First, we counted the occurrences of each possible electrical triplet pattern (Figure 4A). The recorded quadruplets were separated

into triplets for a total of n = 173 triplets. The intersomatic distances measured for each configuration were used to predict the probability of electrical and chemical connections for the nonuniform random model. The occurrences predicted by both random models were counted in the same way as for the data (Supplemental Experimental Procedures). The ratio (data/prediction) indicates the relative occurrence of each of the four possible nonisomorphic patterns, Selisistat ic50 compared to the two random connectivity predictions (Figure 4A). We found that the predictions of both random BLU9931 connectivity models differ from the data. The uniform random prediction shows large deviations compared to the data for most patterns (p values: p1 = 0.003, p2 = 0.022, p3 = 0.0004, p4 = 0.0004), confirming that the model is insufficient to describe the statistics of connections of the MLI network. The nonuniform random prediction also deviates from the data but to a lesser degree, as the occurrence of fully connected triplets (pattern 4) is correctly predicted (p values: p1 = 0.0004, p2 = 0.213, p3 = 0.0004, p4 = 0.202). We separately confirmed that the fully interconnected triplets

(pattern 4) are indeed the result of direct connections and not indirect electrical coupling (Figure S4E). To characterize the electrical connectivity with a single measure and compare it to random connectivity models, we used the clustering coefficient C. C was originally introduced as a measure of the topological organization of networks and used to tuclazepam highlight differences between small-world networks and random networks, whose average C are significantly different ( Watts and Strogatz, 1998). C is usually measured for each node

in a network. Here, we calculate C for the recorded subnetworks of triplets and quadruplets of MLIs and compute the average over the configurations where C could be measured ( Supplemental Experimental Procedures). It should be noted that the average C obtained in this way is not intended to represent the average C of the whole network but is used to compare with C predicted by random connectivity models, where it was also calculated for subnetworks of triplets and quadruplets. For triplets, C effectively measures the likelihood that if neurons A and B, and B and C are connected, then A and C are also connected. The nonuniform random model predicted a higher clustering coefficient for electrical synapses, CE, than the uniform random model. This is expected if the tested neurons are sampled locally, as they were in the experiments ( Figures S2B and S2C). However, CE of the data significantly exceeds even the nonuniform random prediction ( Figure 4B; uniform random p = 0.0001; nonuniform random p = 0.0001).

Similarly, loss of FMRP increased ARC basal expression

( Figure 3C). Furthermore, ARC synthesis triggered by BDNF was much lower in Fmr1 KO neurons compared with wild-type (WT); inhibition of Rac1 activation before BDNF stimulation blocked ARC synthesis in WT as well as the residual synthesis in Fmr1 KO neurons, whereas no effect was observed in Cyfip1-silenced neurons ( Figure 3C). Fmr1 KO neurons silenced for Cyfip1 phenocopied CYFIP1-deficient neurons, further confirming that FMRP and CYFIP1 act in the same pathway ( Figure 3C). We also investigated ARC levels in mice where CYFIP1 expression was genetically reduced. Because Linsitinib clinical trial Cyfip1 KO animals are embryonic-lethal (our observation and Bozdagi et al., 2012), we used heterozygous animals where CYFIP1 levels are reduced by 40% ( Figure 3D). We examined ARC expression in both total brain cortex and cortical synaptoneurosomes and found that Cyfip1+/− mice have elevated ARC levels at synapses ( Figure 3D). These data support the hypothesis that FMRP and CYFIP1 regulate protein synthesis downstream of Rac1 selleck products activation. Activated Rac1 reshapes the CYFIP1-eIF4E complex through a conformational change, so that when translation inhibition is lifted, more CYFIP1 becomes

available for the WRC. Our results suggest that CYFIP1 complexes have a specific function in synaptic protein synthesis and actin polymerization. As proof of principle, we aimed at uncoupling the two complexes and studying their contribution to protein translation and actin polymerization. For this purpose, we designed specific CYFIP1 mutants impairing the interactions with either eIF4E or NCKAP1. To reduce the CYFIP1-eIF4E interaction, we used a mutant replacing Lys743 with a Glu (mutant E), which has been shown over to reduce the interaction with eIF4E (Napoli et al., 2008). To interfere with the CYFIP1-NCKAP1 complex, we studied the large surface of interaction between the two proteins (Chen et al., 2010), and found two hydrophobic patches on CYFIP1 that fit to corresponding sites on NCKAP1

