Different isoforms of fasciclin II are expressed by a subset of developing olfactory receptor neurons and by olfactory-nerve glial cells during formation of glomeruli in the moth Manduca sexta.

During development of the primary olfactory projection, olfactory receptor axons must sort by odor specificity and seek particular sites in the brain in which to create odor-specific glomeruli. In the moth Manduca sexta, we showed previously that fasciclin II, a cell adhesion molecule in the immunoglobulin superfamily, is expressed by the axons of a subset of olfactory receptor neurons during development and that, in a specialized glia-rich "sorting zone," these axons segregate from nonfasciclin II-expressing axons before entering the neuropil of the glomerular layer. The segregation into fasciclin II-positive fascicles is dependent on the presence of the glial cells in the sorting zone. Here, we explore the expression patterns for different isoforms of Manduca fasciclin II in the developing olfactory system. We find that olfactory receptor axons express transmembrane fasciclin II during the period of axonal ingrowth and glomerulus development. Fascicles of TM-fasciclin II+ axons target certain glomeruli and avoid others, such as the sexually dimorphic glomeruli. These results suggest that TM-fasciclin II may play a role in the sorting and guidance of the axons. GPI-linked forms of fasciclin II are expressed weakly by glial cells associated with the receptor axons before they reach the sorting zone, but not by sorting-zone glia. GPI-fasciclin II may, therefore, be involved in axon-glia interactions related to stabilization of axons in the nerve, but probably not related to sorting.

Immunolocalization of synaptotagmin for the study of synapses in the developing antennal lobe of Manduca sexta.

In the mature olfactory systems of most organisms that possess a sense of smell, synapses between olfactory receptor neurons and central neurons occur in specialized neuropil structures called glomeruli. The development of olfactory glomeruli has been studied particularly heavily in the antennal lobe of the moth Manduca sexta. In the current study, we address the development of synapses within the antennal lobe of M. sexta by reporting on the localization of synaptotagmin, a ubiquitous synaptic vesicle protein, throughout development. A cDNA clone coding for M. sexta synaptotagmin was characterized and found to encode a protein that shares 67% amino acid identity with Drosophila synaptotagmin and 56% amino acid identity with human synaptotagmin I. Conservation was especially high in the C2 domains near the C-terminus and very low near the N-terminus. A polyclonal antiserum (MSYT) was raised against the unique N-terminus of M. sexta synaptotagmin, and a monoclonal antibody (DSYT) was raised against the highly conserved C-terminus of D. melanogaster synaptotagmin. In Western blot analyses, both antibodies labeled a 60 kD protein, which very likely corresponds to synaptotagmin. On sections, both antibodies labeled known synaptic neuropils in M. sexta and yielded similar labeling patterns in the developing antennal lobe. In addition, DSYT detected synaptotagmin-like protein in three other insect species examined. Analysis of synaptotagmin labeling at the light microscopic level during development of the antennal lobe of M. sexta confirmed and extended previous electron microscopic studies. Additional synapses in the coarse neuropil and a refinement of synaptic densities in the glomeruli during the last one-third of metamorphic development were revealed.

Localization of cGMP immunoreactivity and of soluble guanylyl cyclase in antennal sensilla of the hawkmoth Manduca sexta.

The intracellular messenger cGMP (cyclic guanosine monophosphate) has been suggested to play a role in olfactory transduction in both invertebrates and vertebrates, but its cellular location within the olfactory system has remained elusive. We used cGMP immunocytochemistry to determine which antennal cells of the hawkmoth Manduca sexta are cGMP immunoreactive in the absence of pheromone. We then tested which antennal cells increase cGMP levels in response to nitric oxide (NO) and to long pheromonal stimuli, which the male encounters close to a calling female moth. In addition, we used in situ hybridization to determine which antennal cells express NO-sensitive soluble guanylyl cyclase. In response to long pheromonal stimuli with NO donors present, cGMP concentrations change in at least a subpopulation of pheromone-sensitive olfactory receptor neurons. These changes in cGMP concentrations in pheromone-dependent olfactory receptor neurons cannot be mimicked by the addition of NO donors in the absence of pheromone. NO stimulates sensilla chaetica type I and II, but not pheromone-sensitive trichoid sensilla, to high levels of cGMP accumulation as detected by immunocytochemistry. In situ hybridizations show that sensilla chaetica, but not sensilla trichodea, express detectable levels of mRNA coding for soluble guanylyl cyclase. These results suggest that intracellular rises in cGMP concentrations play a role in information processing in a subpopulation of pheromone-sensitive sensilla in Manduca sexta antennae, mediated by an NO-sensitive mechanism, but not an NO-dependent soluble guanylyl cyclase.

