Having identified a mechanism of cocaine-dependent regulation of HDAC5, the authors seized the opportunity to test the biological requirements for HDAC5 regulation in behavioral adaptations to cocaine. Using stereotaxic injection of viruses into the NAc of adult mice, the authors found that overexpression of the S279A HDAC5 mutant, which cannot be phosphorylated at S279, inhibited CPP.

These findings are consistent with previous evidence implicating HDAC5 Navitoclax mouse in the inhibition of reward (Renthal et al., 2007). However, they further suggest that regulation of HDAC5 phosphorylation at S279 is an essential part of this mechanism. Unfortunately, how the S279A mutation disrupts HDAC5 function in CPP is not entirely clear, since the authors uncovered no differences in nucleocytoplasmic shuttling between this mutant and wild-type HDAC5 in cultured striatal neurons. Though it remains possible that mutation of S279 to alanine could selectively affect HDAC5 trafficking in adult striatal neurons in vivo, an alternative explanation is that this mutation affects the ability of HDAC5 to act as a corepressor through mechanisms that remain to be identified. The work of Taniguchi and colleagues substantially enhances understanding of the molecular players that lie between exposure to cocaine Ion Channel Ligand Library in vitro and a key enzyme

that regulates histone acetylation. However, the specific findings of this study also raise important new questions about the downstream consequences of HDAC5 regulation for behavior. For example, Renthal and collaborators identified a large set of gene transcripts that were dysregulated in Hdac5 knockout mice compared with their wild-type littermates ( Renthal et al., 2007); however, whether these are direct or indirect targets of HDAC5 regulation remains

unknown. Taniguchi and colleagues Phosphoprotein phosphatase propose that repression of MEF2-dependent transcription is an essential function of HDAC5 and point out that the phenotype of the HDAC5 S279A mutant in CPP is opposite of that seen upon viral overexpression of a constitutively active MEF2 ( Pulipparacharuvil et al., 2008). However, Renthal reported that deletion of the MEF2 binding domain in HDAC5 had no effect on HDAC5-dependent inhibition of CPP ( Renthal et al., 2007). Thus, further experiments will be needed to clarify the gene regulatory pathways that require HDAC5. It will also be important to determine which striatal neuron classes utilize HDAC5 regulation. Given the requirement for cAMP elevation in the cascade that leads to S279 dephosphorylation, it is likely that the D1-class dopamine receptor-expressing medium spiny neurons are a major site of HDAC5 regulation in this study.

As in AD, the role of inflammation in HD pathogenesis may

similarly involve both peripheral and CNS-resident components of the innate immune system. In patients with HD, increased production of inflammatory cytokines can be detected many years prior to symptom onset, and plasma levels of proinflammatory cytokines correlate with symptom progression (Björkqvist et al., 2008). Circulating monocytes from HD patients are more responsive to a proinflammatory signal than monocytes from control patients, a finding that has been recapitulated in multiple HD mouse models (Björkqvist et al., 2008). This hyperreactivity of monocytes may reflect functional alterations triggered by the presence of mutant huntingtin protein. Whether such functional alterations directly contribute to neurodegeneration in HD remains to be determined. One mechanism whereby peripheral innate immune function Alectinib clinical trial could potentially influence neuron survival or

degeneration in HD involves the tryptophan catabolism pathway, which has been shown to be altered by the expression of mutant huntingtin in yeast (Giorgini et al., 2005). One upstream metabolite in this pathway, L-kynurenine is neuroprotective, while downstream metabolites, 3-hydroxykynurenine and quinolinic acid, are neurotoxic (Zádori et al., 2009). MSNs are preferentially susceptible to the toxicity of quinolinic acid (Roberts et al., click here 1993). A recent study reported that pharmacological inhibition of the rate-limiting enzyme in this pathway, kyneurenine 3-monooxygenase (KMO), markedly slowed disease progression in HD mice (Zwilling et al., 2011). Since the KMO inhibitor employed in this study does not cross the blood-brain-barrier, the authors suggest that inhibition of KMO in the peripheral innate immune system is sufficient to increase levels of neuroprotective metabolites from the tryptophan catabolism pathway in the Chlormezanone CNS. Since KMO expression is promoted by proinflammatory stimuli (Connor et al., 2008), the increased inflammatory responses reported in HD peripheral monocytes may enhance