(Figure S5B). The second patch shows a higher complementarity to NCKAP1, in particular in a stretch of eight consecutive hydrophobic amino acids (Ala1003–Ile1010), which was predicted as an essential binding site for NCKAP1. We therefore designed two mutants: mutant Δ, lacking the C-terminal domain that harbors the hydrophobic patch (aa 922–1251), and mutant H, in which the eight hydrophobic residues were replaced by glycines. WT and mutant proteins tagged with the yellow fluorescent protein (EYFP) were expressed in HEK293T cells (Figure S5C) and displayed correct cytoplasmic localization (data not shown). To promote the incorporation of the exogenous proteins into functional complexes, we silenced the endogenous Cyfip1 with siRNAs directed against its 3′UTR ( Figures 4A and S5D).

This work extends our prior findings, which suggest that aggregate flux may occur in the setting of intracellular pathology, raising the possibility of therapies that can assist in aggregate clearance by targeting

extracellular click here species. This work has important implications for the design of therapeutic antibodies and suggests that targeting seeding activity in particular may produce the most effective agents. Several prior active and passive peripheral immunotherapy approaches against tau have also reduced tau pathology and improved behavioral deficits, but the underlying rationale for antibody choice was based either on a phospho-epitope, reactivity with neurofibrillary tangles, or was not stated (Asuni et al., 2007, Bi et al., 2011, Boimel et al., 2010, Boutajangout et al., 2010, Boutajangout et al., 2011, Chai et al., 2011 and Troquier et al., 2012). One tau immunization study, performed by vaccinating mice with full-length tau, induced pathology in wild-type mice (Rosenmann et al., 2006). However, subsequent active immunization approaches with phospho-tau peptides in tau transgenic models reduced tau pathology (Bi et al.,

2011 and Boimel et al., 2010) and showed behavioral improvement (Asuni et al., 2007, Boutajangout et al., 2010 and Troquier et al., ABT-737 price 2012). In a passive immunization study, JNPL3 tau transgenic mice were administered the PHF1 antibody intraperitoneally at 2–3 months of age, prior to the onset of tauopathy. PHF-1 targets a pathological form of abnormally phosphorylated tau (Otvos et al., 1994). Treatment reduced tau pathology and improved behavior (Boutajangout et al., 2011). However, while it decreased insoluble phosphorylated tau, isothipendyl total insoluble tau

did not change. In another passive immunization study, JNPL3 and P301S mice (at age 2–3 months, prior to the onset of tauopathy) were peripherally administered the PHF1 or MC1 antibody, which targets an aggregate-associated epitope (Jicha et al., 1999). Both treatments improved tau pathology and delayed the onset of motor dysfunction (Chai et al., 2011). In these prior studies, the mechanism of action of the antibodies was not clear, and none was explicitly tested. Indeed, some proposed an intracellular mechanism (Sigurdsson, 2009). Moreover, no study appears to have produced the magnitude of reduction in tau pathology that we observed here, with the caveats that we infused antibodies into the CNS, while the other studies utilized peripheral infusion and different animal models were utilized. We designed this study explicitly to test a prediction that extracellular tau seeds are a key component of pathogenesis. We began with a selection process to pick antibodies capable of blocking tau seeding in vitro, purposely testing agents with a range of predicted activities.

, 2009 and Kabayama et al., 2011), stimulus dependent endocytosis in the growth cone has mostly been shown to occur through clatherin mediated endocytosis (CME). CME is a known regulator of the surface buy Alpelisib expression of receptors involved in outgrowth and clatherin activity is necessary for both guidance and desensitization to guidance cues (Tojima et al., 2011). CME occurs downstream of Ca2+ elevation and is an essential mediator of Ca2+ induced chemorepulsion (Tojima et al., 2010). It is highly likely that CME is one of the first downstream events following Ca2+ elevation as it precedes any cytoskeletal remodeling associated with the turning response (Tojima et al., 2010). Recently,

it has become evident that asymmetric CME is essential for mediating guidance responses within the growth cone during repulsion. During myelin-associated glycoprotein (MAG) induced repulsion, there is a rapid spatial remodeling of cell adhesion components, including the surface receptor β1-integrin, with their distribution shifting toward the side that is opposite to the one stimulated by MAG (Hines et al., 2010). This is achieved through CME surface removal of the β1-integrin on the side of the growth cone undergoing repulsion. CME also occurs following local application of Semaphorin 3A (Tojima et al., 2010). Furthermore, local inhibition of CME through application of the clatherin inhibitor MDC was sufficient tuclazepam to