The novel guanylyl cyclase MsGC-I is strongly expressed in higher-order neuropils in the brain of Manduca sexta.

Guanylyl cyclases are usually characterized as being either soluble (sGCs) or receptor (rGCs). We have recently cloned a novel guanylyl cyclase, MsGC-I, from the developing nervous system of the hawkmoth Manduca sexta that cannot be classified as either an sGC or an rGC. MsGC-I shows highest sequence identity with receptor guanylyl cyclases throughout its catalytic and dimerization domains, but does not contain the ligand-binding, transmembrane or kinase-like domains characteristic of receptor guanylyl cyclases. In addition, MsGC-I contains a C-terminal extension of 149 amino acid residues. In this paper, we report the expression of MsGC-I in the adult. Northern blots show that it is expressed preferentially in the nervous system, with high levels in the pharate adult brain and antennae. In the antennae, immunohistochemical analyses show that it is expressed in the cell bodies and dendrites, but not axons, of olfactory receptor neurons. In the brain, it is expressed in a variety of sensory neuropils including the antennal and optic lobes. It is also expressed in structures involved in higher-order processing including the mushroom bodies and central complex. This complicated expression pattern suggests that this novel guanylyl cyclase plays an important role in mediating cyclic GMP levels in the nervous system of Manduca sexta.

Neuron-glia communication via nitric oxide is essential in establishing antennal-lobe structure in Manduca sexta.

Nitric oxide synthase recently has been shown to be present in olfactory receptor cells throughout development of the adult antennal (olfactory) lobe of the brain of the moth Manduca sexta. Here, we investigate the possible involvement of nitric oxide (NO) in antennal-lobe morphogenesis. Inhibition of NO signaling with a NO synthase inhibitor or a NO scavenger early in development results in abnormal antennal lobes in which neuropil-associated glia fail to migrate. A more subtle effect is seen in the arborization of dendrites of a serotonin-immunoreactive neuron, which grow beyond their normal range. The effects of NO signaling in these types of cells do not appear to be mediated by activation of soluble guanylyl cyclase to produce cGMP, as these cells do not exhibit cGMP immunoreactivity following NO stimulation and are not affected by infusion of a soluble guanylyl cyclase inhibitor. Treatment with Novobiocin, which blocks ADP-ribosylation of proteins, results in a phenotype similar to those seen with blockade of NO signaling. Thus, axons of olfactory receptor cells appear to trigger glial cell migration and limit arborization of serotonin-immunoreactive neurons via NO signaling. The NO effect may be mediated in part by ADP-ribosylation of target cell proteins.

Expression of nitric oxide synthase and soluble guanylyl cyclase in the developing olfactory system of Manduca sexta.

The gaseous messenger nitric oxide (NO), with its ability to mediate both intercellular and intracellular communication, can play important roles in mediating cellular communication in both the development and the function of the nervous system. The authors investigated the possible role of NO signaling in the developing olfactory system (antennal lobe) of the moth Manduca sexta. NO synthase (NOS), the enzyme that generates NO, was localized by using immunocytochemistry, in situ hybridization, and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. Although NADPH-d staining appears to be a poor indicator of the presence of NOS in this system, immunocytochemistry and in situ hybridization reveal that NOS is expressed in the axons of olfactory receptor neurons throughout development and in the perineurial sheath that covers the brain early in development. NOS is present in axon terminals as they form protoglomeruli, raising the possibility that NO mediates cell-cell interactions during antennal lobe development. NO-sensitive soluble guanylyl cyclase (sGC), one of the best characterized targets of NO, was localized in the developing olfactory system by using in situ hybridization and immunocytochemistry for the Manduca sexta sGCalpha1 subunit. The ability of the developing olfactory system to respond to exogenous NO also was examined by using cyclic guanosine monophosphate immunocytochemistry. sGC is expressed in mechanosensory neurons in the developing antenna and in many antennal lobe neurons in both the medial and lateral cell body clusters. Thus, NOS and sGC are expressed in a pattern that suggests that this signaling pathway may mediate intercellular communication during development of the olfactory system in Manduca sexta.

Identification of a novel guanylyl cyclase that is related to receptor guanylyl cyclases, but lacks extracellular and transmembrane domains.