KMO expression and/or activity and exacerbate neurodegeneration. Interestingly, KMO inhibition also ameliorated pathology in a murine AD model (Zwilling et al., 2011), suggesting that a similar metabolic mechanism may comprise another facet of CNS-innate immunity cross-talk involved in AD neurodegeneration. Both pathological and positron emission tomography (PET) studies have shown that patients with PD exhibit a robust inflammatory response in brain regions undergoing neurodegeneration (Gerhard et al., 2006, McGeer et al., 1988, Ouchi et al., 2009 and Wersinger and Sidhu, 2002). Furthermore, as in AD, epidemiological studies suggest that chronic users of nonsteroidal anti-inflammatory drugs (NSAIDs) may have a decreased risk of PD (Samii et al., 2009).

This property of morphogen signaling could be particularly useful in the context of developing circuits. During development, many circuits undergo activity-dependent synaptic refinement, where plasticity in each circuit is restricted to specific times during development, often referred to as critical periods (Hensch, 2004). Thus, these forms of critical period plasticity occur in a switch-like manner, opening and closing during specific developmental time windows. We speculate that morphogens and their antagonists may provide a biochemical mechanism for spatial and temporal patterning of synaptic plasticity during development. Antiplasticity molecules could also stabilize

circuit function. selleck compound It has long been proposed that mechanisms must exist that oppose correlation based rules for activity-dependent plasticity (e.g., LTP and LTD) (Miller, 1996). These correlation based plasticity rules are thought to confer instability on circuits because repeated potentiation or depression would systematically shift all synapses to higher or lower activities. Homeostatic plasticity (or metaplasticity) has been proposed as a potential solution to this problem (Pratt et al., 2003). We propose that antiplasticity molecules may also perform this stabilizing function. Inappropriate changes in circuit activity could be prevented by expression of molecules such as RIG-3,

whose function is to prevent expression of plasticity. Conversely, mutations Ibrutinib chemical structure in antiplasticity

molecules would perturb circuit activity, and may contribute to cognitive and behavioral disorders. Strains were maintained as described previously at 20°C (Brenner, 1974). OP50 Escherichia coli were used for feeding. The wild-type reference strain was N2 Bristol. Descriptions of allele lesions can be found at www.wormbase.org. The mutant strains used were: eri-1(mg366), lin-15B(n744), rig-3(ok2156), acr-16(ok789), cam-1(ak37), mig-14(ga62), cwn-1(ok546), and egl-20(n585). over RNAi assays were performed in the eri-1; lin-15b background ( Wang et al., 2005). RNAi clones utilized were previously described ( Kamath and Ahringer, 2003 and Kamath et al., 2003). Acute aldicarb assays were performed in triplicate on young adult worms by an experimenter unaware of the identity of the RNAi clone utilized, all as described ( Lackner et al., 1999). Aldicarb (Sigma and Roche) concentration was 1 mM. All quantitative imaging was done using a Olympus PlanAPO 100× 1.4 NA objective and an ORCA100 CCD camera (Hamamatsu). Worms were immobilized with 30 mg/ml BDM (Sigma). Imaging was done in either untreated animals or after a 60 min exposure to 1 mM aldicarb. Line scans of dorsal cord fluorescence were analyzed in Igor Pro (WaveMetrics) using custom-written software (Burbea et al., 2002 and Dittman and Kaplan, 2006).

, 1997). FGF-2 loss also resulted in a decrease in the slow-dividing stem cell pool and less neurogenesis (Zheng et al., 2004). EGFR is primarily expressed on type C cells and a limited number of type B1 cells, and studies of the EGFR-expressing population www.selleckchem.com/products/BMS-777607.html have indicated that most neurospheres arise from the C cell population (Vescovi et al., 1993 and Doetsch et al., 2002). Exogenous stimulation of the EGFR by ventricular infusion of EGF has striking effects within the adult VZ-SVZ. First, an increased number of type B1 cells contacting the ventricle are visible by electron microscopy (Doetsch et al., 2002). Second,

