31, p = 0.43). The arithmetic sum of all the 13 gluEPSPs shown in Figure 7B is 6.1 mV (see Figures 7C and 7D). Assuming the dendritic membrane potential DAPT to be −86 mV this would correspond to a dendritic peak depolarization to −35 mV. The depolarization

by the average single gluEPSP of 0.48 mV at the soma corresponds to a dendritic depolarization to −82 mV. Assuming a synaptic reversal potential of 0 mV the loss of driving force is around 60%. Thus, the expected linear sum gluEPSP at the soma corrected for driving force loss is just 2.6 mV. Data analysis was performed using Igor Pro (Wavemetrics). Distance measurements were performed on image stacks collected at the end of recordings buy PD-0332991 using ImageJ (NIH). The distance between the soma and the input site was measured from the center of the soma to the approximate midpoint of the input site in the case of gluEPSPs evoked by multisite uncaging. Distances in double-patch and modeling experiments are Euclidean distances. All values are given as

mean ± standard error of mean unless otherwise noted. This work was supported by the Deutsche Forschungsgemeinschaft (SFB TR3), Nationales Genomforschungsnetzwerk NGFNplus EmiNet, EPICURE, ERANET Neuron ‘EpiNet’, Ministry for Innovation, Research, Science, Research, and Technology NRW, and the BONFOR program of the University of Bonn Medical Center. We thank J.C. Magee and D. Dietrich for suggestions on the manuscript, T. Nevian for technical help with unless the dendritic recordings, and F. Helmchen for support. “
“Abrupt changes in an organism’s environment precipitate requisite and rapid adaptive changes in neural circuits. In particular, synapses in hypothalamic nuclei that form the neural network underlying energy balance and food intake are remarkably

susceptible to variations in the availability of food. The dearth of food is of such importance to an organism that it triggers both direct changes in food-related signals and the immediate activation of the stress response that increases circulating corticosteroids (CORT) (Bligh et al., 1990, Dallman et al., 1999 and McGhee et al., 2009). The dorsomedial nucleus of the hypothalamus (DMH) regulates food intake and serves as a center for the integration of food and stress signals (Bellinger and Bernardis, 2002 and DiMicco et al., 2002). More recently, the DMH has also been implicated as being the key food entrainable oscillator in the brain that exhibits synchronous activity in response to food deprivation (Gooley et al., 2006 and Mieda et al., 2006). Although both of these roles are key to an organism’s survival, surprisingly little is known about synaptic processing in the DMH and even less is known about the effects of food deprivation on synaptic function and plasticity in this nucleus.

, 2000). We observed clear differences between wild-type and knockout mice 6 days after crush injury in distal segments of the nerve. CB-839 order Wild-type nerve revealed robust

axonal YFP at 2 mm distal to the crush site, while the signal in knockout nerve was much reduced (Figures 7D and 7E). Thus, both behavioral and histological parameters show delayed regeneration of sensory neurons that specifically lack Importin β1 in the axonal compartment. Our results reveal a central role for locally translated Importin β1 in retrograde axonal signaling after nerve injury. The cell body response to axonal injury in sensory neurons is dependent on the transport of injury signals from lesion site to soma (Rishal and Fainzilber, 2010). Three different types of signaling modalities have been suggested to act in this pathway, including growth factor and receptor complexes (Brock et al., 2010), jun kinase and associated molecules together with the adaptor Sunday Driver ( Cavalli et al., 2005), and importin-dependent signals ( Rishal and Fainzilber, 2010). The complexity and robustness of this system was recently emphasized by a study implicating

approximately hundreds of signaling proteins and thousands of genes in the retrograde injury response in rat sciatic nerve ( Michaelevski et al., 2010). The fact that axonal loss of Importin β1 affects over 60% of the genes activated in the cell body response to injury is striking and supports find more a major role for importin-dependent

transport in the injury response mechanism, as is indeed reflected in the delayed recovery from peripheral nerve lesion seen in the knockout mice. Although injury-regulated expression of the affected genes and Idoxuridine subsequent regeneration are not completely repressed in the Importin β1 long 3′ UTR knockout, the largely attenuated gene regulation and delayed functional recovery we observe most likely reflects the fact that cargo proteins can still bind Importin αs at lower affinity in the absence of Importin β1 ( Lott and Cingolani, 2011). Partial redundancy of multiple retrograde signaling pathways might also play a role ( Abe and Cavalli, 2008; Ibanez, 2007; Michaelevski et al., 2010), and the fact that approximately one-third of the injury-responsive transcripts in our arrays were regulated similarly in wild-type and knockout animals highlights the participation of both Importin β1-dependent and -independent pathways in retrograde injury signaling. Local protein synthesis in axons has been proposed as a critical aspect of importin-dependent retrograde injury signaling. At least four components or regulators of the complex are thought to be locally translated in axons, including Importin β1 itself (Hanz et al.

