Disruption of these proteins can silence neurons by preventing the release of neurotransmitter-containing vesicles. Expression of the light chain of tetanus toxin (UAS-TNT or Tet or TeTxLc) cleaves nSyb and blocks vesicle release ( Sweeney et al., 1995). Cell Cycle inhibitor UAS-TNT has been used to study the role of transmitter release in axon guidance and synapse formation ( Tripodi et al., 2008) as well as to determine the role of many types of neurons in different behaviors ( Kong et al., 2010b) although some neurons seem to be less susceptible to TNT ( Thum et al., 2006). UAS-TNT was originally tested in glutamatergic motor neurons; the release machinery for biogenic amines,

including serotonin, dopamine, octopamine, tyramine, and neuropeptides may differ. Some aminergic neurons do show phenotypes with UAS-TNT ( Friggi-Grelin et al., 2003). UAS-TNT can also affect peptide release of the Eclosion Hormone releasing cells ( McNabb and Truman, 2008), but it may not be fully effective on Pigment Dispersment Factor ( Kaneko and Hall, 2000, Blanchardon et al., 2001 and Umezaki et al., 2011) or Crustacean Cardiac Activating Peptide release ( Luan et al., 2006a). A UAS-FRT-stop-FRT-TNT is available for

intersectional experiments ( Keller et al., 2002). UAS-TNT has major virtues: it targets a neural-specific protein, and thus should only block vesicle release in neurons. Moreover, as it is a potent toxin, even low levels of expression are effective. Since Fossariinae UAS-TNT is constitutively active, chronic expression may lead to some circuit-level this website form of compensation for the silenced neurons, or cell damage within them. A way to silence neurons acutely can bypass developmental roles, reduce pleiotropic effects, and minimize the opportunity for compensation. UAS-Shibirets1, a temperature-sensitive dominant-negative form of dynamin, a GTPase required for vesicle recycling,

blocks chemical neurotransmission acutely (Kitamoto, 2001). Although UAS-Shibirets1 affects vesicle recycling in many cell types, it may act most quickly in neurons where vesicle recycling is a rate-limiting step for neurotransmission. UAS-Shibirets1 is effective in many different neuronal types, including photoreceptors and cholinergic neurons (Kitamoto, 2001), as well as peptidergic and aminergic neurons (Krashes et al., 2009 and Alekseyenko et al., 2010). UAS-Shibirets1 has been used to identify neurons involved in courtship, sleep, color vision, and taste discrimination (Kitamoto, 2002, Broughton et al., 2004, Pitman et al., 2006, Gao et al., 2008b and Masek and Scott, 2010). The acute temporal control afforded by UAS-Shibirets1 allows investigation of neurons in adult behavior and even discrimination between neurons involved in learning and memory retrieval (Waddell et al., 2000, Dubnau et al., 2001, McGuire et al., 2001 and Kasuya et al., 2009).

An approximately 50% reduction in blood calcitriol was observed during eldecalcitol treatment in the clinical trial. In the present study, we demonstrated by using VDRKO mice that the calcemic actions of calcitriol and eldecalcitol were mediated solely by VDR. Administration of small amounts of eldecalcitol in rats markedly reduced serum concentration of calcitriol, which fell to below the limit of detection at 0.1 μg/kg eldecalcitol. Plasma concentration of eldecalcitol increased dose-dependently and reached 3820 pmol/L by 0.1 μg/kg eldecalcitol Pifithrin �� administration. These observations indicate that, after administration

of eldecalcitol, the eldecalcitol rapidly replaces calcitriol in blood and exerts biological activities in target organs. It was observed in an earlier study that the binding activity of CP-673451 price eldecalcitol to VDR is approximately 1/8 of that of calcitriol in vitro [26] and that the distribution capacity of eldecalcitol to target organs is much lower than that of calcitriol in rats. In this study, based on the concentration of each compound in the blood, the relative biological activities of eldecalcitol, such as its activity in increasing serum calcium, FGF-23, and urinary calcium excretion, and in suppressing plasma PTH in vivo were only 15–26% of that of calcitriol ( Table 1). Eldecalcitol stimulated the expression of target genes in the kidneys (VDR, TRPV5, and calbindin-D28k)

and bone (VDR, FGF-23, and RANKL) much less than did calcitriol. Stimulation of target genes in the intestine by eldecalcitol treatment was comparable to that of calcitriol. These results indicate that eldecalcitol is primarily a weak agonist of VDR as compared with calcitriol in vivo. Thus, we conclude that administration of eldecalcitol rapidly suppresses endogenous calcitriol and replaces

