Neuropeptide F regulates courtship in Drosophila through a male-specific neuronal circuit
- WeiweiLiu
- AnindyaGanguly
- JiaHuang
- YijinWang
- Jinfei DNi
- Adishthi SGurav
- Morris AAguilar
- CraigMontell
Abstract
Male courtship is provoked by perception of a potential mate. In addition, the likelihood and intensity of courtship are influenced by recent mating experience, which affects sexual drive. Using Drosophila melanogaster, we found that the homolog of mammalian neuropeptide Y, neuropeptide F (NPF), and a cluster of male-specific NPF (NPFM) neurons, regulate courtship through affecting courtship drive.  We found that NPFM neurons make synaptic connections with P1 neurons, which comprise the courtship decision center. Activation of P1 neurons elevates NPFM neuronal activity, which then act through NPF receptor neurons to suppress male courtship, and maintain the proper level of male courtship drive.
Introduction
To mate is a critical decision that sexually reproductive animals must make to ensure propagation of their species. Although courtship is an innate behavior that can be evoked mostly by stimulatory cues emitted from a conspecific of the opposite sex, the intensity of courtship is largely under control of animal’s internal drive state, which is dictated in part by sexual satiation or deprivation.
The fruit fly, Drosophila melanogaster, has served as an animal model to decipher the genetic and neural basis of courtship behavior (Sturtevant, 1915; Hotta and Benzer, 1976; Dickson, 2008; Villella and Hall, 2008; Robinett et al., 2010; Pavlou and Goodwin, 2013; Yamamoto and Koganezawa, 2013). Distinct male and female courtship rituals (Dickson, 2008) are orchestrated by sexually dimorphic neuronal circuits, which are specified by the master transcriptional regulators – Fruitless and Doublesex (Burtis and Baker, 1989; Ito et al., 1996; Ryner et al., 1996; Lee et al., 2000; Demir and Dickson, 2005; Manoli et al., 2005; Stockinger et al., 2005; Rideout et al., 2010).
 Besides integrating external stimuli, P1 neuronal activity is also influenced by the male’s internal drive state, in part through dopaminergic neurons (Zhang et al., 2016).
 Conversely, courtship decreases once males become sexually satiated, following mating with an abundance of female partners (Zhang et al., 2016). Sexual deprivation induces higher excitability of P1 neurons, while excitability of P1 neurons is down-regulated in sexually satiated flies (Inagaki et al., 2014). Proper activation of P1 neurons allows male flies to display courtship when external sensory cues from potential mates match with their internal drive states. However, we have a poor understanding concerning the identities of the neuromodulators and associated neurons that impact on the courtship decision center, which responds to mating experience.
 However, it is not known whether NPF is essential for male courtship, and the subsets of NPF neurons critical for courtship regulation have not been identified. Moreover, the neurons that interact with the sexually dimorphic NPF neurons to regulate courtship have not been defined.
Here, we show that a cluster of male-specific NPF neurons (NPFM) are essential for regulating male courtship. Disrupting NPF signaling, either by knocking out npf, or by suppressing the activity of NPF neurons, reduces inhibition on courtship behavior in sexually satiated males, and evokes hypersexual activity in deprived males towards inappropriate targets. By combining anatomical, chemogenetic manipulation and Ca2+ imaging approaches, we found that P1 neurons directly activate NPFM neurons, which then act through NPF receptor (NPFR) neurons to suppress male courtship.
Results
Disruption of NPF signaling elevates male courtship
To explore the potential function of NPF in modulating male sex drive, we first inhibited NPF neurons, and assayed its effects on male-female (M–F) and male-male (M–M) courtship. To inactivate NPF neurons, we employed three approaches in conjunction with the Gal4/UAS binary system (Brand and Perrimon, 1993). First, we used flies expressing Shibirets (Shits),which prevents synaptic transmission above 28 °C, due to depletion of synaptic vesicles (Grigliatti et al., 1973; Poodry and Edgar, 1979; van der Bliek and Meyerowitz, 1991; Kuromi and Kidokoro, 1998). We conducted the analysis using males that were group-housed with females, and experienced courtship and mating. Consequently, the males were sexually satiated and had lower courtship drive. When we disrupted synaptic transmission from NPF neurons, a higher percentage of males initiated courtship towards female targets, and their courtship index (ratio of time displaying courtship) was elevated significantly (Figure 1A and B). Courtship was not increased simply by raising the temperature, because flies expressing only the npf-Gal4 or the UAS-Shits exhibited similar courtship at both 23°C and 31°C (Figure 1A and B). At the restrictive temperature (31°C), npf-Gal4/+;UAS-Shits/+ males also vigorously engaged in courting other males and frequently formed courtship chains in which multiple males simultaneously court the preceding male (Huang et al., 2016) (Figure 1C). In contrast, we rarely detected chaining events among males kept at the permissive temperature (23°C; Figure 1C). We observed similar increases in M–M courtship when we inhibited the activity of NPF neurons by expressing a gene encoding either a hyperpolarizing K+ channel (Kir2.1) (Baines et al., 2001) or the diphtheria toxin gene, DTI (Han et al., 2000) (Figure 1—figure supplement 1A). Inhibition of NPF neurons with Kir2.1 or DTI also eliminated M–M lunges, indicating suppression of aggression (Figure 1—figure supplement 1B). Conversely, when we increased NPF activity by constitutively expressing a Na+ channel gene (NaChBac) (Nitabach et al., 2006) or by overexpressing the npf-cDNA (Wu et al., 2003) in NPF neurons, tester males exhibited elevated aggression (Figure 1—figure supplement 1B), but very few M–M courtship events (Figure 1—figure supplement 1A).
To clarify if it is the molecule NPF, rather than just NPF neurons, which is responsible for regulating sex drive, we generated two npf null mutants. To create npfLexA, we replaced the npf gene with the LexA reporter using CRISPR-HDR (Figure 1D and Figure 1—figure supplement 2A,B and C) . We also used the CRISPR-NHEJ method to generate an allele with a single nucleotide deletion, thereby changing the reading frame within codon 19, resulting in a null npf allele (npf1; Figure 1E and Figure 1—figure supplement 2B and E).
The courtship index of isolated control males reaches a ceiling level when they were exposed to mature active female targets (Huang et al., 2016). Therefore, to test whether npf mutant males exhibit an increase in courtship, we used mixed sex group-housed males, which in control flies showed a moderate level of courtship activity due to sexual satiation in the presence of an abundance of females. We found that mixed sex group-housed npf mutant males (npfLexA and npf1 and the trans-heterozygous npfLexA/npf1) retained high levels of courtship towards mature active female targets (Figure 1F and Figure 1—figure supplement 2F), indicating the mutants were resistant to sexual satiety induced by group-housing.
We found that the hypersexual activity in npf mutant males was generalized towards normally undesirable targets. These include females of other Drosophila species such as D. simulans females (Clowney et al., 2015) (Figure 1G), and male target flies (Figure 1H and Figure 1—figure supplement 2G). The increased courtship towards males was not due to an inability to discriminate between males and females. When the mutant males were allowed to choose between a decapitated male and a decapitated female, they showed a strong preference for female targets, similar to control males (Figure 1I).
To determine if this increase in courtship is an outcome of sensitized pheromone detection, we introduced newly-eclosed females, which carry negligible cuticular hydrocarbons and are therefore odorless/tasteless targets to tester males (Liu et al., 2011). We found that compared to control males, npf mutant males (npfLexA/npf1) exhibited significantly higher levels of courtship towards these females (Figure 1J). Thus, elevated courtship exhibited by npf mutant males did not appear to be caused by sensitized perception of attractive female pheromones. To test the possibility that the higher courtship levels was due to higher visual alertness in the mutants, we combined the males with motionless decapitated females as targets. Compared to control males, npf mutants exhibited increased courtship towards decapitated females (Figure 1K). To further establish that the courtship phenotype was due to loss of npf, we performed phenotypic rescue experiments with a wild-type npf genomic transgene (P[g-npf+], which restored npf expression to the npf mutant (Figure 1—figure supplement 2B—D). This genomic transgene also rescued normal levels of male courtship behavior to the npf mutant males (Figure 1F—H,J and K). Together, these experiments indicate that loss of npf function stimulates a sexually hyperactive state in males.
Sexually dimorphic NPFM neurons suppress male courtship
To examine the spatial distribution of the NPF neurons, we expressed lexAop-IVS-mVenus under the control of the LexA that we knocked into the npf gene (npfLexA/+). Among the neurons that were labeled by the npf reporter, was a bilaterally symmetrical cluster of NPF neurons that was male specific (NPFM; Figure 2A and B). The cell bodies of these sexually-dimorphic neurons are dorso-lateral to the antennal lobes and arborize extensively in the superior brain (Figure 2A and B). NPFM neurons can be differentiated from other NPF neurons based on their position in the anterior brain region that is immediately adjacent to antennal lobe. Moreover, NPFM form a cluster of 3— 5 neurons and their cell bodies are smaller than the pair of dorsal medial and the pair of dorsal lateral large NPF neurons. We used anti-NPF antibodies to immunostain the brain and found that the reporter expression pattern recapitulates the spatial distribution of the NPF protein (Figure 2C, c1-c6), confirming that NPFM neurons express NPF.
The fruitless (fru) gene is a master regulator of male courtship behavior, and its role is mediated through expression of a male-specific protein, FruM, which is produced through alternative mRNA splicing (Ito et al., 1996; Ryner et al., 1996; Lee et al., 2000; Demir and Dickson, 2005; Manoli et al., 2005; Stockinger et al., 2005). We examined whether NPFM neurons expressed the FruM protein by performing double labeling using anti-FruM, and anti-GFP, which marks the cells expressing mVenus driven by the npfLexA reporter.
To determine if FruM is essential for determining the fate of NPFM neurons, we used anti-NPF antibodies to stain the brains of fruFLP mutant (Yu et al., 2010) males. We found that staining of NPFM neurons was eliminated in fruFLP males (Figure 2E and F), indicating that specification of NPFM neurons depends on FruM.
To distinguish the projection pattern of NPFM neurons from the remaining NPF neurons, we used the FlpOut method (Wong et al., 2002) to specifically label NPFM neurons with mCitrine. In the absence of both fruFLP and npfLexA, neither mCherry nor mCitrine is expressed (Figure 3A). If the flies contain the fruFLP transgene but not the npfLexA transgene, the mCherry gene is removed due to expression of Flp (FlpOut), but mCitrine is not expressed (Figure 3B). In flies with npfLexA but no fruFLP, mCherry is expressed, but mCitrine is not expressed due to the transcriptional stop cassette downstream of the coding region for mCherry (Figure 3C). If flies harbor both the fruFLP and npfLexA transgenes, then mCherry is removed by FlpOut in fru-expressing neurons, and mCitrine is expressed (Figure 3D). Therefore, NPFM are the only neurons labeled with mCitrine.  In contrast, there were no mCitrine-labeled neurons in the female brain (Figure 3H). Rather, the NPF neurons in females were labeled with mCherry only (Figure 3I and J).
To address whether NPFM neurons are exclusively responsible for regulating male courtship, we expressed a conditional repressor or activator specifically in NPFM neurons. To inhibit NPFM neurons, we used the temperature sensitive Shits, which we expressed in NPFM neurons only using the FlpOut method. We employed a transgene that encodes Shits downstream of a 5’ transcriptional stop cassette that is flanked by FRT sites ( UAS > stop > Shits ; note that ‘ >” indicates FRT sites), and removed the stop cassette specifically in fru neurons by expressing flippase exclusively in fru neurons with the fruFLP. After the stops are removed, we expressed UAS-Shits under control of the npf-Gal4, thereby restricting Shits to NPFM neurons only. When we performed courtship assays at the non-permissive temperature for Shits (31 °C), the males showed elevated courtship relative to flies with the same genotype that were assayed at the permissive temperature (23°C) for Shits (Figure 4A). We then tested the effects of inhibiting neurons except for NPFM neurons, using npf-Gal4/+;fruFLP/ UAS > Shits > stop flies, which removes Shits just in NPFM neurons. These males displayed similar levels of courtship at both the permissive temperature and non-permissive temperatures for Shits (Figure 4A).
To activate NPFM neurons, we employed a similar FlpOut approach, using UAS > stop > trpA1/npf-Gal4; fruFLP/ +flies, to express the thermally-activated TRPA1-A isoform in NPFM neurons only. This TRPA1 isoform is a Na+ and Ca2+-permeable channel, which is activated at temperatures above ~ 27 °C (Viswanath et al., 2003). To perform these assays, we used decapitated females since intact females stimulate ceiling levels of male courtship, which are resistant to down-regulation, while decapitated females induce moderate levels, which facilitate detecting subtle decreases in male courtship. We found that courtship levels in UAS > stop > trpA1/npf-Gal4;fruFLP/+ males were suppressed at 29°C relative to 23°C (Figure 4B). In contrast, none of the three types of control flies exhibited lower male courtship at 29°C (Figure 4B). Nevertheless, because the CI exhibited by the UAS > stop > trpA1/npf-Gal4;fruFLP/+ males at 29°C was not elevated relative to the CIs displayed by the control males at 29°C, the results preclude the conclusion that activation of sexually dimorphic NPFM neurons inhibits male courtship.
To test whether the NPF produced in NPFM neurons is responsible for inhibiting male courtship, we knocked down NPF expression in distinct groups of neurons. To conduct these experiments, we used UAS-npf-RNAi, which was effective as it greatly reduced NPF levels (Figure 4C—E). We found that knocking down npf expression with the fru-Gal4 induced a dramatic increase in M–M courtship, and did so to a similar extent as when we used a pan-neuronal (elav) Gal4 or the npf-Gal4 (Figure 4F).
To specifically interrogate a requirement for NPF in NPFM neurons, we used FlpOut to introduce Gal80 (which binds and inhibits Gal4 activity) in either fru+ or fru- neurons, thereby confining UAS-npf-RNAi expression to fru- NPF neurons or NPFM neurons, respectively. To knockdown npf specifically in NPFM neurons, we used the following flies that cause excision of Gal80 in fru neurons only, thereby allowing Gal4 expression and RNA knockdown in NPFM neurons: npf-Gal4/tub > Gal 80 >stop;UAS-npf-RNAi/fruFLP flies. Conversely, to prevent npf knockdown in NPFM neurons, we expressed Gal80 specifically in these neurons using npf-Gal4/tub > stop > Gal80;UAS-npf-RNAi/fruFLP flies. We found that knocking down npf exclusively in NPFM neurons elevated M–M courtship while npf knock down in fru- NPF neurons did not change the level of male courtship (Figure 4F). These results indicate that sexually dimorphic NPFM neurons are the subset of NPF neurons that are exclusively required for suppressing male courtship, and the effect is dependent on NPF produced in NPFM neurons.
P1 neurons directly activate NPFM neurons
To address how NPFM neurons are integrated into the fru circuit, we adopted the GRASP (GFP Reconstitution Across Synaptic Partners) (Feinberg et al., 2008; Gordon and Scott, 2009) method to detect potential contact loci between NPF neurons and fru neurons. This approach employs a dual binary expression system to synthesize two complementary but non-functional parts of GFP (spGFP1-10 and spGFP11) on the cell membranes of distinct neurons. When the neurons are in close proximity, GFP is reconstituted and fluorescence is produced. We expressed spGFP1-10 and spGFP11 in fru and NPF neurons, respectively and detected strong bouton-shaped GFP signals in the male brain (Figure 5A) but only sparse signals in the female brain (Figure 5B) and no specific reconstituted GFP signals in control male brains missing the driver for the LexAop-spGFP11 (Figure 5C). The reconstituted GFP signals in the male brain reconstruct a distinctive male-specific brain structure – the lateral protocerebral complex (LPC), which includes several neuropils: the lateral junction, superior medial protocerebrum (SMPr) arch, lateral crescent and the ring structure (Figure 5—figure supplement 1A) (Yu et al., 2010).
To compare the projection patterns of NPF and P1 neurons, we expressed GFP and RFP in NPF and P1 neurons, respectively, using two binary expression systems. We found that the projections from NPF neurons overlapped with the LPC structure in the male brain (Figure 5—figure supplement 2A—C). However, the female brain does not include an LPC structure (Figure 5—figure supplement 2D—F). We further combined the FlpOut method and dual binary expression systems to exclusively label NPFM and P1 neurons, and found that the projections from these two clusters of neurons overlapped intensely in LPC region (Figure 5D—F and Figure 5—figure supplement 1 and Video 2).
To address if the projections of NPF and P1 neurons form direct connections, we used the R71G01-Gal4 (which is expressed in P1 neurons and a few other neurons) to drive expression of spGFP1-10, and npfLexA to drive expression of spGFP11.  The GRASP GFP signals appear to be due to expression of the two parts of the split GFP in NPFM and P1 neurons for the following reasons. First, NPFM and P1 neurons are both male-specific, and the GRASP signals are primarily in the male brain and not in the female brain (Figure 5G and H). Second, the GRASP signals label two LPC structures: the lateral junction and SMPr arch (Figure 5G). Third, the projections of NPFM and P1 overlap extensively in the lateral junction and SMPr arch (Figure 5D—F and Video 2), while fru- NPF projections do not innervate the LPC region (Figure 3F and G and Video 1). Thus, the GRASP signals in the LPC structure appear to be formed by connections between NPFM and P1 neurons.
