Inhibition of Nitric Oxide and Soluble Guanylyl Cyclase Signaling Affects Olfactory Neuron Activity in the Moth, \u3cem\u3eManduca sexta\u3c/em\u3e

Nitric oxide is emerging as an important modulator of many physiological processes including olfaction, yet the function of this gas in the processing of olfactory information remains poorly understood. In the antennal lobe of the moth, Manduca sexta, nitric oxide is produced in response to odor stimulation, and many interneurons express soluble guanylyl cyclase, a well-characterized nitric oxide target. We used intracellular recording and staining coupled with pharmacological manipulation of nitric oxide and soluble guanylyl cyclase to test the hypothesis that nitric oxide modulates odor responsiveness in olfactory interneurons through soluble guanylyl cyclase-dependent pathways. Nitric oxide synthase inhibition resulted in pronounced effects on the resting level of firing and the responses to odor stimulation in most interneurons. Effects ranged from bursting to strong attenuation of activity and were often accompanied by membrane depolarization coupled with a change in input resistance. Blocking nitric oxide activation of soluble guanylyl cyclase signaling mimicked the effects of nitric oxide synthase inhibitors in a subset of olfactory neurons, while other cells were differentially affected by this treatment. Together, these results suggest that nitric oxide is required for proper olfactory function, and likely acts through soluble guanylyl cyclase-dependent and -independent mechanisms in different subsets of neurons

A Characterization of the Manduca sexta Serotonin Receptors in the Context of Olfactory Neuromodulation

Neuromodulation, the alteration of individual neuron response properties, has dramatic consequences for neural network function and is a phenomenon observed across all brain regions and taxa. However, the mechanisms underlying neuromodulation are made complex by the diversity of neuromodulatory receptors expressed within a neural network. In this study we begin to examine the receptor basis for serotonergic neuromodulation in the antennal lobe of Manduca sexta. To this end we cloned all four known insect serotonin receptor types from Manduca (the Ms5HTRs). We used phylogenetic analyses to classify the Ms5HTRs and to establish their relationships to other insect serotonin receptors, other insect amine receptors and the vertebrate serotonin receptors. Pharmacological assays demonstrated that each Ms5HTR was selective for serotonin over other endogenous amines and that serotonin had a similar potency at all four Ms5HTRs. The pharmacological assays also identified several agonists and antagonists of the different Ms5HTRs. Finally, we found that the Ms5HT1A receptor was expressed in a subpopulation of GABAergic local interneurons suggesting that the Ms5HTRs are likely expressed heterogeneously within the antennal lobe based on functional neuronal subtype

Reverse and Conventional Chemical Ecology Approaches for the Development of Oviposition Attractants for Culex Mosquitoes

Synthetic mosquito oviposition attractants are sorely needed for surveillance and control programs for Culex species, which are major vectors of pathogens causing various human diseases, including filariasis, encephalitis, and West Nile encephalomyelitis. We employed novel and conventional chemical ecology approaches to identify potential attractants, which were demonstrated in field tests to be effective for monitoring populations of Cx. p. quinquefasciatus in human dwellings. Immunohistochemistry studies showed that an odorant-binding protein from this species, CquiOBP1, is expressed in trichoid sensilla on the antennae, including short, sharp-tipped trichoid sensilla type, which house an olfactory receptor neuron sensitive to a previously identified mosquito oviposition pheromone (MOP), 6-acetoxy-5-hexadecanolide. CquiOBP1 exists in monomeric and dimeric forms. Monomeric CquiOBP1 bound MOP in a pH-dependent manner, with a change in secondary structure apparently related to the loss of binding at low pH. The pheromone antipode showed higher affinity than the natural stereoisomer. By using both CquiOBP1 as a molecular target in binding assays and gas chromatography-electroantennographic detection (GC-EAD), we identified nonanal, trimethylamine (TMA), and skatole as test compounds. Extensive field evaluations in Recife, Brazil, a region with high populations of Cx. p. quinquefasciatus, showed that a combination of TMA (0.9 µg/l) and nonanal (0.15 ng/µl) is equivalent in attraction to the currently used infusion-based lure, and superior in that the offensive smell of infusions was eliminated in the newly developed synthetic mixture

Neuroarchitecture of Peptidergic Systems in the Larval Ventral Ganglion of Drosophila melanogaster