it. However, eldecalcitol may not fully compensate for the action of calcitriol in the kidneys and bone. “
“Epidemiological studies demonstrate an inverse correlation between calcium and vitamin D intake and risk of tumor development [1] and [2]. The calcium-sensing receptor (CaSR) is a putative tumor suppressor gene in the colon, which partially mediates the anti-proliferative and pro-differentiating actions of calcium in colonocytes (for review, see [3] and [4]). However, in colon cancer anti-proliferative effects of Ca2+ are lost [5] and [6], and this could be due to loss of CaSR expression Carnitine dehydrogenase during colorectal tumorigenesis [7]. Very little is known about the factors that regulate the expression of CaSR in the colon. The CaSR gene contains 6 coding exons and two 5′-untranslated exons (exons 1A and 1B), which are under the control of promoter 1 and 2, respectively, yielding alternative transcripts but coding for the same protein [8] and [9]. Several studies performed in rat parathyroid, thyroid, and kidney have mapped binding sites of numerous transcription factors, including NF-κB, STAT, SP1, and vitamin D response elements in both CaSR promoters ( Fig.

26 ± 0.03, n = 4; DR + L 2.05 ± 0.03, n = 4; one-way ANOVA, F2,10 = 273.61, p < 0.001, Figure 5C; NARP−/− DR 1.29 ± 0.02, n = 6; DR + L 2.12 ± 0.04, n = 6; one-way ANOVA, F2,14 = 72.947, p < 0.001, Figure 5D). In both

NARP−/− and wild-type mice, the experience-dependent regulation of VEP contralateral bias was mediated by changes in the amplitude of the contralateral eye VEP (Figure S5). Thus, the expression of a form of synaptic plasticity that is dependent on early visual experience is intact in NARP−/− mice. To ask how the absence of NARP affects ocular dominance plasticity, we examined the response to brief (3 days) and prolonged (7 days) monocular deprivation (MD) on the VEP contralateral bias initiated at P25, the peak of the critical period (Fagiolini et al., 1994, Gordon and Stryker, 1996 and Fagiolini and Hensch, LGK-974 2000). As expected, both brief and prolonged monocular deprivation of the dominant contralateral eye significantly decreased the VEP contralateral bias in juvenile

wild-type mice find more (VEP amplitude contralateral eye/ipsilateral eye average ± SEM: no MD 2.19 ± 0.03, n = 5; 3 days MD 1.32 ± 0.05, n = 4; 7 days MD 1.18 ± 0.04, n = 5; Figure 6). In contrast, no shift in ocular dominance was observed in juvenile NARP−/− mice following either brief or prolonged monocular deprivation (no MD 2.16 ± 0.10, n = DNA ligase 5; 3 days MD 1.91 ± 0.07, n = 6; 7 days MD 1.92 ± 0.07, n = 6). Importantly, enhancing inhibitory output with diazepam (15 mg/kg, 1×/day) enabled ocular dominance plasticity in juvenile NARP−/− mice (5 days MD + DZ 1.09 ± 0.08, n = 5). No shift in ocular dominance was observed following diazepam

alone (VEP amplitude contralateral eye/ipsilateral eye, average ± SEM: NARP−/− + DZ no MD, 2.08 ± 0.11, n = 3, t test versus NARP−/− no MD, p = 0.61). Ocular dominance plasticity persists into adulthood in wild-type mice (Sawtell et al., 2003 and Sato and Stryker, 2008) and may utilize mechanisms distinct from those recruited by monocular deprivation earlier in development (Pham et al., 2004, Fischer et al., 2007 and Ranson et al., 2012). To ask if adult NARP−/− mice could express ocular dominance plasticity, we examined the response to monocular deprivation for 7 days beginning at P90 (Figure 7). However, this manipulation did not induce a shift in ocular dominance in NARP−/− mice (VEP amplitude contralateral eye/ipsilateral eye average ± SEM: adult NARP−/− no MD 2.15 ± 0.13, n = 5; 7 days MD 1.93 ± 0.09, n = 7). To confirm the absence of ocular dominance plasticity in NARP−/− mice, we examined the VEP contralateral bias after chronic monocular deprivation (80 days beginning at P21).