To clarify the directionality of the synaptic connections between NPFM and P1 neurons, we employed genetically encoded markers to label the dendritic (UAS-DenMark) and axonal (UAS-syt::eGFP) branches of NPF and P1 neurons (Wang et al., 2007; Nicolaï et al., 2010). The P1 neurons that extend processes to the lateral junction and SMPr arch within the LPC structure were stained with both Denmark and Syt::eGFP, suggesting that P1 neurons send and receive signals within these neuropils (Figure 5—figure supplement 3A, a1-a6). However, in the corresponding lateral junction and SMPr arch within the LPC region, NPF neurons were labeled with DenMark only (Figure 5—figure supplement 3B, b1-b3), suggesting that NPF neurons mainly receive signals within this region. The NPF axons that stained with Syt::eGFP occurred in several brain regions other than the LPC region (Figure 5—figure supplement 3B, b1-b6).
To distinguish the boutons formed by NPFM neurons from other NPF neurons, we used the FlpOut approach to specifically label projections of NPFM neurons. We stained the brains of male UAS > stop > mCD8::GFP/+;fruFLP/npf-Gal4 flies (Yu et al., 2010) with anti-GFP and anti-NPF so that the boutons formed by NPFM neurons would be double labeled. We found that the double-labeled boutons were concentrated in the medial anterior brain, but not in the lateral superior brain (Figure 5—figure supplement 3C, c1-c6), indicating that the release site of NPFM neurons was outside the LPC region. These results demonstrate that NPFM neurons do not directly act on P1 neurons. Rather, the synaptic connections between NPFM and P1 neurons in the LPC region are formed by pre-synaptic P1 neurons and post-synaptic NPFM neurons.
To determine the impact of activation of P1 neurons on the activity of the NPFM neurons, we combined chemogenetics and GCaMP imaging to monitor Ca2+ dynamics (Yao et al., 2012) as an indicator of neural activation. We expressed P2X2 (encoding an ATP-gated cation channel) (Lima and Miesenböck, 2005) in P1 neurons, and expressed GCaMP3 in NPF neurons. We used R71G01-LexA, which is expressed in P1 neurons and a few other neurons, to drive P2X2 expression, and npf-Gal4 to drive UAS-GCaMP3. In a complementary experiment, we switched the two binary systems, and used the R71G01-Gal4 and npfLexA to drive P2X2 and GCaMP3, respectively. Because the diffusion rate and final concentration of ATP that reaches the brain varies across samples, we calculated the maximum fold changes of the GCaMP3 responses after ATP application relative to the basal levels of GCaMP3 before ATP application. We found that ATP-induced activation of P1 neurons led to robust GCaMP3 signals in NPFM neurons (Figure 6A—C and Figure 6—figure supplement 1 and Videos 3 and 4).
In order to exclude the impact from other neurons, we expressed P2X2 in P1 neurons only using a split-P1-Gal4 comprised of R15A01-AD (activation domain) and R71G01-DBD (DNA-binding domain). We imaged Ca2+ dynamics in NPFM neurons in response to ATP application, and detected large increases in GCaMP3 fluorescence in response to activation of P1 neurons (Figure 6D—F), further supporting the conclusion that P1 neurons directly activate NPFM neurons.
Increase in courtship by inhibiting NPFM neurons depends indirectly on P1 neurons
To determine whether the function of NPFM neurons in courtship regulation is dependent on P1 neurons, we tested if silencing P1 neurons would prevent the courtship elevation induced by disruption of NPF neurons. We expressed UAS-Shits in both NPF and P1 neurons (npf-Gal4 and R71G01-Gal4) and assayed male courtship at both permissive and non-permissive temperatures. We found that the courtship dis-inhibition caused by disrupting NPF neurons was eliminated by simultaneous disruption of P1 neurons (Figure 7A—C). The results suggest that NPFM neurons appear to act through P1 neurons to regulate male courtship. Alternatively, NPFM and P1 neurons may act in parallel and serve opposing inputs onto a common neuronal target.
NPF binds to a G protein-coupled receptor—the NPF receptor (NPFR), which couples to a Gi signaling pathway to inhibit npfr-expressing neurons (Garczynski et al., 2002). To address the roles of the npfr gene and NPFR neurons in regulating male courtship, we replaced a portion of the npfr coding region with LexA, thereby generating an npfr mutant and a reporter (Figure 7—figure supplement 1). We then used the R71G01-Gal4 and npfLexA/+ to label P1 neurons and NPFR neurons with GFP and mCherry, respectively. We found that they primarily stain distinct neuronal populations (Figure 7D—F), indicating that P1 neurons are not the npfr-expressing neurons. These results further support our data suggesting that NPFM axons do not send signals directly to P1 dendrites, but that P1 neurons signal to NPFM neurons.
We assayed courtship behavior of npfrLexA mutant flies, demonstrating that these mutant animals raised in isolation exhibit significantly higher M–M courtship than control males (Figure 7G). We observed similar results with npfrLexA/npfrc01896 trans-heterozygous flies (Figure 7G). RNAi-mediated knockdown of npfr using a pan-neuronal Gal4 (elav) also increased M–M courtship behavior (Figure 7H). In contrast, knocking down npfr expression in P1 neurons had no effect (Figure 7H).
We took advantage of the GRASP method to investigate whether NPFR and P1 neurons make direct connections. We used R71G01-Gal4 and npfrLexA drivers to express spGFP1-10 and spGFP11 respectively. We detected GRASP signals in the lateral crescent within the LPC region of the male brain (Figure 8A,a1,a2). In contrast, we did not detect GRASP GFP fluorescence in female brains or in control male brains (Figure 8B,b1,b2 and Figure 5I).
To examine whether activation of NPFR neurons affects the activity of P1 neurons, we expressed P2X2 in NPFR neurons, and GCaMP3 in P1 neurons. We found that activation of NPFR neurons with ATP application induced robust GCaMP3 responses in the LPC structure (Figure 8C—E and Video 5). In control flies that did not express P2X2 , application of ATP did not induce elevation of GCaMP3 fluorescence (Figure 8E). The preceding results indicate that at least a subset of NPFR neurons anatomically connect and functionally activate P1 neurons. Together, our results indicate that NPFM, NPFR and P1 neurons form intricate interactions, and ensure proper courtship output in accordance with a male’s internal drive state.
 In contrast, much less is known about how the P1 neurons are regulated by the male’s prior mating experience (Inagaki et al., 2014) and how courtship is affected by the internal drive state. An exception is a recent study that identified a group of dopaminergic neurons that changes in activity in proportion to male mating drive, and which directly activates P1 neurons to promote male courtship (Zhang et al., 2016). In the current study, we characterized a cluster of male-specific NPFM neurons which functions antagonistically to dopamine neurons by serving to suppress courtship by responding to sexual satiation. Disruption of NPFM neurons causes dis-inhibition of courtship in satiated males. The internal drive state of males is encoded by opposing excitatory and inhibitory inputs, which enable a male to make an appropriate mating decision in accordance with its internal drive state.
Suppression of NPF neurons or elimination of npf counters sexual satiation
Elimination of npf or knocking down npf expression exclusively in male-specific NPFM neurons causes male flies to exhibit maladaptive, hypersexual activity. In contrast to control males, which are sexually satiated when exposed to an abundance of females, and consequently display very low courtship levels, we found that flies overcome the sexual satiation imposed by mating if we introduce a loss-of-function mutation in npf or inhibit NPF neurons. Thus, satiation of courtship is dis-inhibited by disrupting NPF signaling.
Our findings that suppressing or eliminating NPF neurons elevates male courtship is in contrast to a previous report that genetic disruption or feminization of NPF neurons reduces male courtship activity (Lee et al., 2006). Maintaining males in the presence or absence of females profoundly affects sexual satiation levels, and the housing conditions were not clearly defined in this previous study. Our conclusions are supported by multiple lines of evidence. First, we found that when we inhibit neurotransmission from NPF neurons, using a temperature sensitive dynamin (Shits), the males showed a dramatic increase in courtship towards female conspecifics. This occurred using group-housed males which normally are sexually satiated. Second, introduction of a genetically encoded toxin, or inhibition of NPF neurons by overexpression of a K+ channel, also increases courtship activity. Third, when we disrupted the npf gene, the mutant males displayed a remarkable increase in courtship. This effect was so profound that the males courted females of another species and also displayed a great increase in M–M courtship, even though their gender preferences remained unchanged. Fourth, disruption of the npfr gene resulted in significant elevation in courtship, consistent with the effect of disrupting npf. Fifth, when we specifically silenced male-specific fru+ NPF (NPFM) neurons, male courtship behavior was elevated. In contrast, silencing fru- NPF neurons had no impact on male courtship. Sixth, knocking down npf expression exclusively in NPFM neurons increased male courtship, while knocking down npf in fru- NPF neurons had no effect.
Neuronal circuit models entail P1 neurons activating NPFM neurons
Our anatomical, physiological and functional evidence demonstrate that P1 neurons activate NPFM neurons, and suggest potential models through which these neurons coordinate to regulate male courtship drive. According to one model, P1 and NPFM neurons form a recurrent inhibitory neuronal circuit (Figure 8F). Stimulation of P1 neurons activates NPFM neurons, which act through an intermediate group of NPF receptor (NPFR neurons) and feedback to inhibit P1 neurons. This recurrent inhibitory model posits that P1 neurons are strongly activated when males are exposed to many females, inducing NPFM neurons to release NPF. This neuropeptide acts on the Gi-coupled NPF receptor and inhibits NPFR neurons, leading to a suppression of P1 activity, and attenuation of male courtship. When the activity of P1 neurons is reduced, stimulation of NPFM neurons and NPF release are diminished. This attenuates the feedback inhibition from NPFM to P1 neurons, leading to a return of P1 neuronal activity, and male courtship drive.
We suggest that the recurrent inhibitory neuronal motif proposed here is important for maintaining proper activities of P1 neurons, thus ensuring appropriate behavioral choices that are critical for a male’s reproductive success, depending on the level of sexual satiety.
The recurrent inhibitory neural motif proposed here is dedicated to ensure appropriate activation of P1 neurons. Disruption of the inhibitory NPF afferents leads to excessive courtship behavior in the male fly that is maladaptive, as it overrides the courtship inhibition normally imposed by recent mating with females, other males, or females of other Drosophila species.
Recurrent inhibitory neural motifs are important in the central nervous system. In the mammalian spinal cord, motor neurons send collateral branches to Renshaw cells, which in turn send inhibitory signals back to motor neurons (Alvarez and Fyffe, 2007). The function of this recurrent inhibition is assumed to restrict excessive activation of motor neurons and contribute to precise recruitment of muscle fibers in order to generate proper force for different tasks (Alvarez and Fyffe, 2007). Recurrent inhibitory loops also occur in the hippocampus and entorhinal cortex. In these systems, principal cells send excitatory outputs to fast-spiking, parvalbumin-positive interneurons, and at the same time receive inhibitory inputs from these interneurons, thus, closing the feedback inhibition loop (Pouille and Scanziani, 2004; de Almeida et al., 2009; Pastoll et al., 2013).
While NPFM, P1 and NPFR neurons are essential for regulating courtship by responding to prior mating experience, and may do so through a recurrent inhibitory loop (Figure 8F), our data do not exclude other models. Part of the argument in favor of the recurrent inhibitory loop model is that the GRASP analysis suggests that NPFR neurons make direct connections with P1 neurons. Moreover, by coupling chemogenetic manipulation and Ca2+ imaging, we found that activation of NPFR neurons activate P1 neurons. However, NPFR neurons are widely distributed, and our data do not resolve whether the NPFR neurons that activate P1 neurons are the same subset of NPFR neurons that are the direct downstream target of NPFM neurons. Thus, one alternative to the recurrent inhibitory motif is a feedforward parallel model, in which target neurons (X neurons) control courtship drive by receiving parallel input from P1 neurons and NPFR neurons (Figure 8G). This latter model posits that P1 neurons activate X neurons, and at the same time, send axonal branches to activate NPFM neurons, which then act through NPFR neurons and suppress the target neurons through a feedforward mechanism. Future experiments that resolve the anatomical and functional diversity of NPFR neurons should distinguish between the recurrent inhibitory versus feedforward parallel model, which ensure proper courtship output in accordance with a male’s internal drive state.
Impact of NPF activity on courtship versus aggression
Courtship and aggression are closely interrelated social behaviors. If males are housed in isolation, they exhibit elevated courtship and aggression (Wang et al., 2008; Liu et al., 2011). This positive relationship is consistent with the observation that the presence of a potential mate promotes a male fly’s propensity to fight a competitor to win a mating competition (Kravitz and Fernandez, 2015). Though the tendency to fight or to court is positively related, the behavioral choice between courtship and aggression is mutually exclusive.
We found that when we disrupt the activity of NPF neurons, M–M courtship is dominant over aggression. We suggest that loss of NPF function diminishes inhibition of P1 neurons. As a result, even sub-optimal stimuli strongly activate P1 neurons and induce male courtship behavior even towards inappropriate targets. Conversely, when we increase the activity of NPF neurons or over-express the npf-cDNA in NPF neurons, M–M aggression is dominant over courtship.
The precise contribution of NPF neurons in regulating aggression is unresolved. One group found that activation of NPF neurons elevates male aggression (Asahina et al., 2014) while another reported that silencing or feminizing NPF neurons elevates aggression (Dierick and Greenspan, 2007). We found that when we overexpressed either the Na+ channel NaChBac or the npf-cDNA in NPF neurons, the males exhibited increased aggression. We propose that excessive NPF activity suppresses P1 neurons, thereby setting a high threshold for P1 activation. Our observations are consistent with previous report that weaker activation of P1 neurons favors aggression while stronger activation of P1 neurons favors courtship (Hoopfer et al., 2015). It remains to be determined if NPF neurons also impact on the aggression modulatory or arousal center (Asahina et al., 2014; Watanabe et al., 2017), independent of its effect on P1 neurons.
Possible relationship of NPF to courtship regulation by mammalian NPY
NPF is the Drosophila counterpart of mammalian NPY, which regulates feeding, reproduction, aggression, anxiety, depression and the alcohol addiction (Nässel and Wegener, 2011).  The discovery that Drosophila NPF regulates courtship depending on the internal drive state raises questions as to whether NPY may serve similar functions in mammals.
Materials and methods
Key resources
Descriptions of the key fly strains, antibodies, plasmids, chemicals, kits, services and software are provided in the Supplementary file 1.
Fly stocks
The following strains were obtained from Bloomington Stock Center (Indiana University): npf-Gal4 (#25681, and #25682 have identical promoters, but are inserted on the 2nd and 3rd chromosomes, respectively), elav-Gal4 (#8765), fru-Gal4 (NP21 #30027), R71G01-Gal4 (P1-Gal4 #39599), R71G01-LexA (P1-LexA #54733), UAS-NaChBac (#9468), UAS-Kir2.1 (#6596), UAS-DTI (#25039), UAS-mCD8::GFP (#5137), UAS-npf-RNAi (VDRC108772), UAS-npfr-RNAi (VDRC107663), UAS-DenMark,UAS-syt::eGFP (#33064), LexAop-mCherry (#52271), LexAop(FRT.mCherry)ReaChR-mCitrine (#53744), UAS-IVS-mCD8::RFP, LexAop-mCD8::GFP (#32229), UAS-CD4-spGFP1-10,LexAop-CD4-spGFP11 (#58755), LexAop-IVS-CsChrimson.mVenus (#55139), Lexop(FRT.stop)myr::smGdP-V5 (#62107) npfrc01896 (#10747), tub(FRT.Gal80)stop (#38880), tub(FRT.stop)Gal80 (#38878).
UAS-npf was a gift from Dr. Ping Shen (Wu et al., 2003) (University of Georgia), UAS- P2X2,LexAop-GCaMP3 and UAS-GCaMP3, LexAop P2X2 were from Dr. Orie Shafer (Yao et al., 2012) (University of Michigan), UAS-Shibirets was from Dr. Christopher Potter (Kitamoto, 2001) (Johns Hopkins University School of Medicine), fruFLP, UAS-(FRT.stop)mCD8::GFP, UAS-(FRT.stop)Shibirets and UAS-(FRT.Shibirets)stop, UAS-(FRT.stop)dTRPA1 were from Dr. Barry Dickson (Yu et al., 2010) (Janelia Research Campus), R71G01-DBD;R15A01-AD was from Dr. David Anderson (California Institute of Technology).
The npfLexA and npfrLexA mutants were outcrossed into a w1118 background for five generations. The controls for comparison to these mutants were w1118 flies in which we exchanged the X chromosome with Canton-S so the flies are w + on the X chromosome (w1118-CS flies). The full genotypes of the flies used in each figure and video are listed in Supplementary file 2.
Behavioral assays
The behavioral assays were recorded using a Samsung SCB-3001 camera. All behavioral analyses were performed using these videos.
M–F, and M–M courtship assays
To perform courtship assays, we added 3 ml of 1.5 % agarose into each well of 24-well cell culture plates (Corning Incorporated, REF353847). 2 mm diameter holes were drilled on the cover over each well. Custom silicone plugs were prepared (435570, StockCap) for blocking the holes. The cover and the plate were taped together to avoid gaps that might allow flies to escape.
Unless otherwise specified, 5—7 days old mixed sex, group-housed males (10 males raised together with 30 virgin w1118 females for 3 days) were used for the courtship assays. Three types of female targets were used: 1) mature active females, 2) newly-eclosed females, or 3) decapitated females. In experiments in which the targets were either grouped-housed w1118 males or Drosophila simulans females, we used 5—7 day old isolation-housed males as the testers. One tester male and one target were ice anesthetized, and transferred together into courtship chambers. The flies were allowed to recover for 10 min, and then male courtship was scored over the next 10 min. The courtship index is the fraction of time that a tester male performs courtship towards the target.
To test the effects of inhibiting npf neurons with Shits, a single tester male (npf-Gal4/+;UAS-Shits/+) and a target female (mature, active w1118, 5—7 days old) were ice-anesthetized, and the pair was transferred into courtship chambers. The assays were performed at 23°C and 31°C, which are the permissive and non-permissive temperatures for Shits, respectively. Courtship indexes were calculated based on 20—30 min observation during a 30 min incubation period.
Male chaining assays