Institutions and Selleckchem Panobinostat interests will likely play important roles, but a review of introducing HPV vaccine highlights the contested nature of ideas around vaccines, sexuality, and young people. HPV vaccination meets the standard criteria for policy uptake including epidemiological burden, safety and cost-effectiveness of the intervention. Such criteria are likely to be met for other high-burden STIs. However, such criteria may not be sufficient to ensure policy uptake – importantly, HPV vaccine was framed as a ‘cancer vaccine’ in some settings [30] and [31] and this may have assisted its

widespread policy uptake. Thus, the first policy opportunity for other STI vaccines is to identify similar associative and compelling frames – for example, highlighting the role that chlamydia vaccines could play in preventing infertility, or how syphilis vaccines could contribute to significant reductions in the risk of adverse outcomes of pregnancy [63]. Based on the experience of HPV vaccine introduction, two ideational issues which

are deeply rooted in values and prevailing norms will affect the successful introduction and uptake of future STI vaccine policy – both issues centre on the concept of Entinostat mw consent. The first concerns mandatory policy versus opt-in and we conclude that any STI vaccine policy should eschew mandatory approaches. A number of human rights and ethical arguments weigh against a mandatory policy for infections second that are not transmitted through casual contact, for vaccines that have unknown levels of population efficacy over the longer term, and (in the case of most HPV vaccine programmes) are targeted at one sex only. On these grounds alone, there is no human rights or ethical basis for forcing young people to be vaccinated against STIs. Coercive vaccination would not, we believe, meet ethical standards for public health programmes and may even engender increased resistance from adolescents, their parents/guardians and others. If STI vaccines are not mandatory, then the second consideration involves questions around who can give consent for young people to

receive an STI vaccine. As we have seen in this review, adolescents under 18 are recognized under international human rights laws and treaties as competent agents to seek services on their own according to their evolving capacity. In accordance with these evolving capacities, adolescents should have access to confidential counselling and advice, as well as to health care interventions (such as vaccines), without parental or legal guardian consent, where this is assessed by the professionals (whether in educational or health care settings) working with the child to be in the child’s best interests. A similar principle applies in cases where the adolescent does not have an involved parent or a legal guardian protecting their best interests, or is not under official care.

One would think that the oscillation regulating sequence reactivation across the hippocampus would be the high frequency (∼150–200 Hz) ripple oscillation that accompanies sharp waves.

However, high-frequency ripples are not correlated between CA3 and CA1 (Csicsvari et al., 1999). This is problematic because reactivation in CA1 requires properly timed input from CA3 (Nakashiba et al., 2009). Moreover, the large majority of replay events include neuronal activity from both CA1 and CA3 (Carr et al., 2012). In this issue of Neuron, Carr et al. (2012) propose a solution GS-7340 in vivo to this problem. Their results indicate that low frequency (“slow,” ∼20–50 Hz) gamma oscillations regulate the precisely timed reactivation of neuronal sequences in CA3 and CA1. They report that SWRs are accompanied by increases in CA3 and CA1 slow gamma activity. In contrast to ripples, SWR-associated slow gamma oscillations occurred synchronously across CA3 and CA1. Moreover, CA3-CA1 slow gamma synchrony was stronger

during SWRs than when no SWRs were present. Concurrent increases in CA3-CA1 synchrony were not seen in other frequency bands. CA3 slow gamma oscillations entrained spiking GDC-0068 cell line of neurons in both CA3 and CA1, and CA3 slow gamma entrainment of CA1 spiking was stronger during SWRs than when no SWRs were present. The new findings by Carr et al. (2012) also imply that slow gamma oscillations in the hippocampus serve as an internal clock during sequence reactivation. The authors measured slow gamma phase intervals between spikes from pairs of place cells. They found that slow gamma phase intervals across successive gamma cycles were significantly correlated with distance between the neurons’ place fields. Considering that distinctive places like cue-containing walls (Hetherington and Shapiro, 1997) and goal locations (Hollup et al., 2001) are heavily represented by place cell activity, the new findings raise the possibility that discrete locations are