VZ-SVZ cells exhibit increased proliferation, and generate progeny that invade the surrounding parenchyma (Craig et al., 1996, Doetsch et al., 2002, Aguirre et al., 2005, Aguirre et al., 2007 and Gonzalez-Perez et al., 2009). Elevated EGF signaling biases VZ-SVZ cells toward the oligodendrocytic lineage—rather than giving rise to neurons, labeled EGF-stimulated selleck screening library progenitors largely differentiate into oligodendrocytes or oligodendrocyte precursor cells (Gonzalez-Perez et al., 2009). The most likely endogenous ligand for this pathway is transforming growth factor-alpha (TGF-α). TGF-α-deficient mice exhibit decreased proliferation

within the adult VZ-SVZ, and these proliferation defects can be rescued in vitro by administration of EGF (Tropepe et al., 1997). More recently, TGF-α treatment has been suggested to decrease the percentage of highly motile neuroblasts within the RMS (Kim et al., 2009), but EGFR overexpression in NG2-positive progenitors has been reported to increase migration,

suggesting that this pathway may have different functions in distinct cell types (Aguirre et al., 2005). Intriguingly, the related receptor ErbB4 and its ligands, neuregulin 1 and 2, are also expressed in the adult VZ-SVZ and have been implicated in progenitor proliferation and the initiation of neuroblast migration (Ghashghaei et al., 2006). The platelet-derived growth factor (PDGF) signaling pathway also alters stem cell properties and through lineage decisions, although the endogenous source of ligand for this pathway is unknown. The PDGFRα is expressed by most GFAP-positive cells within the adult VZ-SVZ, and PDGF enhances in vitro neurosphere generation in cooperation with bFGF (Jackson et al., 2006). Infusion of PDGF, like EGF, induces elevated proliferation in VZ-SVZ cells, and many of these progenitors give rise to oligodendrocytes after ligand infusion has ended. However, PDGFRα staining and EGFR staining label separate populations of cells within the adult VZ-SVZ, suggesting that they affect stem and transit-amplifying populations respectively.

33 ± 23.07 pA, n = 6) ( Figure 4D). These results indicate that the binding of exogenously delivered s-SOL-1 is sufficient to reconstitute the function of the receptor complex and that SOL-2 is required cell autonomously to recruit s-SOL-1 to the complex. Our results also suggest that in sol-1 mutants the remaining components of the receptor complex are stably located in the plasma membrane. Presumably, muscle-secreted s-SOL-1 diffuses in the extracellular space and binds to neuronal SOL-2 to reconstitute the GLR-1 receptor complex. One might imagine that postsynaptic signaling molecules, such as SOL-1, have critical developmental roles in addition

to their known signaling functions. Thus, the behavioral defects in sol-1 mutants might also be a consequence of developmental defects in synaptic morphology or function. To test whether SOL-1 has an obligate developmental role, we generated transgenic sol-1; lurcher Selisistat and sol-1; sol-2; lurcher mutants that expressed GFP::s-SOL-1 under the control of a heat-shock HIF pathway inducible promoter (Phsp::gfp::s-sol-1). Four hours following heat shock of adult worms, we assessed their behavior. In the absence of the Phsp::gfp::s-sol-1 transgene, or in the absence of heat shock, sol-1; lurcher mutants did not reverse nearly as often as wild-type lurcher worms ( Figure 4E). In contrast, heat shock induction of GFP::s-SOL-1 rescued reversal

behavior in adult sol-1; lurcher mutants, and the rescue was dependent on sol-2 ( Figure 4E). In a complementary set of experiments, we examined whether heat shock driven expression of SOL-2::GFP in adult sol-2 mutants could similarly rescue the behavioral phenotype. Following heat shock induction, reversal behavior in adult sol-2; lurcher mutants was restored to wild-type values ( Figure 4F).

We extended these studies to examine glutamate-gated currents in heat-shocked worms and next found that within 4 hr of heat shock we could record near wild-type glutamate-gated currents from the transgenic sol-2 mutants ( Figure 4G). These experiments demonstrate that the function of s-SOL-1 is dependent on SOL-2, that the remaining components of the receptor signaling complex are stable in the absence of SOL-1, and that SOL-1 and SOL-2 have ongoing roles in synaptic transmission in the adult nervous system. Glutamate-gated currents are significantly reduced in sol-2 mutants, yet paradoxically, SOL-2 is not essential for reconstitution studies in muscle cells or Xenopus oocytes ( Figure 1; Walker et al., 2006a, 2006b). One possibility is that the overexpression of SOL-1 in reconstitution studies partially compensates for the absence of SOL-2. This hypothesis predicts that overexpressing SOL-1 in sol-2 mutants should rescue glutamate-gated currents. Conversely, overexpressing SOL-2 should not rescue sol-1 mutants.