We inserted newly-eclosed tester males into individual vials, and aged them for 5—7 days. We introduced 8—12 males into a 35 mm Petri dish, which was filled with 8 ml 1.5% agarose through a 2 mm diameter hole drilled on the cover. We allowed the flies to recover for 5 min, and then determined the ratio of time over the next 10 min in which ≥ 3 flies engaged in simultaneous courtship (chaining index).
M–M aggression assay
The aggression assays were carried out as described previously (Zhou et al., 2008), using 5—7 day-old isolation-housed tester males, and 5—7 days group-housed w1118 males as the targets. Briefly, one tester was paired with one target in the assay. The custom-designed chambers were based on previous reports (Zhou et al., 2008; Liu et al., 2011), and were fabricated by the Physics Machine Shop at UCSB (Figure 1—figure supplement 3). The chamber consists of two concentric circular chambers. The outer chamber diameter and height are 13 mm and 7 mm, respectively. The inner chamber diameter and height are 8 mm and 3.5 mm, respectively. The outer and inner chambers are separated by 0.5 mm thick, 3.5 mm high walls. 0.3 ml standard corn meal and molasses fly food was added to the inner chamber. 1.5% agarose was used to fill the space between inner and outer chambers. The heights of the food and agarose patches were the same (3.5 mm). We then dissolved 15% sucrose and 15% yeast in apple juice, and added 15 μl liquid to each food patch. Once the liquid mixture has soaked into the food, and the patch is dry at the surface, the aggression chamber is ready to use. w1118 male targets were transferred to the chamber by ice-anesthetization. A 22 × 22 mm microscope cover glass (Fisher Scientific) was used to cover to the chamber. The targets were allowed to recover for 10 min, and the isolation-housed tester males were introduced into the chamber by gentle tapping. After waiting 5 min for the tester males to recover, we scored the number of lunges during the following 15 min.
Male and female preference assay
To test the preference of a male tester for females versus males, we placed one decapitated w1118 virgin female and one decapitated w1118 male in a courtship chamber. The tester males were isolation-housed for 5—7 days since eclosion, and transferred into the chamber by gentle tapping. After 5 min recovery time, we scored the time during which the tester male performed courtship behavior towards either the decapitated female or the decapitated male target over the course of 10 min. The preference index is the ratio of time that male testers spend courting decapitated female targets out of the total courtship time.
Molecular biology
Generation of npf1 strain
To generate the npf1 allele (Figure 1E) we used the CRISPR mediated NHEJ (clustered regularly interspaced short palindromic repeats – non-homologous end joining) method (Kondo and Ueda, 2013; Ren et al., 2013).
We designed the following oligonucleotides:
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npf-gRNA1-f: 5’ CTTCGCCCTTGCCCTCCTAGCCGC 3’
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npf-gRNA1-r: 5’ AAACGCGGCTAGGAGGGCAAGGGC 3’
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npf-gRNA2-f: 5’ CTTCGTTGCCATGGTCGTCTAAAA 3’
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npf-gRNA2-r: 5’ AAACTTTTAGACGACCATGGCAAC 3’
We annealed the oligonucleotides to obtain two independent dimers, and ligated the primer dimers into the BbsI site of pU6-BbsI-ChiRNA BbsI (Addgene #45946). The pU6-BbsI-npf-gDNA1 and the pU6-BbsI-npf-gDNA2 plasmids were co-injected into the BDSC strain #51324 as the Cas9 source (BestGene Plan R). Based on DNA sequencing, we found that npf1 harbored a single nucleotide frameshift deletion that changed the 2nd position of codon 19.
Generation of npfLexA strain
To generate the npfLexA line with an insertion of the LexA reporter (Figure 1D), we used the CRISPR-HDR (clustered regularly interspaced short palindromic repeats – homology directed repair) method (Kondo and Ueda, 2013; Ren et al., 2013). We chose upstream and downstream guide RNAs that targeted the npf coding sequences using the CRISPR Optimal Target Finder: http://tools.flycrispr.molbio.wisc.edu/targetFinder/.
We annealed the following upstream and downstream primer dimers, which we inserted into the BbsI site of pU6-BbsI-ChiRNA (Addgene #45946).
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npf_up_ChiRNA_F: 5’ CTTCCCAAACAATGCGTTGCATCC 3’
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npf_up_ChiRNA_R: 5’ AAACGGATGCAACGCATTGTTTGG 3’
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npf_down_ChiRNA_F: 5’ CTTCAGATTTATGTTACAGGCTCG 3’
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npf_down_ChiRNA_R: 5’ AAACCGAGCCTGTAACATAAATCT3’
We amplified the npf upstream (1359 bp, nucleotides 3 R:16609779 to 16611137, release = r 6.16) and downstream (1388 bp, nucleotides 3 R:16610753 to 16612140, release = r 6.16) homology arms using the following primers:
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npf_LA_KpnI_F: 5’ GACGCATACCAAACGGTACCCATTGTGACACCGTTGCGCTTTCCA 3’
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npf_LA_KpnI_R: 5’ TTTTGATTGCTAGCGAGTTCTATAAATGGCTAATGTATGT 3’
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npf_RA_NdeI_F: 5’ CTAGGCGCGCCCATATGTCGCGGTTTTAATGAGGAGGAGATATTC 3’
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npf_RA_NdeI_R: 5’ GACAAGCCGAACATATGATCGGACTTGGACGTGGTAAGCCAA 3’
We used the In-Fusion cloning kit (Clontech) to clone the upstream and downstream homology arms into the KpnI and NdeI sites of pBPLexA::p65Uw (Addgene #26231), respectively.
The pU6-BbsI-ChiRNA-npf_up, pU6-BbsI-ChiRNA-npf_down, and pBPLexA::p65Uw-npf_LA + RA plasmids were injected into the BDSC #51323 strain, which provided the source of Cas9 (BestGene Plan R).
We used the following primers to genotype the transformants (as shown in Figure 1—figure supplement 2A):
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npf[LexA]LA_GT_F (P1): 5’ CTTTCGGCCAACATTTATTCACG 3’
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npf[LexA]LA_GT_R (P2): 5’ AAAGCCCAGTCGCTGTGCTATCT 3’
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npf[LexA]RA_GT_F (P3): 5’ TCAAATACCCTTGGATCGAAGTA 3’
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npf[LexA]RA_GT_R (P4): 5’ AGGGCTGCTGTAAGTATCGGTTG 3’
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npf_Deletion_F (P5): 5’ CTTCCCAAACAATGCGTTGCATCC 3’
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npf_Deletion_R (P6): 5’ AAACCGAGCCTGTAACATAAATCT 3’
Generation of npfrLexA strain
We employed CRISPR-HDR (Kondo and Ueda, 2013; Ren et al., 2013) to generate the npfrLexA mutant with the LexA knockin. We chose the upstream and a downstream guide RNAs targeting the third exon using the CRISPR Optimal Target Finder.
We annealed the following upstream and downstream primer dimers, which we cloned into the BbsI site of pU6-BbsI-ChiRNA (Addgene #45946).
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npfr_up_ChiRNA_F: 5’ CTTC GCAGATGGGGAGCATCTGAG 3’
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npfr_up_ChiRNA_R: 5’ AAAC CTCAGATGCTCCCCATCTGC 3’
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npfr_down_ChiRNA_F: 5’ CTTC ATTGCGAGCAGTGCGCATGA 3’
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npfr_down_ChiRNA_R: 5’ AAAC TCATGCGCACTGCTCGCAAT 3’
We amplified the npfr upstream (1426 bp, nucleotides 3 R:6190969 to 6192394, release = r 6.16) and downstream (1250 bp, nucleotides 3 R:6192051 to 6193300, release = r 6.16) homology arms using the following primers:
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npfr_LA_KpnI_F: 5’ GACGCATACCAAACGGTACC TGTGCTGCATAAATTACGGCGACGG 3’
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npfr_LA_KpnI_R: 5’ TTTTGATTGCTAGCGGTACC AGATGCTCCCCATCTGCCAGCTGGG 3’
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npfr_RA_NdeI_F: 5’ CTAGGCGCGCCCATATG TGCGCACTGCTCGCAATCTGTTCAT 3’
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npfr_RA_NdeI_R: 5’ GACAAGCCGAACATATG CGCGCCCACGAACTGCAGGC 3’
We used the In-Fusion cloning kit (Clontech) to clone the upstream and downstream homology arms into the KpnI and NdeI sites of pBPLexA::p65Uw (Addgene #26231).
The pU6-BbsI-ChiRNA-npf_up, and pU6-BbsI-ChiRNA-npf_down, pBPLexA::p65Uw-npf_LA + RA plasmids were co-injected into the BDSC #55821 strain (BestGene Plan R), which provided the source of Cas9.
We used the following primers to genotype the transformants (as shown in Figure 7—figure supplement 1):
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npfr[LexA]LA_GT_F (P1): 5’ CATGTCTCGCCTTGATGTGCTGC 3’
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npfr[LexA]LA_GT_R (P2): 5’ AAAGCCCAGTCGCTGTGCTATCT 3’
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npfr[LexA]RA_GT_F (P3): 5’ TCAAATACCCTTGGATCGAAGTAAA 3’
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npfr[LexA]RA_GT_R (P4): 5’ CACAGCGAGAAGATCGAGTAGTAGAA 3’
The following primers were used to amplify npfr cDNA, which we obtained by performing RT-PCR using mRNA extracted from npfrLexA and control flies:
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npfr_Deletion_F1 (P5): 5’ CACCTCGGATCTGAATGAGACTGG 3’
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npfr_Deletion_R1 (P6): 5’ AGACGATTAGCACGCCGTACATG 3’
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npfr_Deletion_F2 (P7): 5’ CACCCTGGTTGTTATAGCCGTCAT 3’
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npfr_Deletion_R2 (P8): 5’ ACGCACAGCGAGAAGATCGAGTAG 3’
Generation of P[g-npf+] transgenic flies
We obtained a plasmid covering 20,306 bp of the npf genomic region from P[acman] Resources (http://www.pacmanfly.org/libraries.html). The P[acman] BAC CH322-163E17 plasmid, and a plasmid source of phiC31 were co-injected into a strain (BDSC #9723) with an attP40 site (BestGene Plan H).
Immunohistochemistry
Fly brains were dissected in ice-cold phosphate-buffered saline (PBS, pH 7.4, diluted from a sterile filtered 10x PBS stock, cat#:119-069-131, Quality Biological, Inc. 1x working concentration contains 137 mM NaCl, 2.7 mM KCl, 2 mM KH2PO4, 8 mM Na2HPO4) and fixed in 4 % paraformaldehyde in PBST (0.3 % Triton X-100 in PBS) at room temperature for ~ 20 min. Brains were washed three times in PBST for 20 min each time, and blocked in 5 % normal goat serum in PBST for 1 hr. The brains were incubated with primary antibodies diluted in 5 % normal goat serum in PBST for 24 hr at 4 °C. Samples were washed three times with PBST before applying secondary antibodies for 3 hr at 25 °C in darkness. After washing three times with PBST, the samples were mounted with VectaShield (Vector Labs) on glass slides.  The secondary antibodies were AlexaFluor 488 goat anti-chicken (1:1000; Invitrogen, A-11039), AlexaFluor 488 goat anti-rat (1:1000; Invitrogen, A-11006), AlexaFluor 568 goat anti-rabbit (1:1000; Invitrogen, A-11011), AlexaFluor 633 goat anti-mouse (1:1000; Invitrogen, A-21050), Rhodamine Red-X goat anti rabbit IgG (1:1000; Molecular Probe, R6394). We adapted a previously described method for anti-V5 and anti-GFP double staining (Nern et al., 2015). Briefly, we first used chicken anti-GFP as the primary antibodies (1:1000, Invitrogen, A-10262) for 24 hr 4 °C. We washed the brains three times with PBST, and then added AlexaFluor 488 goat anti-chicken IgG (1:1000; Invitrogen, A-11039) and DyLight549 tagged mouse anti-V5 antibodies (1:500 DyLight549 tagged, MCA2894D549GA BioRad). The brains were incubated at 25 °C for 3 hr in darkness, washed three times in PBST, and mounted with VectaShield (Vector Labs) on glass slides. We performed the imaging using a Zeiss LSM 700 confocal microscope, and processed the images using ImageJ.
GRASP analysis
To detect native GRASP GFP fluorescence in brains, we used flies aged for ~ 20 days to enhance the reconstructed GFP signals. We dissected the brains in ice-cold PBS, fixed the tissue for 20 min in 4 % paraformaldehyde in PBST at 25 °C, washed three times with PBST, and mounted the brains in PBS for imaging the native fluorescent signals.
Ex vivo Ca2+ imaging
We dissected brains from 7 to 15 day-old males (separated from females for 5 days, raised in ~ 10 male-only group) in cold Drosophila imaging saline (108 mM NaCl, 5 mM KCl, 2 mM CaCl2, 8.2 mM MgCl2, 4 mM NaHCO3, 1 mM NaH2PO45 mM trehalose, 10 mM sucrose, 5 mM HEPES, pH = 7.5 (Inagaki et al., 2014), transferred individual brains to 35 mm plastic Petri dishes (35 3001 Falcon), attached the brain down to the bottom of the dish with a slice harp (SHD-26GH/10, Warner Instruments), and bathed each brain in 2 ml Drosophila imaging saline. We imaged the Ca2+ dynamics using a Zeiss LSM 700 confocal microscope. The images were acquired using a Zeiss 20x water objective (20x/1.0 DIC (uv) VIS-IR, Zeiss) and a 488 nm laser, with the anterior side of the brain facing up to the objective. The images were acquired at a 128 × 128 pixel resolution, and at a frame rate of ~ 10 Hz.~ 10 Z axial sections were imaged in one time-series cycle. The section interval was ~ 1 μm. The time intervals between each cycle were 2 s.
Before stimulating a brain, we imaged the basal GCaMP3 signals for ≥ 10 cycles. We then gently added 200 μl 50 mM ATP (pH adjusted to 7.0, Sigma, A2383-5G) into the Drosophila imaging saline, resulting in a final ATP concentration of 5 mM. We performed a stack registration using the ImageJ Plugins registration module and measured the GCaMP3 intensities using the ImageJ Analyze ROI manager module. ΔF/F0 (%) was calculated as ΔF/F0 (%)=(F-F0)/F0 × 100. Fmax is the maximum fluorescence value following ATP delivery. Fmin is the minimum fluorescence value that occurred during a total of 80 time series cycles after ATP delivery. F0 is the GCaMP3 baseline value averaged for 10 time-series cycles immediately before ATP application.
Statistical analyses
No statistical methods were employed to predetermine sample sizes. Sample sizes were chosen based on previous publications (Demir and Dickson, 2005; Manoli et al., 2005; Stockinger et al., 2005; Pan et al., 2012; Asahina et al., 2014; Clowney et al., 2015; Huang et al., 2016; Zhang et al., 2016). Statistical analysis was performed with Prism5 (GraphPad Software). We performed nonparametric Mann-Whitney test when comparing two groups of data. For comparison of multiple groups of data, we performed Kruskal-Wallis test followed by Dunn’s post hoc test. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. We present the exact number of samples and P values in the figure legends and in the supporting source data files. We present raw data using scatter plots and include exact values in the source data files. When n < 10, individual data points were identified.
Replication
We used only biological replicates throughout this work. To perform the behavioral studies, we defined biological replicates as animals of the same genotype and rearing conditions, exposed to identical treatments. Courtship indexes were calculated using n = 6—27 individual animals. Preference indexes were calculated using n = 12 individual animals. Chaining indexes were calculated using n = 6 groups (8—12 individual animals in each group). Lunging numbers were calculated using n = 10—12 animals. All animals were used once, since their behavioral indexes are sensitive to prior experience. Replicates for the Ca2+ imaging were defined as the number of neurons (Figure 6C and F) or the selected regions (Figure 8E) analyzed per genotype and condition. In all cases we used 3—9 brains/genotype and condition. 2—5 neurons (Figure 6C and F) or 4—7 regions of selection (Figure 8E) were used per brain. Replicates for the immunostaining were defined as brains of the same genotype that underwent identical staining procedures. We stained ≥ 5 brains per experiment. The Gal4/UAS (or LexA/LexAop) binary systems are highly reproducible. Images that were the most intact were selected for display. We did not exclude any data points.
Group allocation
To perform the behavioral assays, the control and experimental groups were reared under the same conditions, collected on the same day, aged in parallel, and assayed on the same day. The control and experimental groups were assayed in an arbitrary order. Behavioral videos were randomly permuted for scoring behavioral indexes. All behavioral analyses were obtained from videos, in which the genotypes were masked. The indexes were calculated blindly.
To perform the Ca2+ imaging, the control and experimental groups were assayed in an arbitrary order. The raw Ca2+ imaging data files were permutated in order and analyzed by Image J software.
Source data files
The raw data for the behavioral assays, Ca2+ imaging assays, summary statistics, and full stacks of the entire brains used in the GRASP experiments are included in the source data files.
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- MCNeville
- SEadie
- SFGoodwin
-
Sex and the single cell. II. there is a time and place for sex
- CCRobinett
- AGVaughan
- JMKnapp
- BSBaker
-
Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene
- LCRyner
- SFGoodwin
- DHCastrillon
- AAnand
- AVillella
- BSBaker
- JCHall
- BJTaylor
- SAWasserman
-
Sexual deprivation increases ethanol intake in Drosophila
- GShohat-Ophir
- KRKaun
- RAzanchi
- HMohammed
- UHeberlein
-
Neural circuitry that governs Drosophila male courtship behavior
- PStockinger
- DKvitsiani
- SRotkopf
- LTirián
- BJDickson
-
Experiments on sex recognition and the problem of sexual selection in Drosophila
- AHSturtevant
-
Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic
- AMvan der Bliek
- EMMeyerowitz
-
Neurogenetics of courtship and mating in Drosophila
- AVillella
- JCHall
-
Opposite thermosensor in fruitfly and mouse
- VViswanath
- GMStory
- AMPeier
- MJPetrus
- VMLee
- SWHwang
- APatapoutian
- TJegla
-
Neuronal control of Drosophila courtship song
- ACvon Philipsborn
- TLiu
- JYYu
- CMasser
- SSBidaye
- BJDickson
-
Drosophila spichthyin inhibits BMP signaling and regulates synaptic growth and axonal microtubules
- XWang
- WRShaw
- HTTsang
- EReid
- CJO'Kane
-
A common genetic target for environmental and heritable influences on aggressiveness in Drosophila
- LWang
- HDankert
- PPerona
- DJAnderson
-
A circuit node that integrates convergent input from neuromodulatory and social Behavior-Promoting neurons to control aggression in Drosophila
- KWatanabe
- HChiu
- BDPfeiffer
- AMWong
- EDHoopfer
- GMRubin
- DJAnderson
-
Spatial representation of the glomerular map in the Drosophila protocerebrum
- AMWong
- JWWang
- RAxel
-
Developmental control of foraging and social behavior by the Drosophila neuropeptide Y-like system
- QWu
- TWen
- GLee
- JHPark
- HNCai
- PShen
-
Genes and circuits of courtship behaviour in Drosophila males
- DYamamoto
- MKoganezawa
-
Analysis of functional neuronal connectivity in the Drosophila brain
- ZYao
- AMMacara
- KRLelito
- TYMinosyan
- OTShafer
-
Cellular organization of the neural circuit that drives Drosophila courtship behavior
- JYYu
- MIKanai
- EDemir
- GSJefferis
- BJDickson
-
Dopaminergic circuitry underlying mating drive
- SXZhang
- DRogulja
- MACrickmore
-
A subset of octopaminergic neurons are important for Drosophila aggression
- CZhou
- YRao
- YRao
-
Central neural circuitry mediating courtship song perception in male Drosophila
- CZhou
- RFranconville
- AGVaughan
- CCRobinett
- VJayaraman
- BSBaker