cause an attractive guidance response (Tojima et al., 2010). While these data support the selleck compound notion that asymmetric alteration of the balance of exo- and endocytosis can elicit growth cone steering, they do not directly demonstrate that endocytosis is sufficient to induce growth cone repulsion. An ultimate test for a sufficient role of local endocytosis in growth cone repulsion would require techniques that can directly and specifically elicit local endocytosis to examine the growth cone’s response. It has become increasingly

clear that directional growth cone motility is controlled by a combination of mechanisms. While each of these processes regulates distinct sets of cellular activities, they must work in concert to enable the growth cone to respond to environmental signals. Remarkably, there is a substantial amount of crosstalk among different pathways (Figure 3). For example, while the actin cytoskeleton plays a predominant role in motility by providing the major force behind cell protrusions, it also has been shown to spatially regulate microtubule dynamics and membrane recycling. This in turn would affect the delivery and retrieval of migration-relevant molecules, whose downstream targets are ultimately the actin cytoskeleton. Similarly, adhesions are both upstream and downstream of signals from the actin cytoskeleton, microtubules, and membrane recycling pathways (Kolodkin and Tessier-Lavigne, 2011 and Myers et al., 2011).

, 1968 and Peters et al., 1968). While the molecular composition of this granular layer is not fully understood, by analogy with nodes of Ranvier it is thought to contain a high density of voltage-gated channels together with specialized anchoring proteins important for action potential generation. In addition, the AIS of some neuronal cell types, such as cortical pyramidal neurons, receives

synaptic input (Figure 1) (Somogyi et al., 1998). Experiments in the 1950s proposed that action potentials (APs) are initiated in the proximal axon, at either the axon hillock (Fuortes et al., 1957) or the initial segment (Araki and Otani, 1955 and Coombs et al., 1957). Aided by advances in electrical and optical recording techniques, recent data have provided direct evidence in support of these early observations,

showing that APs are initiated at the distal end of the AIS Enzalutamide in a large range of neuronal cell types. These studies have in addition revealed that the AIS is not just a trigger zone for AP generation, but also plays a key role in regulating the integration Autophagy inhibitor order of synaptic input, as well as intrinsic excitability and transmitter release. In this review we focus on the detailed electrical properties of the AIS and describe how these unique properties influence synaptic integration and shape neuronal output. We refer the reader to excellent recent reviews on the physiology of the axon proper and the molecular structure of the AIS (Debanne et al., 2011 and Rasband, 2010). While it MycoClean Mycoplasma Removal Kit has long been thought that APs are initiated in the AIS of neurons in the mammalian CNS, this is not the case in all species. For instance, multipipette recording and voltage-sensitive dye imaging indicate that AP initiation in invertebrate neurons can occur at multiple locations, which can act independently (Calabrese

and Kennedy, 1974, Maratou and Theophilidis, 2000, Meyrand et al., 1992, Tauc, 1962 and Zecević, 1996). These studies indicate that invertebrate neurons lack the functional polarization found in neurons of the mammalian CNS (Rasband, 2010). It is therefore relevant to ask why and when in evolution did neurons develop an AIS, thereby defining a single locus for AP generation? Insights into the evolution of the AIS have been obtained by studying the gene sequences of Na+ and K+ channels, which are localized to the AIS via an interaction with the cytoskeletal scaffolding protein Ankyrin G. Ankyrin G, widely used as a marker for the AIS, is restricted in expression to the AIS and nodes of Ranvier and required for targeting of voltage-gated Na+ channels to the AIS (Kordeli et al., 1995 and Zhou et al., 1998). This occurs via an interaction between Ankyrin G and a conserved nine amino acid sequence in the II-III domain of Na+ channels (Garrido et al., 2003).

