Fallen Fruit’s website points out that the video probes “at the correspondence between the public walking on the tour and the anonymous public of the internet.” This year, the group was also invited to do a project with TED Active. They devised “The Banana Hotline,“ wherein the public is invited to follow a set of instructions, record a memory, and email the audio or video to the group, who will then put together “a living monument of sound.” The exhibit at Art+Sci will also include The Loneliest Fruit in the World . This video portrays the human activities, interactions, and explorations that spring up around a stand of arctic berries growing near Tromsö, Norway: “Against a beautiful, spare landscape peppered with tiny blueberries,” according to the website description, “the video follows a group of Norwegians who while picking negotiate the relation between solitude, gleaning and company.” Integration of chemical signals at the peripheral sensory system remains one of the least understood mechanisms of insect olfaction, particularly in mosquitoes. Despite the great progress made in the last 2 decades in understanding how receptors form the basis of chemosensory perception in insects,bato bucket how olfactory signals integrate at the periphery remains an enigma . ‘‘It is as if a new continent has been discovered but only the coastline has been mapped’’ .
In the largest majority of reported cases , antennal neurons of Cx. quinquefasciatus displayed excitatory responses , but evidence for inhibitory responses , already known for Ae. aegypti , is now emerging for Cx. quinquefasciatus . It has been observed in moths , beetles , the fruit fly , and mosquitoes that activation of one neuron interferes with signaling of other olfactory receptor neurons . It has also been reported that a single compound caused reduction of nerve impulse followed by a transient post-stimulus excitation . Although Carlson and collaborators elegantly demonstrated that in the fruit fly lateral inhibition is most likely mediated by ephaptic coupling , the complete ensemble of the molecular mechanism of inhibition at the peripheral olfactory system of mosquitoes remains terra incognita. A simple explanation of the ephaptic coupling is that, upon stimulation of an ORN, the potential declines. Consequently, per channel current generated by a cocompart mentalized neuron is reduced . This scenario argues that the firing of a neuron causes reduced spike frequency by a colocated neuron because of the close apposition of their neuronal processes. Although ephaptic coupling could explain lateral inhibition, other mechanisms of intraneuron inhibition may exist. While de-orphanizing odorant receptors expressed predominantly in Cx. quinquefasciatus female antennae, we serendipitously recorded currents from an OR that generate inhibition in response to certain odorants. Further studies unraveled a hitherto unknown mechanism of peripheral, intrareceptor inhibition in mosquito olfaction.
In our attempts to de-orphanize ORs from the southern house mosquito, Cx. quinquefasciatus, we challenged Xenopus oocytes coexpressing CquiOR32 along with the obligatory coreceptor Orco with a panelof more than 200 compounds, including mostly physiologically and behaviorally relevant compounds . Because CquiOR32 is predominantly expressed in female antennae , we reasoned that this receptor might be involved in the reception of attractants or repellents. CquiOR32- CquiOrco-expressing oocytes generated dose-dependent inward currents when challenged with various odorants, including cyclohexanone, methyl salicylate, and 2-methyl-2-thiazoline . Interestingly, however, eucalyptol, fenchone, DEET, picaridin, IR3535, PMD, and other compounds generated currents in reverse direction thus resembling inverse agonists. These unusual currents of reverse direction were reproducible, and no indication was found of adaptation . No currents were recorded when oocytes alone or oocytes expressing only CquiOR32 or only CquiOrco were challenged either with methyl salicylate or eucalyptol . However, CquiOR32-CquiOrco-expressing oocytes responded to both compounds. Methyl salicylate elicited inward currents, and eucalyptol generated currents in the reverse direction in a dose-dependent manner .