There are several demos exemplifying the use of pyGPs for various Gaussian process () tasks. We recommend to first go through Basic GP Regression which introduces the regression model. Basic regression is the most intuitive and simplest learning task feasable with . The other demos will then provide a general insight into more advanced functionalities of the package. You will also find the implementation of the demos in the source folder under pyGPs/Demo.
The Demos give some theoretical explanations. Further, it is useful to have a look at our documentation on Kernels & Means and Optimizers.
Regression
Classification
Some examples for real-world data


Resume
ENTRY LEVEL ENGINEER
Knowledgeable professional with Bachelor's degree in chemical field. Experience in mass and energy balances and separation. Background in computer-aided design programs. Demonstrated skills in Java, Python, HTML, CSS, PHP and JavaScript. Well-versed in introductory chemistry laboratory techniques such as titrations, pH readings, calorimetry, dilutions, and HPLC.
EDUCATION
LaunchCode, Seattle, WA, 2017
- Relevant Coursework: Introduction to Computer Programming (Python, HTML, CSS)
Bachelor of Science, Chemical Engineering, University of Washington, Seattle, WA, 2014 (3.24 GPA)
- Relevant Coursework: Material and Energy Balance, Transport Pnomanona, Thermodynamic, Unit Operations, Fuel Cell, Process Optimization, Reactor Design, Fuel Cells, Process Dynamics and Control, Process Design, Solidworks/AutoCAD
- Other Coursework: Introduction to Computer Programming (Java), Introduction to Web Programming (HTML, CSS, PHP, SQL, JavaScript)
EXPERIENCES AND ACHIEVEMENTS
Bookkeeper/Clerk, T & T Seafood Restaurant, Edmonds, WA 2007-Present
Responsible for preparing profit and loss statements, monthly closings, and cost accounting reports. Process total
daily transactions. Help manage inventory. Stock and place orders.
- Implemented a social media marking campaign to drive more repeat customer visits.
Undergraduate Researcher Fuel Cell/Syngas, University of Washington Adler Lab, Seattle, 2013-2014
Researched the technical and economic feasibility of using solid oxide electrolyte cells to produce useable product
from the biomass syngas that is produced.
- Completed in Environmental Innovation Challenge by designing usage of solid oxide electrolyte cells to upgrade biomass syngas to better product for subsequent application.
Treasurer, University of Washington American Institute of Chemical Engineering, Seattle, WA 2013-2014
Created and monitored organization's budget. Helped identify and determine fund-raising activities and events to allow
organization to achieve its goals. Coordinated with other officers and assisted with organizing fundraiser events.
- Enhanced inner communication for future members.
- Helped increase budget by $500.
Laboratory Assistant, University of Washington Nanotech User Facility, Seattle, WA 2012-2013
Analyzed graphene using fabrication and testing methods to create a nanoscale electronic devices. Conducted research
and fabrication on Electron Beam Lithography equipped Scanning Electron Microscope and sputtering tools. Trained in
Atomic Force and Raman Microscopy, and other tools to characterize and test fabricated products.
- Produced a photo type using graphene.
FUTURE
From learning programing throughout LaunchCode and classes through my university I have enjoyed it alot. I would like to keep learning the materials that is necessary to apply for an apprenticeship and find a job that is related to what I enjoy the most from LaunchCode. I hope this will help me get my foot in the door at a company.


