Electrophysiological Recording from <em>Drosophila</em> Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

University of California San Diego

Cytokine immunotherapy using interleukin (IL)-2 and IL-15 may be beneficial for patients receiving umbilical cord blood (CB) transplantation by ameliorating post-transplant T-cell apoptosis. The present study compares the differential effect of IL-15 and IL-2 on survival of phytohemagglutinin (PHA)-activated CB and adult peripheral blood (APB) T lymphocytes. In comparison with IL-2, IL-15 preferentially enhanced the survival of CB PHA-activated T cells by decreasing the caspase-3+ population and by increasing the Bcl-2+ population. Activated CB T cells were more susceptible to TNF-alpha-induced apoptosis compared to their adult counterparts. However, the susceptibility could be abrogated by IL-15 but not by IL-2. IL-15 but not IL-2 down-regulated CD28 expression on both activated CB and APB CD8+ T cells, with a much greater effect seen with CB. Western-blot analysis shows that IL-15 Ralpha is deficient in CB compared to APB immediately after PHA stimulation, while culturing with IL-15 significantly enhanced CB IL-15 Ralpha expression to levels comparable to that of adults. Thus, IL-15 may provide a better therapeutic choice for immune reconstitution than IL-2 post-CB transplantation due to its preferential survival enhancing effect on CB T cells.

Differential effect of IL-15 and IL-2 on survival of phytohemagglutinin-activated umbilical cord blood T cells.

Neural coding for olfactory sensory stimuli has been mapped near completion in the Drosophila first-order center, but little is known in the higher brain centers. Here, we report that the antenna lobe (AL) spatial map is transformed further in the calyx of the mushroom body (MB), an essential olfactory associated learning center, by stereotypic connections with projection neurons (PNs). We found that Kenyon cell (KC) dendrites are segregated into 17 complementary domains according to their neuroblast clonal origins and birth orders. Aligning the PN axonal map with the KC dendritic map and ultrastructural observation suggest a positional ordering such that inputs from the different AL glomeruli have distinct representations in the MB calyx, and these representations might synapse on functionally distinct KCs. Our data suggest that olfactory coding at the AL is decoded in the MB and then transferred via distinct lobes to separate higher brain centers.

A map of olfactory representation in the Drosophila mushroom body.

Recent advances in sensory neuroscience using Drosophila olfaction as a model system have revealed brain maps representing the external world. Once we understand how the brain's built-in capability generates the internal olfactory maps, we can then elaborate how the brain computes and makes decision to elicit complex behaviors. Here, we review current progress in mapping Drosophila olfactory circuits and discuss their relationships with innate olfactory behaviors.

Internal representations of smell in the Drosophila brain.

Infection with Helicobacter pylori cagA-positive strains is associated with gastritis, ulcerations, and gastric cancer. CagA is translocated into infected epithelial cells by a type IV secretion system and can be tyrosine phosphorylated, inducing signal transduction and motogenic responses in epithelial cells. Cellular cholesterol, a vital component of the membrane, contributes to membrane dynamics and functions and is important in VacA intoxication and phagocyte evasion during H. pylori infection. In this investigation, we showed that cholesterol extraction by methyl-beta-cyclodextrin reduced the level of CagA translocation and phosphorylation. Confocal microscope visualization revealed that a significant portion of translocated CagA was colocalized with the raft marker GM1 and c-Src during infection. Moreover, GM1 was rapidly recruited into sites of bacterial attachment by live-cell imaging analysis. CagA and VacA were cofractionated with detergent-resistant membranes (DRMs), suggesting that the distribution of CagA and VacA is associated with rafts in infected cells. Upon cholesterol depletion, the distribution shifted to non-DRMs. Accordingly, the CagA-induced hummingbird phenotype and interleukin-8 induction were blocked by cholesterol depletion. Raft-disrupting agents did not influence bacterial adherence but did significantly reduce internalization activity in AGS cells. Together, these results suggest that delivery of CagA into epithelial cells by the bacterial type IV secretion system is mediated in a cholesterol-dependent manner.

Cholesterol depletion reduces Helicobacter pylori CagA translocation and CagA-induced responses in AGS cells.