To gain better insights into possible mechanism of these currents in reverse direction, we obtained current-voltage curves from oocytes expressing wild-type CquiOR32-CquiOrco with voltage clamped at 80,60, 40, 20, 0, +20, and +40 mV and using methyl salicylate and eucalyptol as stimuli . Similar recordings were performed using N-methyl-D-glucamine chloride as a source of bulky, impermeable mono cation or with sodium gluconate buffer instead of NaCl . These data corroborate that OR32-Orco forms nonselective cation channels , as indicated by the reversal potential shift to more negative voltage upon replacement of Na+ by less permeable NMG . An examination of the first and third quadrants for each graphic in Figure 2 shows that I-V curves elicited by methyl salicylate are above baseline and below control I-V curves for positive and negative voltages, respectively , consistent with a potentiation of the control current by methyl salicylate. By contrast, I-V curves generated with eucalyptol, albeit not robust, are below and above control I-V curves for positive and negative holding potentials, respectively , suggesting an inhibition effect. In the presence of eucalyptol, replacing Na+ with NMG had a minor effect on the I-V curves , whereas replacing Cl with gluconate seems to suggest that Cl is implicated in this inhibition .We then surmised that these ‘‘inhibitors’’ might modulate responses to odorants when these two types of stimuli are simultaneously delivered to receptors. First, we challenged a CquiOR32-CquiOrco-expressing oocyte with methyl salicylate and then eucalyptol and recorded regular and reverse currents, respectively . Then we challenged the same oocyte preparation with mixtures of methyl salicylate and eucalyptol. Binary mixtures with higher doses of eucalyptol elicited reverse currents. Inhibition was also observed with lower doses of eucalyptol, which generated dose-dependent reduced inward currents. A continuous trace displayed in Figure S4 shows no difference in the responses to methyl salicylate before and after these tests thus ruling out adaptation. Taking together, these findings suggest that ‘‘intrareceptor’’ inhibition occurred consistently with outward currents previously recorded from antennal neurons of the vinegar fly, Drosophila melanogaster .w1118 flies gave very weak responses to eucalyptol at high doses, but interestingly Orco-Gal4/UAS-CqOR32 flies generated dose-dependent, inverse EAG responses . To further scrutinize this unusual reverse EAG responses, we used gas chromatography with electroantennographic detection . In GC-EAD analyses, injected mixtures are separated by GC and subjected to antennal preparations under the same condition thus ruling out any possibility of mechanical interference and minor sample contamination. Here, methyl salicylate responded with regular EAG responses, i.e., with the first phase , which is referred to as rise of the receptor potential, and the second phase starting at the end of the stimulus, commonly referred to as the decline of the receptor potential . This is analogous to the depolarization, repolarization, and hyperpolarization of a nervous impulse. As opposed to methyl salicylate, eucalyptol consistently gave inverse EAD responses thus corroborating what we observed in EAG analyses . Next, we recorded EAG responses when flies were challenged with odorants and an inhibitor. First, we compared the response of w1118 and Orco-Gal4/UAS-CqOR32 flies to -2-hexenal when it was delivered alone or in combination with eucalyptol. EAG responses from w1118 flies to 0.1% -2-hexenal alone or in combination with 10% eucalyptol did not differ significantly .
By contrast,dutch bucket hydroponic EAG responses from Orco-Gal4/UAS-CqOR32 flies to 0.1% -2-hexenal plus 10% eucalyptol were significantly lower than those elicited by 0.1% -2-hexenal alone . We then examined the dose-dependent effect of this inhibition by using Orco-Gal4/UAS-CqOR32 flies. Robust responses to 0.1% methyl salicylate were reduced in a dose-dependent manner with the addition of eucalyptol but remained unchanged at the end of the tests. Likewise, EAG responses to 0.01% -2-hexenal were reduced when coapplied with eucalyptol . Of note, -2-hexenal does not activate CquiOR32 . Such inhibition presumably results from CquiOR32 indirectly inhibiting responses of the fly endogenous receptors to -2-hexenal. In these continuous experiments, a small difference between EAG responses before and after costimulus tests may be due to loss of this volatile semiochemical from the cartridge rather than adaptation. Similar inhibition was observed when 2-heptanone was applied alone or coapplied with eucalyptol . Taken together, these results further suggest that intrareceptor inhibition occurs in vivo as indicated by the inhibitory effect of eucalyptol on methyl salicylate responses. Additionally, the effect of eucalyptol on the response to -2-hexenal suggests that intraneuronal inhibition occurred. A few lines of evidence support this hypothesis. First