PAssist supports you in providing on-call service in conjunction with an SMS-based alerting system.
Features
The application is automatically activated via defined on-call service events in your calendar and performs the following functions during the on-call duties:
• Triggering of an alarm on the receipt of an SMS from the operations center as well as acknowledgment via SMS after the confirmation of the alarm by the user.
• Monitoring of the phone signal level and notification in case of bad or off signal level.
• Automatically control of the ringtone volume based on your day and night profile.
• Logging of alert incidents with start and end time as well as all associated SMS messages.
• Monitoring and acknowledgment of periodic test alarms for testing the alarm chain from end to end.
Other functions that are available regardless of the current on-call state:
• On-call event calender
• History of all alert incidents
SMS permissions
In order to perform the functions of PAssist, PAssist needs to be able to receive and send SMS.
As these SMS permissions are no more granted by Google, a compatible SMS adapter is required.
PAssist is free and open source
PAssist is free. The source code is available as Open-Source under Apache-2.0 licence.
If you find PAssist useful and use it on a regular basis for your on-call duties, a voluntary donation via In-App purchase is warmly welcome.
Disclaimer
The use of PAssist is at your own risk. The author assumes no liability for malfunctions of the application.
Any warranty claims are excluded.
Feedback welcome
Feedback, bug reports or feature requests are very welcome.