The intrinsic turbidity of scaffolds formed by natural biomaterials such as collagen fibers prevents high-resolution light microscopy in depth. In this research, we have developed a new method of using light microscopy for penetrative three-dimensional (3-D) visualization of scaffolds formed by collagen, chitosan, or cellulose. First, we applied an optical-clearing solution, FocusClear, to permeate and reduce the turbidity of the scaffolds. The improved photon penetration allowed fluorophores for efficient excitation and emission in the FocusClear solution. Confocal microscopy was applied to achieve cellular-level resolution up to 350 microm for both the fibroblast/collagen and the osteoblast/chitosan constructs and micrometer-level resolution up to 40 microm for the cellulose membrane. The depth of imaging of the cellulose membrane was further improved to 80 microm using two-photon microscopy. Significantly, these voxel-based confocal/two-photon micrographs allowed postrecording image processing via Amira projection algorithms for 3-D visualization and analysis of the scanned region. Although this optical method remains limited in viewing block scaffolds in thin sections, our approach provides a noninvasive way to microscopically examine the scaffold structure, which would be a valuable tool to studying biomaterials and their interactions with the molecule/cell of interest within the scaffold in an integrated fashion.

Integration of optical clearing and optical sectioning microscopy for three-dimensional imaging of natural biomaterial scaffolds in thin sections.

Animal behavior is governed by the activity of interconnected brain circuits. Comprehensive brain wiring maps are thus needed in order to formulate hypotheses about information flow and also to guide genetic manipulations aimed at understanding how genes and circuits orchestrate complex behaviors.

Three-dimensional reconstruction of brain-wide wiring networks in Drosophila at single-cell resolution.

Memory is initially labile and gradually consolidated over time through new protein synthesis into a long-lasting stable form. Studies of odor-shock associative learning in Drosophila have established the mushroom body (MB) as a key brain structure involved in olfactory long-term memory (LTM) formation. Exactly how early neural activity encoded in thousands of MB neurons is consolidated into protein-synthesis-dependent LTM remains unclear. Here, several independent lines of evidence indicate that changes in two MB vertical lobe V3 (MB-V3) extrinsic neurons are required and contribute to an extended neural network involved in olfactory LTM: (i) inhibiting protein synthesis in MB-V3 neurons impairs LTM; (ii) MB-V3 neurons show enhanced neural activity after spaced but not massed training; (iii) MB-V3 dendrites, synapsing with hundreds of MB α/β neurons, exhibit dramatic structural plasticity after removal of olfactory inputs; (iv) neurotransmission from MB-V3 neurons is necessary for LTM retrieval; and (v) RNAi-mediated down-regulation of oo18 RNA-binding protein (involved in local regulation of protein translation) in MB-V3 neurons impairs LTM. Our results suggest a model of long-term memory formation that includes a systems-level consolidation process, wherein an early, labile olfactory memory represented by neural activity in a sparse subset of MB neurons is converted into a stable LTM through protein synthesis in dendrites of MB-V3 neurons synapsed onto MB α lobes.

Drosophila ORB protein in two mushroom body output neurons is necessary for long-term memory formation.

Different stimulus intensities elicit distinct perceptions, implying that input signals are either conveyed through an overlapping but distinct subpopulation of sensory neurons or channeled into divergent brain circuits according to intensity. In Drosophila, carbon dioxide (CO2) is detected by a single type of olfactory sensory neuron, but information is conveyed to higher brain centers through second-order projection neurons (PNs). Two distinct pathways, PN(v)-1 and PN(v)-2, are necessary and sufficient for avoidance responses to low and high CO2 concentrations, respectively. Whereas low concentrations activate PN(v)-1, high concentrations activate both PN(v)s and GABAergic PN(v)-3, which may inhibit PN(v)-1 pathway-mediated avoidance behavior. Channeling a sensory input into distinct neural pathways allows the perception of an odor to be further modulated by both stimulus intensity and context.

Parallel neural pathways mediate CO2 avoidance responses in Drosophila.

During the lifespans of most animals, reproductive maturity and mating activity are highly coordinated. In Drosophila melanogaster, for instance, male fertility increases with age, and older males are known to have a copulation advantage over young ones. The molecular and neural basis of this age-related disparity in mating behavior is unknown. Here, we show that the Or47b odorant receptor is required for the copulation advantage of older males. Notably, the sensitivity of Or47b neurons to a stimulatory pheromone, palmitoleic acid, is low in young males but high in older ones, which accounts for older males' higher courtship intensity. Mechanistically, this age-related sensitization of Or47b neurons requires a reproductive hormone, juvenile hormone, as well as its binding protein Methoprene-tolerant in Or47b neurons. Together, our study identifies a direct neural substrate for juvenile hormone that permits coordination of courtship activity with reproductive maturity to maximize male reproductive fitness.

Hormonal Modulation of Pheromone Detection Enhances Male Courtship Success.

We demonstrate transcutical structural and functional imaging of neurons labeled with genetically encoded red fluorescent proteins and calcium indicators in the living Drosophila brain with cellular and subcellular resolution.

Hui-Hao Lin

Division of Biological Sciences

University of California San Diego

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Hui-Hao Lin

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Biological Sciences

University of California San Diego

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Division of Biological Sciences