We have identified a novel guanylyl cyclase, named MsGC-I, that is expressed in the nervous system of Manduca sexta. MsGC-I shows highest sequence identity with receptor guanylyl cyclases throughout its catalytic and dimerization domains but does not contain the ligand-binding, transmembrane, or kinase-like domains characteristic of receptor guanylyl cyclases. In addition, MsGC-I contains a C-terminal extension of 149 amino acids that is not present in other receptor guanylyl cyclases. The sequence of MsGC-I contains no regions that show similarity to the regulatory domain of soluble guanylyl cyclases. Thus, MsGC-I appears to represent a member of a new class of guanylyl cyclases. We show that both a transcript and a protein of the sizes predicted from the MsGC-I cDNA are present in the nervous system of Manduca and that MsGC-I is expressed in a small population of neurons within the abdominal ganglia. When expressed in COS-7 cells, MsGC-I appears to exist as a soluble homodimer with high levels of basal guanylyl cyclase activity that is insensitive to stimulation by nitric oxide. Western blot analysis, however, shows that MsGC-I is localized to the particulate fraction of nervous system homogenates, suggesting that it may be membrane-associated in vivo.

Identification and characterization of a novel beta subunit of soluble guanylyl cyclase that is active in the absence of a second subunit and is relatively insensitive to nitric oxide.

Previously characterized soluble guanylyl cyclases form alpha-beta heterodimers that can be activated by the gaseous messenger, nitric oxide. In mammals, four subunits have been cloned, named alpha1, alpha2, beta1, and beta2. We have identified a novel soluble guanylyl cyclase isoform from the nervous system of the insect Manduca sexta that we have named M. sexta guanylyl cyclase beta3 (MsGC-beta3). It is most closely related to the mammalian beta subunits but has several features that distinguish it from previously identified soluble cyclases. Most importantly, MsGC-beta3 does not need to form heterodimers to form an active enzyme because guanylyl cyclase activity can be measured when it is expressed alone in COS-7 cells. Moreover, this activity is only weakly enhanced in the presence of the nitric oxide donor, sodium nitroprusside. Several of the amino acids in rat beta1 subunits, previously identified as being important in heme binding or necessary for nitric oxide activation, are substituted with nonsimilar amino acids in MsGC-beta3. There are also an additional 315 amino acids C-terminal to the catalytic domain of MsGC-beta3 that have no sequence similarity to any known protein. Northern blot analysis shows that MsGC-beta3 is primarily expressed in the nervous system of Manduca.

The nitric oxide-cGMP pathway may mediate communication between sensory afferents and projection neurons in the antennal lobe of Manduca sexta.

The nitric oxide (NO)-cGMP signaling system is thought to play important roles in the function of the olfactory system in both vertebrates and invertebrates. One way of studying the role of NO in the nervous system is to study the distribution and properties of NO synthase (NOS), as well as the soluble guanylyl cyclases (sGCs), which are the best characterized targets of NO. We study NOS and sGC in the relatively simple and well characterized insect olfactory system of the hawkmoth, Manduca sexta. We have cloned Manduca sexta nitric oxide synthase (MsNOS) and two sGCs (MsGCalpha1 and MsGCbeta1), characterized their basic biochemical properties, and studied their expression in the olfactory system. The sequences of the Manduca genes are highly similar to their mammalian homologs and show similar biochemical properties when expressed in COS-7 cells. In particular, we find that MsGC functions as an obligate heterodimer that is stimulated significantly by NO. We also find that MsNOS has a Ca2+-sensitive NO-producing activity similar to that of mammalian neuronal NOS. Northern and in situ hybridization analyses show that MsNOS and the MsGCs are expressed in a complementary pattern, with MsNOS expressed at high levels in the antennae and the MsGCs expressed at high levels in a subset of antennal lobe neurons. The expression patterns of these genes suggest that the NO-sGC signaling system may play a role in mediating communication between olfactory receptor neurons and projection neurons in the glomeruli of the antennal lobe.

Progress in understanding the Drosophila dnc locus.

The genetic dissection of learning and memory in Drosophila is two decades old. Recently, a great deal of progress has been made towards isolating new mutants as well as towards a better understanding of the originally isolated ones. This paper reviews the recent developments in the understanding of the structure and function of the gene identified by the first and best-characterized of these mutants, the Drosophila dunce mutant.

The cyclic AMP phosphodiesterase encoded by the Drosophila dunce gene is concentrated in the mushroom body neuropil.

Drosophila dunce (dnc) flies are defective in learning and memory as a result of lesions in the gene that codes for a cAMP-specific phosphodiesterase (PDE). Antibodies to the dnc PDE showed that the most intensely stained regions in the adult brain were the mushroom body neuropil--areas previously implicated in learning and memory. In situ hybridization demonstrated that dnc RNA was enriched in the mushroom body perikarya. The mushroom bodies of third instar larval brains were also stained intensely by the antibody, suggesting that the dnc PDE plays an important role in these neurons throughout their development. The role of the dnc PDE in mushroom body physiology is discussed, and a circuit model describing a possible role of the mushroom bodies in mediating olfactory learning and memory is presented.