reactivated on separate slow gamma cycles. Replay occurring during pauses in exploratory activity matches activation patterns from earlier experiences more accurately than replay occurring during extended periods of quiescence Thalidomide (Karlsson and Frank, 2009). Carr et al. (2012) found that quiescent SWR replay (i.e., relatively low-quality replay) was not associated with increases in slow gamma entrainment of cell spiking, a finding that supports the conclusion that enhanced slow gamma entrainment is necessary for high-fidelity replay. This conclusion received further support from their finding that large increases in CA3-CA1 slow gamma synchrony during SWRs were predictive of high fidelity replay events. Why would slow gamma entrainment of place cell spikes increase during some SWRs (i.e., waking SWRs) but not others (i.e.

The protrusion of microtubules is essential for neurite formation as low levels of the microtubule-destabilizing drug nocodazole attenuated the neurite-restoring effects of cytochalasin D and latrunculin click here B ( Figure S5). Instead, manipulations that were targeted either to stimulate

integrin signaling or to bundle actin filaments, which restore neurite formation in Mena/VASP/EVL KO neurons ( Dent et al., 2007), did not enable neurite formation in AC KO neurons ( Figure S6). We conclude that the drastic F-actin disorganization in AC KO neurons obstructs intracellular space and misdirects microtubule growth patterns. Furthermore, a pharmacological depolymerizing activity bypasses the need for AC proteins, allowing microtubules to coalesce and to radially protrude to generate neurites. Although in some instances the function of ADF and Cofilin are overlapping (Hotulainen et al., 2005), ADF depolymerizes actin filaments better than Cofilin, whereas Cofilin severs filaments better than ADF (Bernstein and Bamburg, 2010). Therefore, we determined the individual contributions of ADF and Cofilin to neuritogenesis. First, we examined the development of neurons with either monoallele ADF

Alpelisib solubility dmso expression (NesCre+/−, ADF+/−, cofilinflox/flox) or monoallele cofilin expression (NesCre+/−, ADF−/−, cofilinflox/+). ADF monoallele expression resulted in defective neuritogenesis with a significant increase in the percentage of cells in stage 1 ( Figures S7A and S7B). In contrast, cofilin monoallele expression conferred wild-type-like neuronal development, with the majority of neurons in stage 2 or stage 3 ( Figures S7C and S7D). Consistently, reintroduction

of ADF into AC KO neurons only partially restored neurite however formation in AC KO neurons, whereas Cofilin reintroduction almost completely reversed the neuritogenesis defect, resulting in cells with wild-type morphology in cell culture ( Figures 7A and 7B). Moreover, Cofilin re-expression restored normal neuronal development in AC KO cortical slices ( Figures 7C, 7D, and S7E). Analysis of the F-actin organization revealed that both ADF and Cofilin restored the gross organization of actin architecture. The percentage of cells extending filopodia increased 2-fold in ADF or Cofilin-transfected AC KO neurons ( Figures 7E and 7F). However, kymograph analysis of live-cell imaging of AC KO neurons cotransfected with Lifeact-GFP revealed that only Cofilin expression increased actin retrograde flow to 4.0 ± 1.0 μm/min, nearly a complete rescue, while ADF only partially increased actin retrograde flow to 2.9 ± 1.2 μm/min, a 65% rescue ( Figures 7E and 7G). Thus, while ADF and Cofilin are equally adept at stimulating filopodia formation, Cofilin has a higher propensity for driving actin retrograde flow and neuritogenesis.

These data indicate that the LRR domain is critical for promoting excitatory synapse formation in vitro and confirm that the lack of rescue we observed in vivo is not taking place due to dominant-negative effects of the domain deletion mutant proteins. To try to further understand the role of the specific interaction with netrin-G2, we obtained a mutant protein that we termed NGL1(NGL2LRR), in which 20 residues of the NGL-1 LRR domain have been swapped for NGL-2 residues (Seiradake et al., 2011). These

mutations cause NGL1(NGL2LRR) to bind to its normal receptor, netrin-G1, with very low affinity and instead to bind netrin-G2 with high affinity (Seiradake et al., 2011). We coelectroporated shNGL2 and NGL1(NGL2LRR) into a subset of CA1 pyramidal cells and analyzed spine density in SR and SLM. We found that NGL1(NGL2LRR) could fully Androgen Receptor Antagonist in vivo rescue the spine density in SR (Figures 5F and 5G) but had no effect on spine density in SLM (Figures 5F and 5H), indicating that the interaction between NGL-2 and netrin-G2

