Drosophila Pheromone-Sensing Neurons Expressing the ppk25 Ion Channel Subunit Stimulate Male Courtship and Female Receptivity

As in many species, gustatory pheromones regulate the mating behavior of Drosophila. Recently, several ppk genes, encoding ion channel subunits of the DEG/ENaC family, have been implicated in this process, leading to the identification of gustatory neurons that detect specific pheromones. In a subset of taste hairs on the legs of Drosophila, there are two ppk23-expressing, pheromone-sensing neurons with complementary response profiles; one neuron detects female pheromones that stimulate male courtship, the other detects male pheromones that inhibit male-male courtship. In contrast to ppk23, ppk25, is only expressed in a single gustatory neuron per taste hair, and males with impaired ppk25 function court females at reduced rates but do not display abnormal courtship of other males. These findings raised the possibility that ppk25 expression defines a subset of pheromone-sensing neurons. Here we show that ppk25 is expressed and functions in neurons that detect female-specific pheromones and mediates their stimulatory effect on male courtship. Furthermore, the role of ppk25 and ppk25-expressing neurons is not restricted to responses to female-specific pheromones. ppk25is also required in the same subset of neurons for stimulation of male courtship by young males, males of the Tai2 strain, and by synthetic 7-pentacosene (7-P), a hydrocarbon normally found at low levels in both males and females. Finally, we unexpectedly find that, in females, ppk25 and ppk25-expressing cells regulate receptivity to mating. In the absence of the third antennal segment, which has both olfactory and auditory functions, mutations in ppk25 or silencing of ppk25-expressing neurons block female receptivity to males. Together these results indicate that ppk25 identifies a functionally specialized subset of pheromone-sensing neurons. While ppk25 neurons are required for the responses to multiple pheromones, in both males and females these neurons are specifically involved in stimulating courtship and mating

Nonthermal plasma processing for nanostructured biomaterials and tissue engineering scaffolds : a mini review

Traditional wet chemistry offers a great magnitude of methods for surface modification and surface coatings for nanostructured materials. However, most methods require solvents and purification steps, and generate waste and byproducts. An interesting alternative set of methods involves the use of nonthermal or low-temperature plasmas (LTP) toward making and modifying nanostructured biomaterials. In this current opinion piece, some of the recent literature in this area is highlighted, and current perspectives are given. Emphasis is noted for the role of LTP for surface modification of nanofibrous scaffolds and plasma electrolytic oxidation (PEO) for surface-nanostructuring of metallic implants. The morphological nanofeaturing in fibrous mats as an extracellular matrix mimicking scaffold is presented along with recent perspectives of using LTP and plasma electrolytic oxidation for surface structuring for enhanced biointegration via cell/surface interactions with plasma processed implants/scaffolds constructs

Antenna-less females mutant for <i>ppk25, ppk23 or ppk29</i> are unreceptive to males.

<p>A. The percentage of females that mated within 30(“Receptivity”) was calculated for females with homozygous null mutations in <i>ppk25</i>, <i>ppk23</i> or <i>ppk29 </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Liu1" target="_blank">[11]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Thistle1" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Lin1" target="_blank">[16]</a>, and for control females with one normal copy of <i>ppk25</i>, <i>ppk23</i> and <i>ppk29</i>. N = 30–34. For receptivity in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen-1004238-g005" target="_blank">Fig. 5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen-1004238-g006" target="_blank">6</a>, error bars indicate the SEM. B. Antenna-less females mutant for <i>ppk25</i>, <i>ppk23 or ppk29</i> show reduced receptivity to males. Receptivity was measured for mutants and control females as in A but after removal of antennae. N = 30–38; ***p<0.001. (Fisher's exact test). C. Arista-less females mutant for <i>ppk23 or ppk25</i> show reduced receptivity to males. Receptivity was measured for mutants and control females as in A but after removal of arista. N = 21–32; ***p<0.001. (Fisher's exact test). D. <i>wt</i> males find <i>ppk25</i> mutant and control females equally attractive. CI measured for Canton-S males toward females that were either alive (left) or decapitated (right). CI of males toward live females was quantified by measuring male courtship behavior during the first 10 minutes of the receptivity assay shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen-1004238-g005" target="_blank">Fig. 5B</a>. Females were either homozygous null mutants for <i>ppk25</i> or contained one normal copy of <i>ppk25</i>. N = 20. E. Introduction of a <i>ppk25</i> transgene increases receptivity of <i>ppk25</i> mutants but not of <i>ppk29</i> mutants. Receptivity was measured for antenna-less females carrying an <i>UAS-ppk25</i> transgene in either a <i>ppk25</i> mutant or <i>ppk29</i> mutant background and compared to <i>ppk25</i> mutant and controls shown earlier (panel A). N = 30–38; ***p<0.001 (Fisher's exact test).</p

Male courtship of immature males requires <i>ppk25</i> function.

<p>A. <i>ppk25</i> expression in gustatory neurons defined by <i>ppk25-Gal4</i> is required for male courtship of immature males. CI, fraction of males initiating courtship toward decapitated immature males and Total Behavioral Index (TBI) were calculated for <i>ppk25</i> mutant males, control males with one <i>wt</i> copy of <i>ppk25</i> and for <i>ppk25 null</i> mutant males where <i>ppk25</i> expression has been restored in cells expressing <i>ppk25-Gal4</i>. N = 32–39; Mean ± SEM; ***p<0.001; **p<0.01 (Error bars indicate the SEM for the fraction of males initiating courtship and statistical significance was determined by Fisher's exact test. Error bars and statistical significance for TBI was determined as described previously for CI). Newly eclosed Canton-S males with light body pigmentation and unfurled wings were used as immature male targets. B. Targeted RNAi knockdown of <i>ppk25</i> in gustatory neurons with <i>Poxn-Gal4</i> reduces courtship toward <i>Tai2</i> males. CI, fraction of males initiating courtship toward decapitated <i>Tai2</i> males and TBI were calculated for males expressing <i>ppk25</i> RNAi in gustatory neurons. Control males expressed eGFP or a control RNAi targeting <i>CG13895</i>, a gene with no known involvement in mating behavior <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Benchabane1" target="_blank">[58]</a> in all gustatory neuron. N = 32–34; Mean ± SEM; ***p<0.001.</p

<i>ppk25</i> function in gustatory neurons is required for courtship stimulation by <i>Tai2</i> males or 7-pentacosene.

<p>A. <i>ppk25</i> function in cells defined by <i>ppk25-Gal4</i> expression is required for male courtship directed at <i>Tai2</i> males. CI, fraction of males initiating courtship toward decapitated <i>Tai2</i> males and TBI were calculated for <i>ppk25 null</i> mutant males, control males with one wild-type copy of <i>ppk25</i>, and <i>ppk25</i> mutant males where <i>ppk25</i> expression had been restored in <i>ppk25</i> cells. N = 36–40; Mean ± SEM; ***p<0.001. B. Targeted RNAi knockdown of <i>ppk25</i> in all gustatory neurons reduces courtship toward <i>Tai2</i> males. CI, fraction of males initiating courtship toward decapitated <i>Tai2</i> males and TBI were calculated for males expressing <i>ppk25</i> RNAi in gustatory neurons using <i>Poxn-Gal4</i>. Control males expressed eGFP or a control RNAi targeting <i>CG13895</i> under control of <i>Poxn-Gal4</i>. Error bars are SEM; N = 31–34; ***p<0.001. C. The number of wing extensions performed by males with normal or mutant copies of <i>ppk25</i>, or by <i>ppk25</i> mutant males in which <i>ppk25</i> function is restored in <i>ppk25</i> cells, was measured in the presence of oe- females painted either with solvent alone or with 7-Pentacosene. oe- females were pierced through the head with forceps, contributing to the low courtship background and assays were conducted in the light. n = 20–24; Mean ± SEM; ***p<0.001.</p

<i>ppk25</i> is required for courtship stimulation by 7,11HD but not for inhibition of courtship by 7-T.

<p>A. The number of wing extensions performed by males with normal or mutant copies of <i>ppk25</i>, or by <i>ppk25</i> mutant males in which <i>ppk25</i> function is restored specifically in <i>ppk25</i> cells, was measured in the presence of oe- females painted either with solvent alone or with a single female pheromone, 7,11HD. oe- females were pierced through the head with forceps, contributing to the low courtship background. These and all following assays involving perfumed oe- targets were conducted under normal laboratory lights <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Thistle1" target="_blank">[13]</a>. n = 19–24; Mean ± SEM; ***p<0.001 (perfumed relative to solvent control). Error bars indicate the SEM for number of wing extensions and statistical significance was determined using the Kruskal-Wallis test followed by Dunn's post hoc test. B. The number of wing extensions displayed by males with normal or mutant <i>ppk25</i> was measured in the presence of oe- males painted either with solvent alone or with a single male pheromone, 7T. In this experiment, males were raised in groups as this resulted in higher baseline courtship toward oe- targets, thereby allowing inhibition to be measured. n = 24–28; Mean ± SEM; **p<0.01; (Kruskal-Wallis test followed by Dunn's post hoc test to solvent control). C. The Courtship Index (CI, percentage of a ten minute observation time during which the male is courting <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Lin1" target="_blank">[16]</a>) was measured for control <i>w1118</i> males, males mutant for <i>ppk23</i>, and <i>ppk23</i> mutant males in which expression of <i>ppk23</i> is targeted to F cells using the <i>ppk25-Gal4</i> driver. Male-female courtship was measured in the presence of decapitated w1118 females to reduce behavioral feedback. Male-male courtship was performed with intact male targets in the light since <i>ppk23</i> mutants display robust male-male courtship under these conditions <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Thistle1" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Toda1" target="_blank">[14]</a>. n = 30–40; Mean ±SEM; ***p<0.001 to <i>wt</i>. Error bars indicate the SEM for CI and statistical significance was determined using the Kruskal-Wallis test followed by Dunn's post hoc test.</p

Female <i>ppk25-Gal4</i> neurons represent a subset of <i>ppk23-Gal4</i> neurons and are involved in female receptivity.

<p>A. Segment of female front legs showing expression of <i>ppk23-Gal4</i> (green) in each of the two <i>fru</i>-positive cells (<i>fru-LexA</i>, magenta) under each chemosensory bristle. Scale 10 µ<i>m</i>. B. Segment of female front legs showing expression of <i>ppk25-Gal4</i> (green) in only one of the two <i>fru</i>-positive cells (<i>fru-LexA</i>, magenta) present under each chemosensory bristle. Scale 10 µ<i>m</i>. C. Silencing of <i>ppk25-Gal4</i> or <i>ppk23-Gal4</i> neurons reduces female receptivity. Receptivity was calculated for females expressing active (<i>UAS-TNT</i>) or inactive (<i>UAS-IN-TNT</i>) forms of TNT under control of either <i>ppk23-Gal4</i> or <i>ppk25-Gal4</i>. Additional control females contained <i>UAS-TNT</i> driven by <i>Or22b-Gal4</i>, which is expressed in a neuronal subset not associated with mating behaviors <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Hallem1" target="_blank">[36]</a>. N = 30–35; ***p<0.001 (Fisher's exact test). D. Transient inactivation of <i>ppk25-Gal4</i> neurons inhibits female receptivity. Females expressing the temperature-sensitive dominant <i>Dynamin</i> allele <i>shi^ts</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Kitamoto1" target="_blank">[37]</a> under control of <i>ppk25-Gal4</i> were incubated at either permissive (23°C) or non-permissive (30°C) temperature for 20 minutes prior to, and during the test. Receptivity for females expressing GFP in <i>ppk25-Gal4</i> neurons or females with <i>UAS-Shi^(ts)</i> alone was measured under identical conditions. N = 30–45; ***p<0.001;**p<0.01;*p<0.05 (Fisher's exact test).</p

Calcium imaging reveals that <i>ppk25</i> cells respond specifically to female pheromones.

<p>A. Solutions (100 ng/µl in 10%hexane, 90% water) of 7,11-HD (HD), 7,11-ND (ND), 7T, cVA, a mixture of all pheromones (mix) or 10% hexane, 90% water solution alone (hex) were applied to single leg bristles of <i>ppk25-Gal4</i>, <i>20×UAS-GCaMP3</i> flies. “<i>wt</i>” flies contained one copy of the normal <i>ppk25</i> gene. <i>ppk25 null</i> mutants were heterozygous for two different deletions of the <i>ppk25</i> locus <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Lin1" target="_blank">[16]</a>, and “<i>ppk25</i> rescue” flies are <i>ppk25</i> mutants carrying <i>UAS-ppk25</i> and <i>ppk25-Gal4</i> transgenes to target <i>ppk25</i> expression to <i>ppk25</i> cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Starostina1" target="_blank">[10]</a>. “#” denotes pheromones that did not elicit responses significantly higher than hexane alone in “<i>wt</i>” flies and were not tested further. n = 7–10; Mean ± SEM; ttest to <i>wt</i>, *p<0.05, **p<0.01. B. The same pheromone solutions as in A were applied to single leg bristles of <i>ppk25</i> mutants carrying the <i>ppk23-Gal4</i> and <i>20×UAS-GCaMP3</i> transgenes. As previously observed in flies with normal <i>ppk25</i> genes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Thistle1" target="_blank">[13]</a>, one population of <i>ppk23</i> cells, the M cells, respond specifically to male pheromones. In contrast to <i>wt</i> males however, in <i>ppk25 null</i> mutants the second population of <i>ppk23</i> cells, corresponding to F cells does not respond to any pheromone. n = 8; Mean ± SEM; ttest to <i>wt</i>,*p<0.05,**p<0.01.</p

Synaptic Contacts Enhance Cell-to-Cell Tau Pathology Propagation

Accumulation of insoluble Tau protein aggregates and stereotypical propagation of Tau pathology through the brain are common hallmarks of tauopathies, including Alzheimer's disease (AD). Propagation of Tau pathology appears to occur along connected neurons, but whether synaptic contacts between neurons are facilitating propagation has not been demonstrated. Using quantitative in vitro models, we demonstrate that, in parallel to non-synaptic mechanisms, synapses, but not merely the close distance between the cells, enhance the propagation of Tau pathology between acceptor hippocampal neurons and Tau donor cells. Similarly, in an artificial neuronal network using microfluidic devices, synapses and synaptic activity are promoting neuronal Tau pathology propagation in parallel to the non-synaptic mechanisms. Our work indicates that the physical presence of synaptic contacts between neurons facilitate Tau pathology propagation. These findings can have implications for synaptic repair therapies, which may turn out to have adverse effects by promoting propagation of Tau pathology.publisher: Elsevier articletitle: Synaptic Contacts Enhance Cell-to-Cell Tau Pathology Propagation journaltitle: Cell Reports articlelink: http://dx.doi.org/10.1016/j.celrep.2015.04.043 content_type: article copyright: Copyright © 2015 The Authors. Published by Elsevier Inc.status: publishe

Synaptic Contacts Enhance Cell-to-Cell Tau Pathology Propagation

Accumulation of insoluble Tau protein aggregates and stereotypical propagation of Tau pathology through the brain are common hallmarks of tauopathies, including Alzheimer’s disease (AD). Propagation of Tau pathology appears to occur along connected neurons, but whether synaptic contacts between neurons are facilitating propagation has not been demonstrated. Using quantitative in vitro models, we demonstrate that, in parallel to non-synaptic mechanisms, synapses, but not merely the close distance between the cells, enhance the propagation of Tau pathology between acceptor hippocampal neurons and Tau donor cells. Similarly, in an artificial neuronal network using microfluidic devices, synapses and synaptic activity are promoting neuronal Tau pathology propagation in parallel to the non-synaptic mechanisms. Our work indicates that the physical presence of synaptic contacts between neurons facilitate Tau pathology propagation. These findings can have implications for synaptic repair therapies, which may turn out to have adverse effects by promoting propagation of Tau pathology