Cross-hatching GLSL shader Blinn-Phong shading and rim lighting.
Based on Microsoft Research "Real-Time Hatching" paper.
Click and drag to move model, scroll to zoom. Play with the controls!
Coded by Jaume Sanchez @thespite using three.js.


Dear friend, RE save our soul I am Mrs. Deborah Kabila, one of the wives of Late President Laurent D. Kabila of Democratic Republic of Congo (DRC). Consequent upon the assassination of my husband, I am in possession of USD 58,000,000 (Fifty Eight Million US Dollars Only) being funds earlier earmarked for special projects. This fund has since been deposited in a security company in West African Country of Togo where I am residing now with my children. It is now my intention to move the said fund out of this place (Togo) to a safer place for the benefit of my children and I, for immediate investment. Based on this that I solicited for your assistance to enable me take this money out of Togo. However, note that my children and I have agreed to give you 20% of the total fund if you can accept the offer of assisting us. Also it will be your responsibility in directing us on a viable business. It is also my intention to relocate and probably take a temporarily resident in your country pending when all the troubles in my country will be resolved. We advised that you look for a house we will buy as soon as we arrived. To conclude this transaction, you will be required to come to Togo to open an account in any bank here in Togo where the security company will deposit the total sum in your favor. From this bank the money will be remitted into your original bank in your country. Immediately this done, all of us will depart Togo to your country, where my children and I are expected to take a temporary resident. Please note that I can not open any Bank account with my name because, My late husband's first son-JOSEPH-who took over power in our country, don't want to see me and my children, He claimed that when our husband was alive, that I was very close to him than any other of his wives including his (Joseph) mother. He also claimed that because of my closeness to him that I was able to get things from him more than others. As a result he has been monitoring me. Infact this one of the main reasons I want to take my children out of our country and any nearby country. Thanking you in advance. Yours faithfully, Deborah Kabila (Mrs.) N/B: Kindly give me your direct telephone,fax and your mobile telephone numbers for more explanations. ------------------------------------------------------------ http://Game.37.com/ <--- Free Games http://newJoke.com/ <--- J O K E S ! ! ! --------------------------------------------------------------------- Express yourself with a super cool email address from BigMailBox.com. Hundreds of choices. It's free! http://www.bigmailbox.com ---------------------------------------------------------------------


Package org.apache.juneau.marshaller
package org.apache.juneau.marshaller
Marshallers
-
ClassDescriptionA subclass of
Marshaller
for character-based serializers and parsers.A pairing of aCsvSerializer
andCsvParser
into a single class with convenience read/write methods.A pairing of aHtmlSerializer
andHtmlParser
into a single class with convenience read/write methods.A pairing of aJsonSerializer
andJsonParser
into a single class with convenience read/write methods.A pairing of aJson5Serializer
andJsonParser
into a single class with convenience read/write methods.Top-level class for a pairing of aSerializer
andParser
into a single class with convenience read/write methods.A pairing of aMsgPackSerializer
andMsgPackParser
into a single class with convenience read/write methods.A pairing of aOpenApiSerializer
andOpenApiParser
into a single class with convenience read/write methods.A pairing of aPlainTextSerializer
andPlainTextParser
into a single class with convenience read/write methods.A subclass ofMarshaller
for stream-based serializers and parsers.A pairing of aUonSerializer
andUonParser
into a single class with convenience read/write methods.A pairing of aUrlEncodingSerializer
andUrlEncodingParser
into a single class with convenience read/write methods.A pairing of aXmlSerializer
andXmlParser
into a single class with convenience read/write methods.


Just a simple timestamp server
In this demo, the server sends the current timestamp over a WebSocket once per second, using the awesome Socket.io library. You can see the received timestamps below.
- Nothing received from the server yet...
In this demo, the server sends the current timestamp over a WebSocket once per second, using the awesome Socket.io library. You can see the received timestamps below.