Slight changes in the direction of motion occurred at all three locations, but the trial ended without reward if the animal responded to a change at an uncued location. To measure the effect of normalization

for each neuron (Figure 1A), we collected data while the animal was cued to attend to the location outside of the receptive field, so that spatial attention did not modulate the neuron’s rate of firing. To prevent feature attention from modulating the response, the Gabors presented at the cued location always drifted in the same direction, which was intermediate between the preferred and null directions of the neuron. While attention was directed outside the receptive field, series of Gabors were presented at the two locations within the receptive field. Whenever a pair of Gabors appeared in the receptive field, one drifted in the preferred direction of the DNA Damage inhibitor neuron and the other drifted in the null direction, PCI 32765 but the locations of the preferred and null stimuli were pseudorandomly selected on each presentation. Additionally, each receptive field stimulus had a pseudorandomly selected contrast of 0%, 50%, or 100%. Using 0% contrast meant that stimuli sometimes briefly

appeared alone in the receptive field. The stimulus presentations were short (200 ms; Figure 1C) so that the animal did not have time to adjust its attention based on the contrast or number of Gabors that appeared from (Williford and Maunsell, 2006, Lee and Maunsell, 2009 and Lee and Maunsell, 2010). To measure the effect of spatial attention for each neuron (Figure 1B),

the animal’s attention was directed to one of the two locations within the receptive field. The drifting Gabors within the receptive field were independently and pseudorandomly set to a contrast of 0% or 100% on each presentation. One Gabor within the receptive field drifted in the preferred direction and the other drifted in the null direction. For most neurons (72 of 117) drift direction was pseudorandomly assigned to the receptive field locations for each short stimulus presentation. If the animal responded to a direction change from preferred to null or vice versa (i.e., 180° direction change) the trial was terminated without reward. For the remaining neurons (45/117) the locations of the preferred and null directions were fixed, but results from those neurons were not significantly different. By presenting the Gabors at 0% or 100% contrast, we could measure attention with one or two stimuli in the receptive field. Different MT neurons showed different degrees of normalization. Figure 2A shows responses from a neuron with pronounced normalization. The average response to a preferred direction alone (in either receptive field location; thick black line) was substantially reduced when a null stimulus was added to the other receptive field location (dashed line).

Typically-developing children (n = 38, 24 girls) completed data collection at two visits (separated by 36 ± 10 months), and provided high-quality fMRI and behavioral data at both time points (average age = 10.0 ± 0.57 and DAPT nmr 13.0 ± 0.67 years at T1 and

T2, respectively). Participants and their parents provided written informed assent/consent according to guidelines specified by the Institutional Review Board at UCLA. Participants had no history of significant medical, psychiatric, or neurological disorders. As a group, participants were ethnically and socioeconomically diverse. Fifty percent of participants were White, 25% Hispanic, 7.5% Asian, 5% Black, 5% Native American, and 2.5% Pacific Islander; 15% of this sample reported being multiethnic, including one participant (2.5%) who primarily identified as multiethnic. Parent reports of annual household income ranged from <$25,000 to >$400,000 (with an average income Akt inhibitor bracket of $100,000–$120,000). Full-scale IQ assessed by the Wechsler Intelligence Scale for Children (Wechsler, 1949) ranged from 86 to 144

(with an average IQ of 118). The Pubertal Development Scale (PDS; Petersen et al., 1988) was completed at both time points; on this measure, participants self-report visible development of secondary sexual characteristics. There was a highly significant increase from T1 to T2 on the PDS [Ms = 1.58 and 2.56 at T1 and T2, respectively; t(1, 37) = 9.43, p ∼ 0]. According to methods outlined by Shirtcliff et al. (2009),