Fino and selleck compound Yuste (2011) analyzed more than 60

maps and observed a very high occurrence of connections made by sGFPs onto their neighboring pyramidal cells. Almost half of sGFPs within 400 μm and three quarters within 200 μm of a given pyramidal cell were connected. Interestingly, about a fifth of all of the pyramidal cells recorded had input connections from every single interneuron in the field (20 cells on average). The authors interpret this high convergence of interneurons onto a single pyramidal cell as also implying a high divergence of a single interneuron’s connections to a neighboring population of pyramidal cells. While there is no direct evidence

of this, it is a reasonable interpretation given the relatively small number of interneurons compared to pyramidal cells and the random selection of pyramidal cells by the experimenters. To confirm the unexpectedly high degree of connectivity, the authors performed whole-cell patch clamp recordings of randomly selected pairs of sGFPs and pyramidal cells and observed a connectivity probability that closely agreed with that of their uncaging experiments. They also directly validated their method by intracellular electrical stimulation of the putatively presynaptic sGFPs I-BET151 purchase identified by two-photon uncaging and detecting responses in the postsynaptic pyramidal click here cell, verifying that almost all (11 of 12) sGFPs were truly presynaptic to the pyramidal cell. It is worth noting that paired recordings were not performed to test the putatively unconnected sGFPs to determine false negatives, so the number of truly connected interneurons may actually have been underestimated in the uncaging experiments. The authors also demonstrated that the high density of interneuron connections to pyramidal cells was similar for adult and juvenile mice and therefore was not merely a transient pattern arising in immature brain circuits prior to undergoing

synaptic pruning. How unexpected is this high degree of connectivity from sGFPs onto pyramidal cells? It is certainly higher than estimations from previous studies using patch-clamp recordings of pairs or triplets of neurons (Thomson and Lamy, 2007). In layer 2/3 rat somatosensory cortex, the connection probability of somatostatin-containing interneurons onto pyramidal cells was 49% for intersomatic distance ≤ 50 μm (Kapfer et al., 2007), while in layer 2/3 rat visual cortex, the connection probability of adapting interneurons (which includes somatostatin-expressing neurons) onto a pyramidal neuron was 16% for an intersomatic distance of 40–50 μm (Yoshimura and Callaway, 2005).

, 2009 and Ramsey et al., 2009). Disruption of NAD+ oscillation by mutations to NAD+ hydrolase CD38 alters behavioral and metabolic circadian rhythms (Sahar et al., 2011). Specifically, CD38-deficient mice display shortened circadian periodicity and alterations in plasma levels of amino acids. These data illustrate the importance of oscillating NAD+ levels and highlight its importance in amino acid regulation. Oscillating NAD+ levels also have indirect involvement in the regulation of brain function because several amino acids, such as tryptophan, tyrosine, glutamate, aspartate, glycine, and GABA, are either precursors of neurotransmitters or themselves neurotransmitters.

Volasertib concentration Although NAD+ is modulated by many additional processes (e.g., glycolysis, fatty acid synthesis), its regulation via NAMPT reinforces coupling between the circadian clock mechanism and NAD+-dependent metabolic pathways. In addition to the BMAL1/CLOCK and BMAL1/NPAS2 heterodimers that can serve as sensors for the

NAD(P)+/NAD(P)H ratio, the NAD+-dependent enzymes SIRT1 and PARP-1 may also link this metabolic ratio to the circadian clock. Levels of the enzyme SIRT1, which deacetylates histones and several transcription factors Bioactive Compound Library (Blander and Guarente, 2004), fluctuate throughout the day (Asher et al., 2008), and its activity may change as well (Nakahata et al., 2008). This enzyme physically interacts with BMAL1/CLOCK heterodimers, leading to a rhythmic deacetylation of BMAL1, histone H3 (Nakahata et al., 2008), and PER2 (Asher et al., 2008). As a result, the stability and/or activity of these proteins may be affected and lead to changes in circadian

gene expression. Interestingly, SIRT1 also affects the activity of other transcription factors such as PPARα (Purushotham et al., 2009) and coactivator PGC-1α (Rodgers et al., 2005), highlighting another avenue for the modulation of circadian gene expression and metabolism in the liver. Specifically, PGC-1α appears to be tightly linked to the circadian clock mechanism because it is expressed in a circadian fashion and serves as a coactivator of ROR (Liu et al., 2007), an activator of several clock components (see above and Figures 2 and 4). Furthermore, PGC-1α is a coactivator of FOXO1, which is part of a fasting-inducible switch that modulates gluconeogenesis (Liu et al., almost 2008). These relationships illustrate how circadian mechanisms and energy homeostasis could be related. Another potential NAD+ sensor is PARP-1 (Asher et al., 2010), a feeding-dependent factor implicated in the phase entrainment of peripheral oscillators. Illustrating a possible way to orchestrate feeding-induced phase changes and glucose homeostasis, PARP1 acts by binding to FOXO1 and attenuating the transactivation potential of the latter (Sakamaki et al., 2009). Several organic molecules serve as ligands for nuclear receptors, which regulate specific genes in response to ligand binding.