and foremost, eucalyptol does not cause inhibition in control flies and -2-hexenal does not activate CquiOR32 . The simplest explanation is that, in Orco-Gal4/UAS-CqOR32 flies, all endogenous receptors are coexpressed with CquiOR32. Thus, CquiOR32 response to eucalyptol interferes with the response of DmelOR7a to -2-hexenal. In short, inhibitor and agonist are likely to be acting on different receptors in the same neurons, thus an intraneuron inhibition. To further test the notion of intraneuronal inhibition, we turned to single sensillum recordings .The best ligand for ab4A, the neuron in ab4 sensilla with a large spike amplitude, is -2-hexenal , although ab4A is also very sensitive to other ligands, including hexanal . Contrary to ab4B, ab4A houses only one OR, namely, DmelOr7a . Because expression of CquiOR32 was driven by DmelOrco, ab4A neurons in our transgenic flies house both DmelOr7a and CquiOR32. Coexpression was confirmed by a significantly stronger response to methyl salicylate recorded from Orco-Gal4/UAS-CquiOR32 than from WT flies , while retaining response to hexanal . It is known that methyl salicylate is the best ligand for DmelOr10a in ab1D but elicits only very low response in ab4A . The low response of WT flies to methyl salicylate did not differ significantly when the odorant was delivered alone or codelivered with eucalyptol . By contrast, responses recorded from Orco-Gal4/UAS-CquiOR32 flies were significantly lower when the two stimuli were delivered simultaneously from two different cartridges . Next, we tested whether CquiOR32 response to eucalyptol would affect DmelOR7a response to a cognate ligand, hexanal. Responses of WT flies to hexanal did not differ significantly when comparing hexanal alone with hexanal plus eucalyptol . Recordings from ab4 sensilla in the Orco-Gal4/UAS-CquiOR32 flies showed a slight, albeit not significant, increase in response to hexanal. This is unlikely to be due to hexanal activation of CquiOR32 . When hexanal and eucalyptol were delivered simultaneously firing of DmelOR7a was completely abolished . We also recorded from ab7 sensilla, which expresses DmelOR98a, in ab7A and for which butyl acetate is one of the best ligands . In the transgenic flies both methyl salicylate and butyl acetate generated excitatory responses , which were inhibited by eucalyptol . Because methyl salicylate and eucalyptol elicit inward and reverse currents in CquiOR32, this in vivo inhibition is not surprising. However, the consistent observation that eucalyptol inhibits the response of an endogenous receptor to a cognate ligand supports the notion that intraneuronal inhibition occurs when receptors are colocated in a neuron. Specifically, the inhibitory responses of CquiOR32 interferes with the activation of a collocated receptor by a cognate ligand. For example, activation of DmelOR7a in ab4A neuron by hexanal and activation of DmelOR98a in ab7A neuron by butyl acetate were both inhibited by eucalyptol upon interaction with CquiOR32. Contrary to the fruit fly, which expresses only one receptor per neuron , mosquitoes can coexpress multiple ORs in the same neuron .In the traditional view, the skin serves as a protective barrier between the body and the external environment. Yet, more and more evidence suggests that cutaneous function extends far beyond mere protection. In fact, cutaneous function regulates a wide spectrum of cutaneous and systemic functions. Compromised epidermal function has been linked to the development of a variety of cutaneous and extracutaneous disorders. For example, disruption of epidermal permeability barrier not only provokes the release and production of proinflammatory cytokines, but also induces infiltration and activation of inflammatory cells in the skin, suggesting that defective epidermal permeability barrier predisposes to the development of inflammatory dermatoses. But barrier disruption also stimulates barrier homeostasis responses, including epidermal proliferation and lipid synthesis. Moreover, defects in epidermal permeability barrier allow the penetration of microbial pathogens into the skin. Importantly, recent studies showed that the epidermal dysfunction-induced elevations in cutaneous cytokines lead to increased levels of proinflammatory cytokines not only in the skin, but also in circulation, supporting not only a pathogenic role for epidermal function in cutaneous and extracutaneous inflammation, but also suggesting a link between cutaneous function and inflammation-associated systemic disorders. In addition to epidermal permeability barrier homeostasis, other epidermal functions, such as pH and stratum corneum hydration, also regulate cutaneous functions. Accordingly, improvements in epidermal function could benefit multiple cutaneous and extracutaneous functions. Because of the importance of cutaneous function, much recent attention has focused on the identification of active ingredients that could lead to the development of products that improve cutaneous function.