the PDS was transformed into values corresponding with Tanner stages on a gender-specific basis. These transformations suggested that the average level of development was similar between girls and boys in late childhood (T1 Ms = 1.96 and 1.93 for girls and boys, respectively, indicating early pubertal status). By early adolescence, the girls were slightly more advanced (T2 Ms = 3.73 and 3.07 for girls and boys, indicating mid-to-late and midpubertal status, respectively). Participants also filled out the RPI Scale (Steinberg and Monahan, 2007), and indicators of risk behavior and delinquency as utilized in the Lerner 4-H Study of Positive Youth Development Survey Non-specific serine/threonine protein kinase (IRBD; Gestsdóttir and Lerner, 2007). The RPI is a self-report measure of resistance to peer influence that has been validated in nearly 4000 individuals ranging in age from 10–30 and varying in ethnicity and socioeconomic status. Important advantages of the RPI are that it targets primarily neutral, not antisocial or deviant influences, and minimizes socially desirable responding. On average, resistance to peer influence is lowest in late childhood and early adolescence, but then increases linearly beginning around 14 years of age. Our study thus presumably investigated the window of maximum susceptibility.

The proportion of rotavirus positives among surveillance stool samples was 3.1%

(825/27,008) and among diarrheal samples was 17.5% (324/1856). Rotavirus was associated with 15.1% of mild diarrhea, 38.9% of moderate/severe diarrhea and 66.7% of very severe diarrhea. Of all rotavirus diarrheal episodes, 18.6% were moderate/severe and 4% of affected children Selleck MLN0128 were hospitalized. Of the diarrheal episodes which resulted in hospitalizations, 28% were associated with rotavirus compared to 13% of diarrheal episodes treated at home. Rotavirus diarrhea presented more often with vomiting (27% vs 14%, p < 0.001) and fever (25% vs 16%, p < 0.001) than non-rotaviral diarrhea ( Table 3). Children with rotaviral diarrhea were taken to hospital, needed intravenous rehydration and hospitalization more frequently than children with non-rotaviral diarrhea, but these differences were not statistically significant. Rotaviral diarrhea lasted a little longer, 3 (2–5) days (p < 0.001), and the proportion that was severe was greater in rotaviral diarrhea than non-rotaviral diarrhea (p = 0.002). Vesikari score was 6 (5–9) for rotaviral diarrhea and 5 (4–7) for non-rotaviral diarrhea. Of the 373 children in the cohort, 237 (63.5%) children experienced at least one rotavirus infection in the first year. A comparison of the infected children with the non-infected children demonstrated that

developing rotavirus infection in the first year Dabrafenib in vivo was associated with the mother’s educational status, religion and birth order (Table 4). Month of birth was not associated with risk of developing rotavirus infection. Factors associated with risk of developing symptomatic rotavirus were explored by comparing children who ever had a rotavirus diarrhea with children who had a rotavirus infection but never developed rotavirus diarrhea (Table 5). Of the 352 children who were eligible for the analysis, 193 children developed rotavirus diarrhea at least once while the remaining 159 did not develop rotavirus diarrhea but had one or more rotavirus infections. The final model showed that a child was more likely to develop

rotavirus diarrhea if male (odds ratio 1.6, p = 0.03), or had an illiterate mother (odds ratio 1.8, p = 0.04), and less likely much if first-born (odds ratio 0.6, p = 0.09). Genotyping results were available for 582 samples, 309 (53%) from children who had an asymptomatic infection whereas the other 243 (47%) were from children who had diarrhea. The most common G:P combinations observed were G1P[8] (14%), G2P[4] (11.5%), G10P[11] (7.4%), G9P[8] (6.5%), G1P[4] (4.6%), G1P[6] (1.2%), G10P[4] (1.2%), and G9P[4] (1.0%). Other genotypes identified were G3, G4, G8, G11 and G12 and P[3], P[9], P[10] and P[25]. Mixed infections were identified in about 39 (6%) of samples. Both G and P were untypable in samples from 88 (15.1%) infections.

15 were covered. The two NHBA 21 fHbp 1.15 strains not predicted to be covered were from Québec. This study provides the first data on the potential coverage of