is critical for driving spine formation in SR. The specific effect of NGL-2 manipulations on high throughput screening SR synapses suggested that NGL-2 might be localized to the dendritic domain of CA1 neurons where SR synapses form. To address this possibility, we coelectroporated GFP-tagged NGL-2 with pCAG-tdTomato as a cytosolic marker. At P14, brains were perfused, sectioned, stained for GFP, and segments of dendrites were imaged in SR and SLM (Figure 6A). The GFP signal was visible in spines and in the dendritic shaft in

a pattern that was predominantly restricted to SR (Figure 6A, top), consistent with an earlier report old (Nishimura-Akiyoshi et al., 2007). We quantified the intensity of the GFP signal normalized to the intensity of the tdTomato signal and found more NGL-2 in SR (Figure 6A, bottom). These data indicate that localization of NGL-2 to a restricted domain of CA1 pyramidal cells could account for the synapse-specific effect of NGL-2. To test whether this localization depends on the interaction with netrin-G2, we generated a GFP-tagged LRR domain deletion mutant version of NGL-2 because the LRR domain is required for binding to Netrin-G2 (Seiradake et al., 2011). We coelectroporated this mutant with pCAG-tdTomato and analyzed the pattern of GFP immunofluorescence within CA1 dendrites. NGL2ΔLRR-GFP was expressed in a punctate pattern throughout the entire length of the CA1 pyramidal cell apical dendrites (Figure 6B, top). We quantified GFP expression levels in SR and SLM and found no significant difference between these regions (Figure 6B, bottom).

In that case, the potentiation induced in the contralateral side would correspond to de-depression of LTD previously induced in vivo with visual simulation. A complementary set of results was obtained when isoproterenol was injected (n = 4 rats) (Figure 8D). In this case pairing with 0mV potentiated synapses only in the ipsilateral hemisphere (p < 0.0001), whereas pairing with −40mV depressed synapses only in the contralateral hemisphere (p < 0.0001). Altogether, the results support the idea that in vivo each agonist facilitate

synaptic changes in one polarity, suppresses changes in the opposite polarity, and do not affect the reversal of plasticity. Temozolomide mw We have shown that agonists for specific Gq11- and Gs-coupled receptors can bring synapses to LTD-only or LTP-only states. To complement these findings we asked whether the polarity of plasticity can also be controlled using antagonists to alter the Gs/Gq11 balance set by endogenous neurotransmitters. We focused on blocking the basal activity of β-adrenergic receptors (coupled AZD8055 to Gs) because we previously showed that blocking LTD induction requires antagonists against multiple Gq11-coupled receptors (adrenergic, serotonergic, cholinergic, and metabotropic glutamate receptors) (Choi et al., 2005). We first examined the effects of the β-antagonist propranolol (5 μM

at least 30 min before baseline and throughout the experiments) on plasticity induced in vitro. At this concentration propranolol did not affect baseline responses (93% ± 4% at 20 min, p = 0.5, n = 5; data not shown) yet it severely impaired the induction of LTP with 0mV pairing (CTR: 141.1 ± 5.1, p < 0.001, n = 20; Prop: 97.5 ± 7.1, p = 0.662, n = 12) (Figure 9A)

and promoted the induction of LTD with −20mV pairing (82.5 ± 8.1, p = 0.068, n = 12; Prop: 76.8 ± 7.7, p = 0.0039. n = 11) (Figure 9B). Next we examined whether systemic administration of propranolol promotes the induction of LTD in vivo using the experimental design described Cell press in Figure 7, which consist of 1 hr of monocular stimulation followed by ex vivo quantification of mEPSCs in the monocular segments of the cortices contra- and ipsilateral to the stimulated eye. In these experiments, we coinjected propranolol (10 mg/kg) with the norepinephrine re-uptake inhibitor maprotiline (10 mg/kg) to boost the endogenous level of norepinephrine. The results, shown in Figure 9C, indicate that the average amplitude of all EPSCs recorded in the contralateral (stimulated) cortices was smaller than the average amplitude of the mEPSCs recorded in the ipsilateral (nonstimulated) cortices (Contra: 13.3 ± 0.39 pA, n = 23 cells; Ipsi: 15.28 ± 0.40 pA, n = 25 cells, five rats; p < 0.0001) (Figure 9C). The distributions of mEPSC amplitude were significantly different (Wilcoxon test: p < 0.0001).

