No abstract

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 (MsGC␣1 and MsGC␤1), 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 Ca 2ϩ -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 Ms-NOS 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 NOsGC signaling system may play a role in mediating communication between olfactory receptor neurons and projection neurons in the glomeruli of the antennal lobe.

Soluble guanylyl/guanylate cyclase (sGC), the primary biological receptor for nitric oxide, is required for proper development and health in all animals. We have expressed heterodimeric fulllength and N-terminal fragments of Manduca sexta sGC in Escherichia coli, the first time this has been accomplished for any sGC, and have performed the first functional analyses of an insect sGC. Manduca sGC behaves much like its mammalian counterparts, displaying a 170-fold stimulation by NO and sensitivity to compound YC-1. YC-1 reduces the NO and CO offrates for the ϳ100-kDa N-terminal heterodimeric fragment and increases the CO affinity by ϳ50-fold to 1.7 M. Binding of NO leads to a transient six- Nitric oxide (NO)2 regulates numerous vital functions in animal physiology, including blood pressure, memory formation, platelet aggregation, and tissue development (1). The primary NO receptor is soluble guanylyl/guanylate cyclase (sGC), a heterodimeric protein of ϳ150 kDa that binds NO through a ferrous heme. NO binding stimulates cyclase activity, the production of cGMP from substrate GTP, and the subsequent amplification of NO-dependent signaling cascades (2-5). Although NO is the best described allosteric regulator of sGC, numerous other forms of regulation may also be of importance, including phosphorylation (6), nucleotide binding (7-10), calcium binding (11), nitrosylation (12, 13), and protein-protein interactions (14 -17).The primary form of sGC is an ␣1/␤1 heterodimer composed of two evolutionarily related subunits that display several recognizable domains (Fig. 1A) (18). Heme is bound to the protein through proximal ␤1 His-105. NO binding to the heme leads to proximal histidine release and stimulation of cyclase activity, presumably through a change in protein conformation (reviewed in Ref. 19). The heme-binding domain has evolved from a widespread family of bacterial proteins called H-NOX (Heme-Nitric oxide/OXygen) domain proteins, of which the structures of three are known (20 -22). The central portion of sGC contains two PAS domains (18,23), and the C-terminal region contains a catalytic domain that is very similar to that of adenylyl cyclase (24, 25).In the 1990s, the anti-platelet activity of YC-1, a benzylindazole derivative (Fig. 1B), was found to derive from its ability to bind to and stimulate sGC (26, 27), leading to a search for related compounds that might serve as sGC-targeted drugs for human health (28). The YC-1 mechanism of action remains unclear, as does the location of its binding site in the sGC protein. The nucleotide-like structure of YC-1, in conjunction with mutagenesis studies, has led to the suggestion that YC-1 binds to the cyclase domain (29), whereas cross-linking studies with YC-1-related compounds have indicated that they bind to the N-terminal domain of the ␣1 subunit (30).Studies of insect sGC have lagged behind studies of the mammalian enzyme, but, as in mammals, insect sGC plays an important physiological role. In the Manduca sexta larva (tobacco hornworm), sGC is implicated in ante...

The conserved Eph receptors and their Ephrin ligands regulate a number of developmental processes, including axon guidance. In contrast to the large vertebrate Eph/Ephrin family, Drosophila has a single Eph receptor and a single Ephrin ligand, both of which are expressed within the developing nervous system. Here, we show that Eph and Ephrin can act as a functional receptor-ligand pair in vivo. Surprisingly, and in contrast to previous results using RNA-interference techniques, embryos completely lacking Eph function show no obvious axon guidance defects. However, Eph/Ephrin signaling is required for proper development of the mushroom body. In wild type, mushroom body neurons bifurcate and extend distinct branches to different target areas. In Eph mutants, these neurons bifurcate normally, but in many cases the dorsal branch fails to project to its appropriate target area. Thus, Eph/Ephrin signaling acts to guide a subset of mushroom body branches to their correct synaptic targets.

Sensory systems must be able to extract features of environmental cues within the context of the different physiological states of the organism and often temper their activity in a state-dependent manner via the process of neuromodulation. We examined the effects of the neuromodulator serotonin on a well-characterized sensory circuit, the antennal lobe of Drosophila melanogaster, using two-photon microscopy and the genetically expressed calcium indicator, G-CaMP. Serotonin enhances sensitivity of the antennal lobe output projection neurons in an odor-specific manner. For odorants that sparsely activate the antennal lobe, serotonin enhances projection neuron responses and causes an offset of the projection neuron tuning curve, most likely by increasing projection neuron sensitivity. However, for an odorant that evokes a broad activation pattern, serotonin enhances projection neuron responses in some, but not all, glomeruli. Further, serotonin enhances the responses of inhibitory local interneurons, resulting in a reduction of neurotransmitter release from the olfactory sensory neurons via GABA B receptor-dependent presynaptic inhibition, which may be a mechanism underlying the odorant-specific modulation of projection neuron responses. Our data suggest that the complexity of serotonin modulation in the antennal lobe accommodates coding stability in a glomerular pattern and flexible projection neuron sensitivity under different physiological conditions.