Canadian MenB isolates by the investigational 4CMenB vaccine. Using a conservative predictor for coverage, 4CMenB appears to provide good strain coverage (65% for cc41/44 and 82% for cc269) for the most prevalent recent ccs, ABT-263 nmr which include ST-269 and ST-154 predicted covered at 95% and 100%, respectively. Across all age groups, the majority of isolates are predicted to be covered by the 4CMenB vaccine. Of note the vaccine appears to provide coverage across a wide diversity of endemic strains and is not limited to protecting against one or two subtypes. At least 40% of isolates were covered by two or more vaccine

antigens, with fHbp and NHBA contributing the most to vaccine coverage. The 4CMenB antigens are also found in non-MenB isolates thus protection against these other serogroups may be an added bonus, particularly in individuals not immunized with meningococcal conjugate see more vaccines. In terms of prevention, over two-thirds of the recent cases caused by MenB were potentially preventable with this vaccine. Our results are similar to those found in England and Wales where the overall proportion of strains estimated to be covered in 2007–2008 was 73% (57–87%) and the combinations of antigens with MATS RP above the PBT was similar to that observed in Canada [26]. The overall frequency of coverage by at least two antigens was lower (40% vs. 50%) in Canadian than in English and Welsh isolates [26], thus the chance for escape mutants to emerge with vaccine use could differ between the two countries. The last national

characterization of MenB isolates was from 1994 to 1996. In this earlier study the most commonly expressed PorA serosubtypes were P1.14 (13.3%), P1.16 (11.3%), P1.5 (7.9%), P1.7 (7.0%), P1.13 (7.0%), and P1.2 (4.3%); and the only hypervirulent clones were cc32 and cc11 [27]. The much most noticeable differences in our current study were the emergence of the ST-269 clone in Québec and a change in the prevalence of other hypervirulent clones. CC32 decreased from 12.0% in 1994–1996 to 5.1% in 2006–2009 and cc41/44 became a predominant clone, accounting for about 33% of MenB isolates in 2006–2009. Besides these temporal changes, we noted geographical differences in the distribution of common hypervirulent clones from 2006 to 2009 as exemplified by the finding of ST-269 (cc269) and ST-571 (cc41/44) mainly in the province of Québec, and ST-154 (cc41/44) from Ontario and the Atlantic provinces. By province, the predicted coverage of 4CMenB ranged from 43% to 100% and reflected the strains circulating within each region and the level of antigen expression within each isolate.

We found that one published Doc2A shRNA, but not another similarly effective published Doc2A shRNA and two different Doc2B shRNAs, altered the intrinsic properties of neurons and impaired neurotransmitter release (Figure S1

available online). Gemcitabine cost Viewed together, the expression and KD results thus argue against a selective role for Doc2A in Ca2+-triggered asynchronous release in hippocampal neurons, consistent with previous studies (Groffen et al., 2010 and Pang et al., 2011a). As a consequence, we focused on Syt7 as the most abundant candidate Ca2+ sensor that is universally expressed in all neurons and developed multiple shRNAs to knock down Syt7 mRNAs. We identified four shRNAs that suppress Syt7 expression by >80% (Figure 2A). These shRNAs, as well as the Syt7 knockouts (KOs) discussed below, interfered with expression of all Syt7 splice variants. In wild-type (WT) neurons, the Syt7 shRNAs had no effect on release as measured by inhibitory postsynaptic currents (IPSCs) elicited by a 10 Hz, 1 s stimulus train (Figure 2B). In Syt1 KO neurons, however, all four Syt7 shRNAs BMN-673 similarly suppressed the remaining asynchronous release

by ∼70% (Figures 2C and 2D). The shRNAs had no effect on intrinsic neuronal properties (Figure S2). The selective effect of the Syt7 shRNAs on release in Syt1 KO neurons but not in WT neurons could potentially be associated with changes in the expression of Syt7 or other candidate Ca2+ sensors in Syt1 KO neurons. However, quantification of all Doc2 and synaptotagmin mRNAs in WT and Syt1 KO neurons failed to uncover a specific change (Figure S3). KD experiments always give rise to concerns about specificity (Figure S1). Such specificity can be assessed by three tests. First, the same KD phenotype should be obtained with multiple independent shRNAs. Second, the KD phenotype should be rescued by expression

of shRNA-resistant WT mRNA, although this experiment is only valid if the WT rescue—which necessarily involves overexpression of the rescue mRNA—does not in itself produce a phenotype and does not impair others the shRNA-mediated KD by “sponging” up shRNAs. Third, the KD phenotype should be phenocopied by a genetic KO, although there may be genuine differences between the KD and KO phenotypes that could arise from incompleteness of shRNA-mediated KDs or from compensation effects of constitutive KOs. The first criterion for the Syt7 KD phenotype specificity was met in Figures 2C and 2D, where the lack of an effect of the Syt7 KD in WT neurons (Figures 2A and 2B) serves as an additional negative control.