To increase basal rate of NLG1 cleavage, cultures were incubated with bicuculline (50 μM) and 4AP (25 μM) 2 days prior to imaging (Figures 3A and 3B). Interestingly, terminals apposing synapses with GFP-NLG1-ΔSD3 exhibited faster FM4-64 unloading kinetics (τ = 46.1 ± 1.2 s; Figure 6G) than terminals contacting GFP-NLG1-expressing cells (τ = 60.5 ± 1.5 s), indicating that blocking NLG1 cleavage increases presynaptic

release probability. To address whether cleavage of NLG1 is regulated by activity in vivo, we measured NLG1-NTFs generated during SCH727965 pilocarpine-induced status epilepticus (PSE) in mice. Intraperitoneal administration of pilocarpine in P60 mice induced robust seizures and resulted in a 2.2 ± 0.3-fold increase of soluble NLG1-NTFs in the hippocampus after 2 hr PSE (Figures 7A–7C). To test whether MMP9 is involved in PSE-induced NLG1 cleavage, we performed pilocarpine injections in MMP9 KO mice. Notably, 2 hr

PSE characterized by robust behavioral seizures failed to elevate soluble NLG1-NTFs in MMP9 KO hippocampus (1.1 ± 0.1 relatively to control; selleck chemicals llc Figures 7B and 7C). As a control for epileptic activity, both WT and MMP9 KO mice exhibited upregulation of the activity-regulated protein Arc/Arg3.1 after PSE (Figure 7B). Given the enrichment of NLG1-NTFs during the first postnatal weeks (Figures 2G and 2H), we addressed whether NLG1 cleavage is regulated by sensory experience during development. For this, we subjected mice to 5 days of dark rearing (DR) from P21–P26, a period of heightened sensory-evoked refinement of visual cortical circuits (Hensch, 2004), and subsequently re-exposed them to light for a brief period of 2 hr (DR+2hL, Figure 7D). This protocol induces rapid synaptic remodeling in the visual cortex and results Linifanib (ABT-869) in extensive molecular, functional, and structural synaptic changes (Philpot et al., 2001; Tropea et al., 2010). With this paradigm, 2 hr of re-exposure to light after 5 days of DR caused an increase in NLG1

cleavage in the visual cortex of WT mice (DR: 1.0 ± 0.1; DR+2hL: 1.5 ± 0.2, relatively to light-reared (LR) group; Figures 7E and 7F), but not in MMP9 KO animals (DR: 0.9 ± 0.1; DR+2hL: 1.0 ± 0.1; relative to LR group). Together these findings indicate that increased neuronal activity in vivo triggers MMP9-dependent cleavage of NLG1 in both mature and developing circuits. Although implicated in diverse forms of activity-dependent synaptic maturation and plasticity (Choi et al., 2011; Chubykin et al., 2007; Jung et al., 2010), it has been unclear whether neuroligins acutely regulate synapse function and whether the neuroligin-neurexin transsynaptic complex undergoes dynamic dissociation. Here we have shown that increased neuronal activity decreases synaptic NLG1 in minutes.

9 ± 1.7 and the mean nonverbal IQ was

88.4 ± 1.4. Self-reported ancestry was as follows: White non-Hispanic, 74.5%; mixed, 9.3%; Asian, 4.3%; White Hispanic, 4.0%; African-American, 3.8%; other, 4.2%. Additional phenotypic data may be found in recent publications (Fischbach and Lord, 2010) and at www.sfari.org/simons-simplex-collection. DNA samples derived from whole blood (n = 4381), cell lines (n = 68), or saliva (n = 8) were genotyped on the Illumina IMv1 (334 families) or Illumina IMv3 Duo Bead arrays (840 families), which share 1,040,853 probes in common. CNV prediction was performed by PennCNV (PN) (Wang et al., 2007), QuantiSNP (QT) (Colella et al., 2007), and GNOSIS (GN), (www.CNVision.org) (Figure 1). To assess selleck screening library detection accuracy, we evaluated 115 predicted rare CNVs (≤50% of the span of the event found at > 1% in the Database