THE PREACHER'S WIFE A film review by Scott Renshaw Copyright 1996 Scott Renshaw
(Touchstone) Starring: Denzel Washington, Whitney Houston, Courtney B. Vance, Gregory Hines. Screenplay: Nat Mauldin and Allan Scott, based on THE BISHOP'S WIFE screenplay by Robert B. Sherwood and Leonardo Bercovici. Producer: Samuel Goldwyn Jr. Director: Penny Marshall. MPAA Rating: PG (mild profanity) Running Time: 121 minutes. Reviewed by Scott Renshaw.
It won't be difficult for many viewers to accept Denzel Washington as a character sent straight from heaven...particularly female viewers. Washington has been a regular on "sexiest man" lists, dazzling audiences with his confident smile while building an impressive body of work in roles characterized by intensity, integrity and -- frequently -- inscrutability. Yet for all his obvious talent and good looks, he has often seemed unapproachable on screen, so cool that he fails to be warm. THE PREACHER'S WIFE comes as a welcome addition to Washington's resume, a somewhat predictable holiday confection buoyed by Washington's engaging lead performance.
THE PREACHER'S WIFE is set in an inner city New York neighborhood, where local Baptist minister Henry Biggs (Courtney B. Vance) is facing a personal crisis. Confronted by financial problems in the church and social problems in the community which seem beyond his ability to change, Henry drives himself relentlessly, to the point of neglecting his wife Julia (Whitney Houston) and son Jeremiah (Justin Pierre Edmond). Finally Henry asks for help from above, and he gets it in the form of Dudley (Washington), a rookie angel thrilled to be back on earth. Henry naturally has difficulty believing this insistent fellow is divine, especially when Dudley begins spending quality time with Julia and Jeremiah, forcing Henry to consider his priorities more carefully.
A remake of the 1947 Cary Grant-Loretta Young-David Niven vehicle THE BISHOP'S WIFE is a natural notion for a spiritually renewed era when angels are appearing on television ("Touched by an Angel") and in the movies (the upcoming MICHAEL) faster than they can dance on the head of a pin. It is equally natural to expect a certain amount of holy-rolling in such an effort, but THE PREACHER'S WIFE does a fairly good job of walking the fine line between sincere and sanctimonious. Penny Marshall (BIG, AWAKENINGS) has shown herself to be that rare contemporary director who can make a "feel-good" film which doesn't leave you feeling insulted, and she generally avoids saccharine overload. There is a bright good humor to most of THE PREACHER'S WIFE which makes it possible to overlook such manipulations as the fate of an adorable orphan and a conflict with a nasty real estate developer (Gregory Hines, discovering a new career as a heavy after this film and Marshall's 1994 RENAISSANCE MAN).
The major problem with THE PREACHER'S WIFE is the same problem which has characterized most of Marshall's films: it's just too long. Her movies have a tendency to run out of gas with a lot of film left to run, and THE PREACHER'S WIFE could have benefited from plenty of additional editing. Much of it could have come from Whitney Houston's performance, which consists largely of finding reasons to break into song like Elvis Presley in one of his carefully crafted vehicles. Houston certainly has a spectacular voice, and it is nice to hear her using it for gospel standards rather than dreary ballads, but half a dozen musical numbers simply pushes the limit and pads the running time. Little Justin Pierre Edmond should have been another strong candidate for the editor's knife; the button-cute rascal is one of the worst child actors I have seen on screen in years.
Thankfully, Denzel Washington is on hand to provide a jolt whenever THE PREACHER'S WIFE needs it most. His first appearance may be a shock to those who know him best as serious, determined characters in films like MALCOLM X, CRIMSON TIDE and COURAGE UNDER FIRE: dropped from the sky into the snow, looking decidedly dapper in a grey suit, hat and topcoat, Washington's Dudley proceeds to celebrate his return to corporeal form by spinning around on the ground like one of the Three Stooges. It is a giddy and completely unexpected moment, and one of many where Washington shows the mischievous edge to this angel seduced by flesh. His innocence and earnestness are thoroughly charming, and his scenes with Courtney B. Vance (effective as the real principal character of the story) have a particular appeal. If there isn't much chemistry between Washington and Houston, well, that's not really the point of THE PREACHER'S WIFE. Besides, Denzel Washington is a chemical reaction all by himself, and it is a pleasure to see that he can sparkle as well as he smolders.
On the Renshaw scale of 0 to 10 preacher features: 6.
Visit Scott Renshaw's MoviePage http://www-leland.stanford.edu/~srenshaw Subscribe to receive reviews directly via email See details on the MoviePage
The review above was posted to the
rec.arts.movies.reviews newsgroup (de.rec.film.kritiken for German reviews).
The Internet Movie Database accepts no responsibility for the contents of the
review and has no editorial control. Unless stated otherwise, the copyright
belongs to the author.
Please direct comments/criticisms of the review to relevant newsgroups.
Broken URLs inthe reviews are the responsibility of the author.
The formatting of the review is likely to differ from the original due
to ASCII to HTML conversion.
Related links: index of all rec.arts.movies.reviews reviews


Plane crash kills at least 27 in Democratic Republic of Congo
by The New DailyAt least 27 people, including nine members of the same family, have been killed after a passenger plane crashed into houses in the Congolese city of Goma.
The aircraft, operated by local company Busy Bee, hit the densely populated neighbourhood shortly after takeoff en route to Beni, a city 250km north of Goma.
An official from Busy Bee, who asked not to be named, said the 19-seater Dornier 228-200 crashed with 16 passengers and two crew on board.
Nine people from the same family who were in one of the houses hit were among those killed as the plane descended towards the Mapendo District, engulfing the area in giant plumes of black smoke and flames.
News site actualite.cd quoted one of the company’s maintenance workers as saying the crash occurred due to a “technical problem”.
The plane apparently came down about a minute after taking off from the city’s airport, a source at Goma airport told the BBC.
Two people were rescued before the plane exploded, including one member of the crew, according to the National Border Health Program.
A witness, Djemo Medar, said the plane appeared to be “spinning three times in the air and emitting a lot of smoke” before it crash-landed.
“When the plane crashed many of us rushed there, we know the pilot, his name is Didier; he was shouting ‘Help me, help me’, but we had no way to get to him because the fire was so powerful,” he told Reuters.
After taking a taxi to the site to help out, local pilot Placide Kambale said he witnessed the plane catch fire.
“I called other young people from the neighborhood,” he told The Associated Press.
“They helped me to try to remove those who still moved. We have managed to recover two that was quickly sent to the hospital,” he added, but then the fire expanded.
It was unclear how many others on the ground may have been killed or injured.
Air accidents are relatively frequent in Congo because of lax safety standards and poor maintenance. All Congolese commercial carriers, including Busy Bee, are banned from operating in the European Union.
A cargo plane departing from the same airport crashed an hour after take-off in October, killing all eight people aboard.
-with agencies


optimize Optimize action parameter by exhaustive function maximization.
OPTIMACT = optimize(DS, EVALFN, PARAMNAME, PARAMVALS)
returns an optimized prtAction object, with parameter
PARAMNAME set to the optimal value. DS must be a prtDataSet
object. EVALFN must be a function handle that returns a
scalar value that indicates a performance metric for the
prtAction object, for example a prtEval function. PARAMNAME
must be a string that indicates the parameter of the
prtAction that is to be optimized. PARAMVALS must be a
vector of possible values of the parameter that the
prtAction will be evaluated at.
[OPTIMACT, PERF] = optimize(...) returns a vector of
performance values that correspond to each element of
PARAMVALS.
Example:
ds = prtDataGenBimodal; % Load a data set
knn = prtClassKnn; % Create a classifier
kVec = 3:5:50; % Create a vector of parameters to
% optimze over
% Optimize over the range of k values, using the area under
% the receiver operating curve as the evaluation metric.
% Validation is performed by a k-folds cross validation with
% 10 folds as specified by the call to prtEvalAuc.
[knnOptimize, percentCorrects] = knn.optimize(ds, @(class,ds)prtEvalAuc(class,ds,10), 'k',kVec);
plot(kVec, percentCorrects)
Help for prtClassLogisticDiscriminant/optimize is inherited from superclass prtAction


Below you will see a image in the same folder
We can also load images in subfolders
We can also load images in websites


Public Member Functions | |
| douglas_peucker_point (const P &ap) | |
| douglas_peucker_point< P > | operator= (const douglas_peucker_point< P > &other) |
Public Attributes | |
| bool | included |
| const P & | p |
| geometry::strategy::simplify::douglas_peucker_point< P >::douglas_peucker_point | ( | const P & | ap | ) |
| douglas_peucker_point<P> geometry::strategy::simplify::douglas_peucker_point< P >::operator= | ( | const douglas_peucker_point< P > & | other | ) |
| bool geometry::strategy::simplify::douglas_peucker_point< P >::included |
| const P& geometry::strategy::simplify::douglas_peucker_point< P >::p |
|
October 14, 2008 |
Copyright © 1995-2008 Barend Gehrels, Geodan Holding B.V. Amsterdam, the Netherlands Copyright © 2008 Bruno Lalande, Paris, France |


Drag nodes to move them (and their subtrees).
Click a root node to begin joining it. Then click another root to join with a
default new root at the midpoint,
or drag from the other root to place the new root.
Click a non-root node to unlink it from its parent (becomes a new tree).
Click nowhere particular to create a new one-node tree.


Arrange and size cells to create your dashboard.
more info
Show code on hover
Move cell:
Click and drag
to move a cell. Hold
Shift
to drag from anywhere on a cell.
Resize cell:
Click and drag cell edges or corners to resize.
Hide cell:
Click
to hide a cell from the dashboard view.
Show cell:
Click
to return a hidden cell to the dashboard view.
Hide/Show all:
Select the options under
Cell > Dashboard
to hide or show all cells.
Edit cell:
Click
to jump to the Notebook and edit the code.