Recent studies on Drosophila melanogaster and other insects have revealed important insights into the functions and evolution of neuropeptide signaling. In contrast, in- and output connections of insect peptidergic circuits are largely unexplored. Existing morphological descriptions typically do not determine the exact spatial location of peptidergic axonal pathways and arborizations within the neuropil, and do not identify peptidergic in- and output compartments. Such information is however fundamental to screen for possible peptidergic network connections, a prerequisite to understand how the CNS controls the activity of peptidergic neurons at the synaptic level. We provide a precise 3D morphological description of peptidergic neurons in the thoracic and abdominal neuromeres of the Drosophila larva based on fasciclin-2 (Fas2) immunopositive tracts as landmarks. Comparing the Fas2 “coordinates” of projections of sensory or other neurons with those of peptidergic neurons, it is possible to identify candidate in- and output connections of specific peptidergic systems. These connections can subsequently be more rigorously tested. By immunolabeling and GAL4-directed expression of marker proteins, we analyzed the projections and compartmentalization of neurons expressing 12 different peptide genes, encoding approximately 75% of the neuropeptides chemically identified within the Drosophila CNS. Results are assembled into standardized plates which provide a guide to identify candidate afferent or target neurons with overlapping projections. In general, we found that putative dendritic compartments of peptidergic neurons are concentrated around the median Fas2 tracts and the terminal plexus. Putative peptide release sites in the ventral nerve cord were also more laterally situated. Our results suggest that i) peptidergic neurons in the Drosophila ventral nerve cord have separated in- and output compartments in specific areas, and ii) volume transmission is a prevailing way of peptidergic communication within the CNS. The data can further be useful to identify colocalized transmitters and receptors, and develop peptidergic neurons as new landmarks

The role of nitric oxide in memory is modulated by diurnal time

Nitric oxide (NO) is thought to play an important neuromodulatory role in the olfactory system. This modulation has been suggested to be particularly important for olfactory learning and memory in the antennal lobe (the primary olfactory network in invertebrates). We are using the hawkmoth, Manduca sexta, to further investigate the role of NO in olfactory memory. Recent findings suggest that NO affects short-term memory traces and that NO concentration fluctuates with the light cycle. This gives rise to the hypothesis that NO may be involved in the connection between memory and circadian rhythms. In this study, we explore the role of diurnal time and NO in memory by altering the time of day when associative-olfactory conditioning is performed. We find a strong effect of NO on short-term memory, and two surprising effects of diurnal time. We find that (1) at certain time points, NO affects longer traces of memory in addition to short-term memory, and (2) when conditioning is performed close to the light cycle switches—both from light to dark and dark to light—NO does not significantly affect memory at all. These findings suggest an intriguing functional role for NO in olfactory conditioning that is modulated as a function of diurnal time

Nitric oxide has differential effects on currents in different subsets of Manduca sexta antennal lobe neurons.

Nitric oxide has been shown to regulate many biological systems including olfaction. In the moth olfactory system nitric oxide is produced in the antennal lobe in response to odor stimulation and has complex effects on the activity of both projection neurons and local interneurons. To examine the cell autonomous effects of nitric oxide on these cells, we used patch-clamp recording in conjunction with pharmacological manipulation of nitric oxide to test the hypothesis that nitric oxide differentially regulates the channel properties of these different antennal lobe neuron subsets. We found that nitric oxide caused increasing inward currents in a subset of projection neurons while the effects on local neurons were variable but consistent within identifiable morphological subtypes

Eph Receptor and Ephrin Signaling in Developing and Adult Brain of the Honeybee (Apis mellifera)

Roles for Eph receptor tyrosine kinase and ephrin signaling in vertebrate brain development are well established. Their involvement in the modulation of mammalian synaptic structure and physiology is also emerging. However, less is known of their effect

Copper/zinc superoxide dismutase-like immunoreactivity in the metamorphosing brain of the sphinx moth Manduca sexta

Cu/Zn superoxide dismutase (SOD) is part of the defense mechanism that protects cells from being damaged by reactive oxygen species. During metamorphosis of the nervous system, neurons undergo various fates, which are all coupled to high metabolic activities, such as proliferation, differentiation, pathfinding, and synaptogenesis. We describe the pattern of SOD immunoreactivity of identified neurons and neuron groups in the brain of Manduca sexta from the late larva through metamorphosis into adult. We focused on neurons of the developing antennal lobes, the optic lobes, and the central brain. Our results indicate the transient expression of SOD during phases in which the neurons develop their final adult identities. Our data also suggest that the SOD immunoreactivity may be used as an indicator for the period in which developing neurons form their synapses. We also observed SOD immunoreactivity within nitric oxide-sensitive cells as characterized by immunolabeling against 3′5′-cyclic guanosine monophosphate and soluble guanylyl cyclase, a novel finding in insects

Cultured antennal lobe neurons examined in this study after 14 day <i>in vitro</i>.

<p><b>A</b>. Projection neuron (PN) B. RickRack local interneuron (RR LN) showing recording electrode <b>C</b>. Fuzzy compact local interneuron (FC LN) D. Symmetrical local interneuron (SM LN). Scale bar = 50 µm.</p