of Genomic Variation [DGV; Cabozantinib http://projects.tcag.ca/variation/]) by quantitative polymerase chain reaction (qPCR). A higher positive predictive value was observed for CNVs called by PN and QT, with or without GN (PPV = 97% with GN, PPV = 83% without) than for other combinations of algorithms, irrespective of the number of probes mapping within the structural variation (Table S2 and Figure S1); these “high-confidence” criteria were subsequently used to identify all rare transmitted CNVs. Given a particular interest in de novo variation and the relative challenge of accurately detecting these CNVs (Lupski, 2007), we sought to optimize our detection strategy further for this class of structural variation by using the first 585 quartets with complete genotyping data (Figure 1). We identified de novo events from among the predicted rare high-confidence CNVs based on the combination of within-family intensity and genotypic data and used a blinded qPCR confirmation process (Figure S1). Fifty-three percent of de novo predictions based on ≥20 probes (n = 94) were

confirmed compared with 2.6% based on <20 probes (n = 430). Eighty-two percent of failures were false-positive predictions in offspring; 18% were false-negatives in parents. The data from this experiment were then used to further refine de novo prediction thresholds (Supplemental Experimental Procedures). In addition, given the large number of predictions of small CNVs, and the low yield of true positives in the pilot most data set (Figure S1), we elected to restrict all further statistical analysis to those rare de novo events that both encompassed ≥20 probes and were confirmed by qPCR in whole-blood DNA (Figure S1). Subsequently, at the conclusion of our study, we were able to evaluate our methods further via a comparison of confirmed de novo CNVs identified in our study versus those detected by Nimblegen 2.1M arrays from among a total of 1340 overlapping subjects (probands or siblings), as described by Levy and colleagues in this issue (Levy et al., 2011).

There are at least two possible molecular mechanisms through which localization of OBP49a at the cell surface of sugar-responsive GRNs inhibits these neurons. OBP49a binds directly to bitter compounds and either interacts with or lies in close proximity to the sucrose receptor PD0332991 price GR64a. According to one possibility, OBP49a might deliver bitter chemicals to the cell surface of sugar-activated GRs, thereby greatly increasing the

local concentration of bitter chemicals. The bitter chemicals might then bind to sugar-activated GRs, causing them to change from a high-affinity state to a low-affinity state for sugars. Alternatively, the bitter chemicals might not bind directly to sugar-activated GRs, even at very high concentrations. Rather, once bound to bitter tastants, OBP49a might undergo a conformational change that in turn inhibits the GR64a complex. Since GRs may be cation channels (Sato et al., 2011), OBP49a might provide insects a mechanism by which bitter compounds suppress sugar-activated cation conductances. All fly stocks were maintained

on conventional cornmeal-agar-molasses medium under 12 hr light/12 hr dark cycles at 25°C and 60% humidity. 70FLP,70I-SceI/CyO, Sco/CyO,P[w+,Cre], UAS-mCD8::GFP flies were obtained from the Bloomington Small molecule library concentration Stock Center. Gr5a-GAL4 and Gr66a-I-GFP were provided by K. Scott. ASE5-GFP, nompA-GAL4, and UAS-SNMP1-YFP(2) were provided by J.W. Posakony, Y.D. Chung, and L. Vosshall, respectively. To generate pw35loxPGAL4, we modified the pw35GAL4 vector ( Moon et al., 2009). We inserted loxP oligonucleotides into the NotI and Acc65I sites. Each oligonucleotide also included portions of the NotI and Acc65I sites so that these two restriction sites were preserved. The loxP sequences were in the same orientation so that we could remove the floxed mini-white and the GAL4 coding sequences after genetically introducing the Cre recombinase. To generate the Obp19b1, Obp49a1, and Obp56g1 alleles, we PCR amplified 3 kb genomic DNAs encompassing

both the Org 27569 5′ and 3′ ends of the Obp coding sequences from isogenic w1118 flies. The genomic fragments were selected to introduce deletions of 930, 759, and 465 bp, respectively. To produce the OBP57c1 allele, we PCR amplified from isogenic w1118 flies a 3 kb genomic DNA extending from the 5′ end of the start codon, and a 3 kb genomic DNA extending from the 3′ side of the start codon. This latter DNA included a stop codon at codon position one. Each homologous arm was subcloned into the pw35loxPGAL4 vector. The transgenic flies were generated by first obtaining random insertions of the transgenes (BestGene) and then by mobilizing the transgenes and screening for targeted insertions as described previously ( Gong and Golic, 2003). Each Obp mutation was confirmed by genomic PCR.