Toggle navigation
iphone
ipad
imac pro
music
TV
watch
surpport
iphone x
say hello to future
Learn more
buy
ipad
not computer,more than computer
Learn more
buy
iwatch
keep pace with your every movement
Learn more
buy


We want to use our skills in building and shipping software and hardware to make a positive difference. The area that interested us the most was moving towards 100% clean and healthy energy.
We decided to focus on lowering mindless excessive use of electricity. We found from our own experience, we were using 40% more than we needed with no change to our lifestyles. If this is true for us, we figure it is true for many others.
The cleanest and healthiest electricity is the electricity we don’t use. It will be easier to get to 100% clean and healthy energy if we cut down on wasteful consumption of electricity. Besides, we save money and breathe easier. What’s not to like?
BUT…who has time to wade through their electricity bills then figure out from some random web source how to lower their use? Besides, the reward in doing so is not immediately obvious.
This is why we’re doing FitHome. To make it extremely easy to use less electricity while at the same time getting immediate feedback on the amount of electricity being saved and the positive impact this savings is having on our environment.
The FitHome experience is successful if the participants use 15% less electricity than they did before participating. Consuming 15% less electricity is a reachable goal. After all, folks in Germany use 54% less electricity than folks in the US. Of course, there could be many reasons for this difference perhaps it is because:
But still given the 54% difference, we should be able to save 15%. When we took the FitHome challenge, we ended up saving 40%. With no change to our lifestyle.
The ACEE Report, Saving Energy, Saving Lives pointed out…
Nationwide, reducing electricity consumption by 15% for a single year would result in:
If you use 15% less electricity, you would save $200 a year.
In our city (Kirkland, Washington), there are 38,093 housing units. While the weather varies quite a bit between Eastern and Western Washington, for this discussion we’ll use the EIA’s info on the average monthly amount of electricity consumed as 955kWh.
Our electricity cost is (at the time of this writing):
If a household uses 955 kWh/month, their electricity cost is roughly $121.82 /month.
(.1*600)+(.12*355)+7.87 = $110.47
110.47 + (.0604*110.47) + (.03783*110.47) = $121.32
If a household starts using 15% less electricity, their electricity cost is roughly
855*.85 = 812 kWh/month
(.1*600) + (.12*212) + 7.87 = $93.31
93.31 + (.0604*93.31) + (.03783*93.31) = $102.48
a savings of $18.84 / month or $206 / year.
The 38,093 households in Kirkland use 36,378,815 kWh/month (38,093 * 955). Or 436,545,780 kWh / year of electricity. That is 340,288 Tons of greenhouse gas emissions (using the Greenhouse Gas Equivalencies Calculator). This is equivalent to the amount of CO2 2,505 acres of forests can sequester.
If we all used 15% less electricity, we’d be using 65,481,867 kWh less per year. Just 15% means we decrease the amount of greenhouse gas emissions by 51,043 Tons.
Think of FitHome as an interactive article on how to save electricity completely personalized to your home.
Certainly, you can Google for “energy saving techniques”, find recommendations, and follow those. Or you can use FitHome if you’re like we were “I’m so lazy about using less electricity that an app which walks me through all of the possible power saving ideas is just what I need. I usually get home and the last thing I want to do is think.”
FitHome tells you how much electricity you are using. As you start saving using the interactive steps provided, you begin to see the amount of money you are saving as well as the contribution you are making to combating climate change.
With FitHome, we are all improving our home health and combating climate change together.


This is the partial for view 1.
Please select your business unit:
SHOW PRAISE COZ PRAISE SONG LIST WAS SELECTED IN DROPDOWN
SHOW GENERAL, WAS SELECTED in DROPDOWN
Business Unit is required.
Where did the observation take place?
Location is required.
Do not use this form for an injury report. Please inform your supervisor of the incident.


|
Version 2.41c 31-July-2012
What's New
Documentation Set
This toolbox is covered by the BSD License. Copyright (c) 2012, Frédéric Moisy. All rights reserved. Laboratoire FAST. University Paris Sud, University Pierre et Marie Curie, CNRS. 91405 Orsay Cedex, France<EMAIL_ADDRESS>|


This is some text
First Name
Last Name
Comments
TextArea
Phone
Select One:
Mobile
Office
State
AZ
CA


Britney Wilson of American Fork, Utah and I were married on November 19, 2011 in the
Salt Lake City Temple. I graduated from BYU in April of 2015 with my undergraduate degree in Computer Science. We currently live in Pleasant Grove near where we both grew up. Britney finished up her degree in Family Life at BYU and takes care of our son Azure who was born in 2016. Britney and I enjoy hiking, watching movies, eating good food, spending time with family (especially our little boy) and traveling whenever occasion allows.
You can find some of our photos
here.
I thoroughly enjoy reading. I read whenever I can and can often be seen with a book. I grew up with a mother who was nearly obsessed with books which meant we had thousands of books in bookshelves littered throughout our house for us kids to enjoy. My favorite genres are fantasy and science fiction. I also enjoy classics like
The Count of Monte Cristo and
A Tale of Two Cities but I will read just about any good book. Some of my favorites include:
I am also very active on Goodreads if you wish to see what I am reading.
I grew up surrounded by music. My siblings and I played at least two instruments growing up. My parents required us all to play the piano and urged us to pick up at least one more instrument. My extra instruments became the guitar, the trumpet and I also participated in choir. I enjoy playing soft piano and acoustic guitar music. I also enjoy listening to music throughout each day. Lately it has been
Enya,
Coldplay,
The Piano Guys, and occasionally some dubstep.
As I mentioned earlier, I really like to eat. I am not at all a picky eater and will try just about anything. Britney and I love the
Cheesecake Factory and will sometimes make the one hour drive up to Salt Lake City just to go there. We have also been known to go to Park City to eat at the
Eating Establishment. We like to find good food wherever we go. Some local places we like to go include:
I grew up playing every sport I could. These included, Basketball, Football, Baseball, Soccer, Wrestling, Swimming, Gymnastics, and Racquetball. Almost all of those phased out except Soccer, Basketball and Racquetball. I played soccer for my high school team and played recreation basketball throughout and after high school. I still look for opportunities to play any of those sports.
Since graduating from college I have really gotten into running, cycling and now mountain biking. I love trail running in the mountains near Utah Valley. I also enjoy biking the Alpine Loop, South Fork and anywhere else that is challenging and scenic. I recently got into mountain biking and it has been a blast.
I am also a big BYU sports fan. I follow football and basketball primarily but will cheer on anyone wearing the BYU uniform (provided they aren't complete knuckleheads).
My brothers and I played a lot of video games together when we were young. We were often satisfied simply to watch one of the brothers play. We had many of the classic games on NES like
Duck Hunt,
Techmo Bowl and
Mike Tyson Punch-Out. We enjoyed a succession of consoles from Sega to N64 to PS2 to XBox360. We also played PC games like
Myst,
Starcraft, and
Warcraft (though not World of Warcraft). We still sometimes get together to play games and prefer ones we can all play together.
We also all enjoy board and card games. We try to get together regularly to play some kind of game. Some of our favorites include:
We aren't big-time travelers but nevertheless enjoy traveling. We enjoy going to California and visiting Disneyland, the beaches and other random places in the Greater Los Angeles and San Diego areas. St. George is a convenient getaway for us and like to visit
Tuacahn, eat some of the good food down there and visit Zion National Park. Utah has some breathtaking
national and state parks which we like to visit. We made a trip to Manhattan after I graduated as a graduation present to both of us which was a blast. We sometimes visit family in
Alta Wyoming and we spend a week at
Bear Lake every year. We look forward to many opportunities in the future to travel.
My wife and I prioritize family at the top of our lists. I want to spend as much time as I can with family and help our children grow up to achieve their full potential. We want to build a beautiful modern home somewhere in Utah Valley and live there until retirement if not longer.
While my goals in life never were to get into writing code, I have learned that I absolutely love to create things on the computer. It is magic to me and the best part is that I understand it (most of the time). I want to continue to write code the rest of my life. Whether it be small utilities, smartphone apps, or websites, I want to continue to hone my abilities to create.
I have toyed with the idea of getting a PH.D. in Computer Science. When I began my undergraduate education the first thing I wanted to do was to become a physician. Even though I no longer have those wishes I haven't quite given up the idea of being called a doctor and obtaining a PH.D. is one way I could do that. In all seriousness though I believe I would enjoy the learning and research involved in a Computer Science PH.D. program.
I also want to continue learning regardless of how formal it might be. I love general education and want to continue to learn new things every day.
I have had the privilege of being a volunteer teacher for several
programming fundamentals courses at my local library. I have had a great time teaching what I love.
I served a two-year
LDS mission in Atlanta Georgia (
Georgia Atlanta North Mission). I lived in Grayson, Lawrenceville, Sandy Plains, Athens and Washington during my two years there and had many opportunities to provide community service and to share my beliefs regarding
The Church of Jesus Christ of Latter-day Saints. I continue to practice my faith and am always interested in ways I can help my community to be a stronger and more conducive place for families to raise children.


Art direction: The problem whereby you want to serve cropped images for different layouts — for example a landscape image showing a full scene for a desktop layout, and a portrait image showing the main subject zoomed in close for a mobile layout. This can be solved using the


--- title: Komolion Fund jumbotron-title: Empower locals to change lives jumbotron-subtitle: Community members drive social change. We are there to help. jumbotron-give-button-text: Give Now mission-title: We envision a world in which children and adults have opportunities to thrive personally and to make positive contributions to their communities. mission-content: Komolion Human Development Fund encourages and provides funding for projects in local communities that are designed by community members to enable increased education, health, cooperation, poverty reduction and quality of life for the entire community. core-values-title: Core Values donations-title: All administrative costs (besides processing fees) are covered by the Komolion Human Development Fund's Board of Directors, so 100% of your donation goes directly to empowering communities in Kenya. projects-title: Projects & Partnerships video-title: Featured Video video-description: The winds of change are blowing in Komolion, a tiny village in East Pokot, Kenya. A non-governmental organization called Noonkera, supported by the Komolion Fund, has championed the cause of ending female genital mutilation once and for all in this community. ---


{% extends 'main/base.html' %} {% block content %}
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An instance of the {@link java.util.regex.Pattern} class represents a regular expression that is specified in string form in a syntax similar to that used by Perl.
Instances of the {@link java.util.regex.Matcher} class are used to match character sequences against a given pattern. Input is provided to matchers via the {@link java.lang.CharSequence} interface in order to support matching against characters from a wide variety of input sources.
Unless otherwise noted, passing a null argument to a method in any class or interface in this package will cause a {@link java.lang.NullPointerException NullPointerException} to be thrown.
An excellent tutorial and overview of regular expressions is Mastering Regular Expressions, Jeffrey E. F. Friedl, O'Reilly and Associates, 1997.
@since 1.4 @author Mike McCloskey @author Mark Reinhold


In this section, you’ll have a chance to practice the concepts you’ve learned in the videos. First, review the core concepts covered that you’ll need to keep in mind. Then go through the exercises below.
Remember, these are for your own benefit. Feel free to skip them if you don’t find a particular exercise valuable or you get stuck for too long.
When handling errors, we can check for bad values (e.g. None
where a proper string was expected). But Python’s native error handling
approach is exception-based: throwing and catching exceptions.
Below is the minimum code to catch an error in Python.
try:
do_risky_thing1()
do_risky_thing2()
do_risky_thing3()
except Exception as x:
# Deal with error, use x for help on what happened.
The example above is good to catch errors. But it catches them all (well, almost all of them), and it treats them all the same.
Below is code needed to handle different errors as well as unforeseen errors.
try:
do_risky_thing1()
do_risky_thing2()
do_risky_thing3()
except json.decoder.JSONDecodeError:
# Handle malformed JSON error
except FileNotFoundError as fe:
# Handle missing file error
except ValueError:
# Handle conversion error.
except Exception as x:
# Deal with error, use x for help on what happened.
Note: It is important that the most specific errors are
listed first and the most general the last (Exception
).
Python selects the first (not best) match.
Now it’s your turn. In this practice, go back to the tic tac toe game we created back in the chapter on problem solving. Alternatively, if you made it through Connect 4, you can work with that one instead. Your job will be to: