, sensory neurons at first remain closely associated with central glia which lie around the periphery of the lobe anlage. Coincident with the arrival of sensory neurons into the brain, glial precursors undergo mitosis and neural precursors expressing Dachshund appear around the lobe. Sensory neurons and glial cells project into the lobe at around the same time and are likely to coordinate the correct localization of different glomeruli. The influence of sensory neurons on the development of the olfactory lobe could serve to match and lock peripheral and central properties important for the generation of olfactory behavior.     

The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages

The locus elav (ella-vee) of Drosophila melanogaster, which is necessary for the proper development of the embryonic and adult nervous systems, has been characterized both genetically and molecularly. This locus has been shown to be transcribed exclusively within, and ubiquitously throughout, the developing nervous system during Hours 6 to 12 of embryogenesis. We present in situ RNA localization data which demonstrate that elav is expressed in the central nervous system as well as the peripheral nervous system of embryos, larvae, pupae, and adults. We also demonstrate that elav is not transcribed in embryonic or larval neuroblasts (the neuronal progenitor cells), or in at least one type of glial cell. These data provide evidence that the requirement for elav function is not limited to the 6- to 12-hr embryonic nervous system and the adult eye and developing optic lobe, but that its function is required for the development and continued maintenance of all neurons of the organism.     

Visualization of neuropeptide expression, transport, and exocytosis in Drosophila melanogaster.

Neuropeptides affect an extremely diverse set of physiological processes. Neuropeptides are often coreleased with neurotransmitters but, unlike neurotransmitters, the neuropeptide target cells may be distant from the site(s) of secretion. Thus, it is often difficult to measure the amount of neuropeptide release in vivo by electrophysiological methods. Here we establish an in vivo system for studying the developmental expression, processing, transport, and release of neuropeptides. A GFP-tagged atrial natriuretic factor fusion (preproANF-EMD) was expressed in the Drosophila nervous system with the panneural promoter, elav. During embryonic development, proANF-EMD was first seen to accumulate in synaptic regions of the CNS in stage 17 embryos. By the third instar larval stage, highly fluorescent neurons were evident throughout the CNS. In the adult, fluorescence was pronounced in the mushroom bodies, antennal lobe, and the central complex. At the larval neuromuscular junction, proANF-EMD was concentrated in nerve terminals. We compared the release of proANF-EMD from synaptic boutons of NMJ 6/7, which contain almost exclusively glutamate-containing clear vesicles, to those of NMJ 12, which include the peptidergic type III boutons. Upon depolarization, approximately 60% of the tagged neuropeptide was released from NMJs of both muscles in 15 min, as assayed by decreased fluorescence. Although the elav promoter was equally active in the motor neurons that innervate both NMJs 6/7 and 12, NMJ 12 contained 46-fold more neuropeptide and released much more proANF-EMD during stimulation than did NMJ 6/7. Our results suggest that peptidergic neurons have an enhanced ability to accumulate and/or release neuropeptides as compared to neurons that primarily release classical neurotransmitters.     

Postembryonic development of Arg-Phe-amide-like and cholecystokinin-like immunoreactive neurons in the blowfly optic lobe

Summary The adult optic lobes of the blowfly Calliphora erythrocephala were found to be innervated by more than 2000 neurons immunoreactive to antisera raised against the neuropeptides FMRFamide, its fragment RFamide, and gastrin/cholecystokinin (CCK). All of the CCK-like immunoreactive (CCK-IR) neurons also reacted with antisera to RFamide, FMRFamide and pancreatic polypeptide. A few RFamide/FMRFamide-like immunoreactive (RF-IR) neurons did not react with CCK antisera; they reacted instead with antisera to Leu-enkephalin and Met-enkephalin-Arg⁶-Phe⁷. The RF-IR neurons are, thus, heterogeneous with respect to their contents of immunoreactive peptides. Two of the RF-IR neuron types innervating the adult optic lobes could be traced in their entirety only after following their postembryonic development, because of the complexity of the trajectories of the immunoreactive neuronal process in the adult insect. The majority of the cell bodies of the RF-IR and CCK-IR neurons lie within the optic lobes and are derived from imaginal neuroblasts of the inner and outer optic anlagen. Six of the peptidergic neurons are, however, metamorphosing larval neurons with their cell bodies in the central part of the protocerebrum. The full extent of immunoreactivitiy is not attained in some of the neurons until the late pupal or early adult stage. The larval optic center was also found to be innervated by neurons immuno-reactive with both RFamide and CCK antisera. The cell bodies of these RF-IR/CCK-IR neurons are located near the developing lamina (one on each side). In the 24 h pupa, the cell bodies of these neurons are still immunoreactive, but thereafter they cannot be immunolabeled apparently due to cell death or a change in transmitter phenotype.