As anticipated from the results shown in Figure 4D, this decrease was fastest for d3 (black dotted line), followed by d2 (blue dotted line) and d1 (red dotted line). The color plots appearing in the lower panels of Figure 5A

quantify these findings. The colors represent probability values associated GSK1120212 purchase with the null hypothesis that the responses are not different from baseline. Dark red indicates probability values lower than the level required (Student’s t tests, evaluated at Bonferroni-corrected p < 0.05/number of comparisons across time) for rejecting the null hypothesis. Blue and green indicate values higher than that level. Shortly after stimulus onset, responses became significantly higher than baseline for all stimuli and distances. However, after color-change onset, responses to targets Crizotinib in vivo remained significantly higher than baseline, but responses to distracters dropped to baseline levels, losing significance faster for d3, followed by d2 and d1. The results were very different during fixation (Figure 5B). After stimulus onset, responses did not significantly change. The responses to stimuli corresponding to distracters in the main task condition did not significantly depart from baseline during the whole period. Although responses to stimuli corresponding to targets in the main task appear to slightly

increase after the color change, the increase did not reach statistical significance. This result demonstrates that the gradual decrease of responses to distracters in the task condition was dependent on the increase in response preceding the color change. On the other hand, during fixation response decreases were constrained by low firing rates. In order to test whether the decrease in distracter responses as a function of distance following color-cue

onset was related to motor preparation rather than to selecting and allocating attention to the target, we aligned the same normalized responses appearing in Figure 4D to the time CYTH4 of button release. This caused the distance effect to disappear (Figure S3C), suggesting that it was indeed due to processes related to target selection and the allocation of attention triggered by color changes in the RDPs. We also tested whether the distance effect in the units’ response suppression was caused by the existence of universal distracter and target stimuli (“border” stimuli) in the color scale (i.e., gray and turquoise). It is possible that these stimuli evoked a strong change in response when paired with any other color, and because the proportion of pairs containing universal stimuli is larger for d3 followed by d2 and finally d1, data pooling for pairs of the same distance may result in the pattern observed in Figure 4 (larger effects for d3, followed by d2 and d1).

Beclin 1, the mammalian ortholog of yeast Atg6/Vps30, is part of a Class III PI3K complex that

participates in autophagosome formation, mediating the localization of other autophagy proteins to the pre-autophagosomal membrane [54]. It was first identified in a yeast two-hybrid screen as a Bcl-2-interacting protein [55]. Beclin 1 possesses a BH3 domain that dictates its interaction with the BH3 receptor domain of anti-apoptotic proteins of the Bcl-2 family. Association of Beclin 1 with Bcl-2 family proteins blocks the autophagy-promoting function of Beclin 1 [56]. It has been reported that Beclin 1 directly interacts with Bcl-2, Bcl-xL, Bcl-w and to a lesser extent with Mcl-1 [57]. Overexpression of Bcl-2 or Bcl-xL

has been Venetoclax datasheet shown to inhibit autophagy by sequestering Beclin 1 [58]. Accordingly, increased expression Bortezomib ic50 or stabilization of BH3-only proteins (such as Bid, Bad or Bik), which bind to anti-apoptotic proteins through their BH3 domain, reduces the availability of anti-apoptotic proteins, thereby disinhibiting Beclin 1-dependent autophagy [56], [57] and [59]. Of note, emerging evidences reveal that the competitive displacement of Beclin 1 by pro-apoptotic proteins may not be of significance at all in turning on autophagy, for only the endoplasmic reticulum (ER)-targeted but not the mitochondrion-targeted Bcl-2 has been shown to inhibit autophagy [39]. For instance, studies have shown that Bcl-2 targeted to the ER but not mitochondrial outer membrane inhibits starvation-induced autophagy [58]. This suggests that binding of Bcl-2 to Beclin 1 prevents it from assembling the pre-autophagosomal vesicles at an ER-associated location. BH3 mimetics are small molecules that have close structural or functional similarity to BH3-only proteins. Most BH3 mimetics that are currently under preclinical and clinical development bind to and antagonize anti-apoptotic members of the Bcl-2 family of proteins [60]. Several excellent reviews on the apoptosis-promoting

effects of these BH3 mimetics have recently been published [51], [61], [62], [63] and [64]. Here, we review the three BH3 mimetics (obatoclax, (−)-gossypol, Chlormezanone and ABT-263) that have advanced furthest clinically in relation to autophagy in a variety of cancers including gastrointestinal cancers. Obatoclax (GX15-070) is a novel BH3 mimetic pan Bcl-2 inhibitor [65]. In vitro, obatoclax binds to all anti-apoptotic Bcl-2 family members and by fluorescence polarization assays has IC50s in the 1–7 μM range for Bcl-2, Bcl-xL, Bcl-w and Mcl-1 [66]. Given the inhibitory effect of anti-apoptotic Bcl-2 family proteins on Beclin 1, obatoclax is proposed to induce autophagy by disrupting anti-apoptotic Bcl-2 family proteins and Beclin 1 interactions.

The experiment consisted of 672 trials, divided into seven sessions of 96 trials. After each session, participants were presented with a wheel of fortune that randomly selected one trial from the

block; participants won an additional £1 bonus if their response on that trial was correct. The titration procedure ensured that participants typically won £5 in additional bonuses across the seven experimental sessions. A Neuroscan system with SynAmps-2 digital amplifiers was used to PD332991 record EEG signals from 32 Ag/AgCl electrodes located at FP1, FPz, FP2, F7, F3, Fz, F4, F8, FT7, FC3, FCz, FC4, FT8, T7, C3, Cz, C4, T8, TP7, CP3, CPz, CP4, TP8, P7, P3, Pz, P4, P8, POz, O1, Oz, and O2, plus four additional electrodes used in a bipolar montage as horizontal and vertical electro-oculograms (EOGs) and two electrodes located at the mastoids used as reference. All electrode impedances were kept below 50 kΩ. EEG signals were recorded at a sampling rate of 1,000 Hz and high-pass

filtered online at 0.1 Hz. Preprocessing Dinaciclib was carried out using the EEGLAB toolbox for MATLAB (Delorme and Makeig, 2004). The data were downsampled to 250 Hz, band-pass-filtered between 1 and 40 Hz, and then epoched from 500 ms before the onset of the first premask to 1 s following the offset of the postmask (i.e., 1 s following the onset of the response period). We visually inspected these epochs (1) to remove trials containing nonstereotypical artifacts (such as transient muscular activity) and (2) to identify “bad” electrodes showing frequent amplifier “jumps” or other electrical artifacts (e.g., spikes), which were interpolated to the weighted average of neighboring electrodes. A maximum of one electrode was identified as bad per participant, and only for 3 of the 15 recorded participants. Independent component analysis (ICA) was then performed on the epoched data as implemented in EEGLAB—excluding the EOG, reference, and

interpolated electrodes from the analysis—and ICA components were visually inspected to reject the ones capturing stereotypical artifacts (in particular eye blinks Methisazone and sustained high-frequency noise). Finally, single epochs were reinspected visually to ensure that no artifact remained. Rejected trials were excluded from all further analyses, resulting in an average of 565 ± 15 trials per participant (mean ± SEM). Steady-state frequency spectra were estimated using a standard Fourier transform from the onset of the first element (i.e., following the two premasks) until the offset of the last element (i.e., preceding the postmask). Frequency power was defined as the average square amplitude of complex Fourier components, whereas phase locking was defined as the length of the vector average of single-trial phase estimates.

, 1998, Rutledge

et al., 2009 and Shohamy et al., 2005). Thus, our contention that initial learning of a rotation occurs through adaptation but savings results from operant learning predicts that patients with PD would show a selective savings deficit in an error-based motor learning paradigm. This is exactly what has been found: CH5424802 datasheet patients with PD were able to adapt to initial rotation as well as control subjects but they did not show savings (Bédard and Sanes, 2011 and Marinelli et al., 2009). Thus, our framework of multiple learning processes can explain this otherwise puzzling result. A prediction would be that PD patients would show no difference in learning rates between Adp+Rep− and Adp+Rep+ protocols, because only adaptation would occur. Prevailing theories

of motor learning in adaptation paradigms have been fundamentally model-based: they posit that the brain maintains an explicit internal model of its environment and/or motor apparatus that is directly used for planning of movements. When faced with a perturbation, this model is updated based on movement errors and execution of subsequent movements reflects this updated model (Shadmehr et al., 2010). We wish to define adaptation as precisely this model-based mechanism for updating a control policy in response to a perturbation. Adaptation does not invariably result in better task performance. OSI744 For example, in a previous study we showed that adaptation to rotation occurs despite conflicting with explicit task goals (Mazzoni and Krakauer, 2006). In the current study, hyper- or overadaptation occurred to some targets due to unwanted generalization; this was why the steady-state predicted by the state-space model for Adp+Rep+ showed that subjects adapted past the 70° target for near targets and insufficiently adapted for far targets ( Figure 2D). Diedrichsen and colleagues also showed that force-field adaptation occurs at the L-NAME HCl same rate with or without concomitant use-dependent learning ( Diedrichsen et al., 2010). It appears, therefore, that adaptation is “automatic”;

it is an obligate, perhaps reward-indifferent ( Mazzoni and Krakauer, 2006), cerebellar-based ( Martin et al., 1996a, Martin et al., 1996b, Smith and Shadmehr, 2005 and Tseng et al., 2007) learning process that will attempt to reduce prediction errors whenever they occur, even if this is in conflict with task goals. In spite of the fact that most behavior in error-based motor learning paradigms is well described by adaptation, we argue here that there are phenomena in perturbation paradigms that cannot be explained in terms of adaptation alone. Instead, additional learning mechanisms must be present which are model-free in the sense that they are associated with a memory for action independently of an internal model and are likely to be driven directly by task success (i.e., reward).

, 2009 and Yamaguchi and Mori, 2005), which largely overlaps with the period when interneurons become synaptically integrated http://www.selleckchem.com/products/gsk1120212-jtp-74057.html into the olfactory bulb (15–30 days after birth). During this period, interneurons arriving to the olfactory bulb (i.e., roughly born at the same time)

compete for survival, probably because newborn interneurons are more sensitive to the overall activity of nearby circuits than mature olfactory interneurons. In agreement with this idea, interneurons that survived this period tend to persist for life (Winner et al., 2002). Thus, both the synaptic integration and the survival of newborn interneurons seem to depend on sensory activity mechanisms, which are intrinsically linked to the cell excitability. Consistent with this, synaptic development and survival of newly generated neurons are dramatically impaired in anosmic mice selleck chemicals llc (Corotto et al., 1994 and Petreanu and Alvarez-Buylla, 2002), while sensory enrichment promotes the survival of newborn olfactory interneurons (Bovetti et al., 2009 and Rochefort et al., 2002). Moreover, increasing cell-intrinsic excitability in maturing granule cells enhances their synaptic integration and partially rescues neuronal survival in a sensory-deprived olfactory bulb (Kelsch et al., 2009 and Lin et al., 2010), while forced hyperpolarization decreases

survival (Lin et al., 2010). Since most interneurons have already matured and received connections by the time they die, it has been hypothesized that only interneurons connected to active circuits would ultimately survive (Petreanu and Alvarez-Buylla, 2002), an idea that has obtained experimental support in the adult dentate gyrus (Kee et al., 2007). Thus, the death of adult-born interneurons seems to be intimately linked to mechanisms of structural plasticity in the olfactory bulb. It is presently unclear whether

programmed cell death in developing cortical interneurons depends on similar mechanisms than in the olfactory bulb, but recent experiments pointed out an interesting parallel between both structures. Southwell and colleagues (2012) found that heterochronically transplanted interneurons do not influence cell death dynamics in the endogenous population (Figure 7). This seems to suggest that the competition for survival is normally restricted to cortical interneurons born roughly at the same Linifanib (ABT-869) time, as in the olfactory bulb. Thus, it is conceivable that cell death selectively eliminate inappropriately integrated cortical interneurons within specific lineages, although this hypothesis remains to be experimentally tested. In any case, these results reinforce the view that the integration of interneurons into cortical networks critically depends on a maturational program linked to their cellular age. Much progress has been made over the past years regarding our understanding of the mechanisms regulating the migration of embryonic and adult-born GABAergic interneurons.

Surprisingly, there has been little progress

in assigning specific developmental functions to individual pathways (Lemmon and Schlessinger, 2010). Indeed, the many in vitro studies carried out with pharmacological inhibitors clearly predict that these signaling cascades integrate the effects of multiple extracellular signals and that elimination of even a single pathway in vivo would result in complex, difficult to interpret phenotypes. MAPK signaling generally refers to four cascades, each defined by the final tier of the pathway: extracellular signal-regulated kinases 1 and 2 (ERK1 and 2), ERK5, c-Jun N-terminal kinases (JNK), and p38 (Raman et al., 2007). ERK1 and ERK2 (ERK1/2) exhibit a high NVP-AUY922 in vitro degree of similarity and are considered functionally equivalent, although isoform-specific effects have been described. In the nervous system, ERK1/2 and ERK5 are the primary MAPK cascades activated by trophic stimuli and have been shown to mediate proliferation, growth, and/or survival in specific contexts (Nishimoto and Nishida, 2006). Aberrant ERK1/2 signaling plays a primary role in a range of human syndromes that affect the nervous system, particularly the family of

“neuro-cardio-facial-cutaneous” (NCFC) syndromes (Bentires-Alj et al., 2006). The precise role of ERK1/2 in the neurodevelopmental abnormalities that characterize Olaparib these disorders is only now being investigated (Newbern et al., 2008, Samuels et al., 2008 and Samuels et al., 2009). Indeed, most of our understanding of ERK function is derived from in vitro models in the context of isolated trophic stimuli. Such studies provide support for involvement of ERK/MAPK signaling in almost every aspect of neural development and function. However, the physiological relevance of many in vitro findings has not been adequately tested, and much less is known about ERK functions in the context of multiple extracellular signals, as occurs in vivo. The PNS has been the standard model system for defining biological actions

of many neurotrophic molecules. The PNS principally derives from the neural crest, which generates sensory and sympathetic neurons, satellite glia within the DRG, and Schwann cells within the peripheral nerve. Peripheral neuron development requires trophic signaling via neurotrophins and GDNF family members, which Montelukast Sodium act via RTKs that activate ERK1/2 (Marmigere and Ernfors, 2007). Analyses of PNS neuronal development in vitro have shown that ERK1/2 signaling is important for differentiation and neurite outgrowth in response to neurotrophins, other trophic factors, and ECM molecules (Atwal et al., 2000, Klesse et al., 1999, Kolkova et al., 2000 and Markus et al., 2002). ERK1/2 activation by some of these same molecules has been implicated in regulating neurite outgrowth from motor neurons, which also extend axons in peripheral nerves (Soundararajan et al., 2010).

Microtubule-associated proteins from adult flies collected 16 hr after a 1 hr, 37°C heat shock to induce GAL4 expression were purified from fly extracts as described (McGrail et al.,

1995). NMJ analysis was limited to muscle 4 unless stated otherwise. Antibodies used are detailed in Supplemental Experimental Procedures. A synapse was considered to have TB anti-HRP or anti-Dhc accumulation if the fluorescence intensity within the TB was clearly much higher than in proximal boutons. Fluorescent images were acquired by using a Zeiss LSM 510 confocal Proteasome inhibitor microscope using a PLAN-APO 63×, 1.4 NA oil-immersion objective. Maximum-intensity Z projections of confocal stacks were generated and processed by using Adobe Photoshop. Intensity measurements and NMJ TB volume were obtained by thresholding with Imaris software. For scanning electron microscopy, fly heads were coated with gold:palladium by using a vacuum evaporator and imaged immediately by using a LEO/Zeiss Field-Emission SEM. SPAIM experiments were performed as described (Wong et al., 2012). For other live-imaging experiments,

we rinsed wandering third-instar larvae and pinned them in Ca2+-free HL3 on the sylgard insert of a custom-made imaging mount, placed a coverslip over the preparation, and secured it. Imaging of axonal transport was performed on a Zeiss Axio Observer with a 40× oil objective (EC Plan-Neofluar 1.3 NA) and collected on an AxioCAM charge-coupled device camera. Movies were analyzed as described high throughput screening (Louie et al., 2008). For ANF:GFP fluorescence recovery after photobleaching (FRAP) experiments, we acquired spinning-disc confocal images of dense-core vesicles at muscle 6/7 NMJs by using a Zeiss Axio Imager Z1 microscope and 63× 1.4 NA oil-immersion objective and collected them on a QuantEM 512SC camera (Photometrics). ANF:GFP in proximal boutons was bleached by using a 488 nm laser controlled

by a Mosaic Digital Illumination System (Photonic Instruments). Electrophysiological recordings from muscle 6, segment A3, were performed as described (Imlach and SB-3CT McCabe, 2009). Data are expressed as mean ± SEM. A Student’s t test was performed for pairwise comparisons between each genotype and its wild-type control by using GraphPad Prism. We are grateful to Chris Doe, Vladimir Gelfand, Tom Hays, Rod Murphey, Phil Wong, Sangyun Jeong, and Herman Aberle for reagents. We thank Ben Choi for pMad work and Manish Jaiswal and Vafa Bayat for helpful comments. We thank Erik Griffin, Geraldine Seydoux, Norm Haughey, Terry Shelley, Michele Pucak, and the NINDS Multi-photon Core Facility (MH084020) at JHMI for assistance with imaging. We also thank the BDSC, VDRC, and DGRC for fly stocks and reagents. This work was funded by the Packard Center for ALS Research (A.L.K. and T.E.L.), P2ALS (B.D.M.), NINDS K08-NS062890 to T.E.L., R01-NS35165 to A.L.K, and RO1-NS32385 to M.Y.W. and E.S.L. A.L.K.