Strikingly, mutant Doc2B not only rescued minirelease at all Ca2+ concentrations, but even slightly enhanced it (Figure 4D) and reversed the small increase in apparent Ca2+ affinity observed in the DR KD neurons (Figure 4E). Thus, mutant Doc2B is fully active in this functional assay. Spontaneous minirelease probably mediates important information transfer and may be mechanistically distinct from

evoked release (Sara et al., 2005, Fredj and Burrone, 2009, Stacey and Durand, 2000 and Sutton et al., 2006). Most spontaneous release is Ca2+ dependent, and controlled by at least selleck compound two different Ca2+ sensors: a low-affinity, high-cooperativity Ca2+ sensor in wild-type synapses and a high-affinity, low-cooperativity Ca2+ sensor in synaptotagmin- or complexin-deficient synapses (Sun et al., 2007, Xu et al.,

2009 and Yang et al., 2010). For wild-type synapses, two Ca2+ sensors for spontaneous release were proposed: synaptotagmins (Xu et al., 2009) and Doc2A and Doc2B (Groffen et al., 2010). No candidate Ca2+ sensor exists for minirelease in synaptotagmin-deficient synapses, although this Ca2+ sensor may be the same as that for asynchronous release, analogous to the proposed role of synaptotagmin as a Ca2+ sensor for both spontaneous and synchronous release in wild-type BKM120 synapses. Both synaptotagmin and Doc2 are attractive Ca2+ sensor candidates for spontaneous release based on their biochemical properties, but only for synaptotagmin is there evidence linking changes in Ca2+-binding affinity to changes in spontaneous release (Xu et al., 2009). Here, we have examined the potential role of Doc2 proteins as Ca2+ sensors in spontaneous release and their relation to asynchronous release. In doing so, we strove

to avoid potential problems caused by the expression of four closely related isoforms of Doc2 proteins that could produce functional redundancy and developed an approach that allowed simultaneous KD of four different targets with a rescue control (Figures 1A and 1B). Our data confirm KO studies showing that MTMR9 Doc2 proteins are essential for normal minirelease—in fact, the degree of impairment in spontaneous release we observed with a 75% KD of all four isoforms (Figure 1 and Figure S1) is strikingly similar to that described for the Doc2A and Doc2B double KO (Groffen et al., 2010). We show that in DR KD synapses, the apparent Ca2+ dependence of minirelease exhibits a small but significant increase (Figure 1), but that otherwise no change in Ca2+ triggering of either spontaneous or evoked release is detected (Figure 2). Moreover, our results indicate that the DR KD does not alter synchronous or asynchronous evoked release and—importantly—does not impair the enhanced spontaneous release detected in Syt1 KO synapses (Figure 2). This latter result confirms the notion that spontaneous release events in Syt1 KO and wild-type neurons are qualitatively different, consistent with their distinct Ca2+ dependence (Xu et al., 2009).

1; Miesenböck et al., 1998). Ecliptic pHluorin was engineered by introducing six mutations to the wild-type aqGFP (Miesenböck et al., 1998). The pH/fluorescence intensity profile of ecliptic pHluorin is basic-shifted with respect to the parent aqGFP (Miesenböck et al., 1998). While the FP voltage sensor containing ecliptic pHluorin exhibited a small voltage-dependent change in fluorescence intensity (−1.3% ± 0.3% ΔF/F) to a +100mV voltage step (Figures 1A and 1B), we discovered in one stable HEK293 cell line an unintended point mutation, A227D (following the numbering of wild-type aqGFP residues; Figure S1A available online), within the inserted

ecliptic pHluorin. buy Torin 1 The mutant sensor produced a 14-fold increase (−18.1% ± 0.3%, n = 6) in ΔF/F (Figures 1A and 1B) per 100mV. This fractional fluorescence change is ∼3 times larger

than current CiVS-based FP voltage sensors (Lundby et al., 2008; Tsutsui et al., 2008; Akemann et al., 2010). We sought to determine whether the large response magnitude imparted by the A227D mutation could be reproduced in other FP voltage sensors. We examined the effects of the A227D mutation on FP voltage sensors containing either super ecliptic pHluorin (Sankaranarayanan et al., 2000) or eGFP. These two FPs are closely related and were both derived from the wild-type aqGFP. Super ecliptic pHluorin contains two eGFP-like mutations, F64L and S65T, in addition to mutations found in ecliptic pHluorin (Sankaranarayanan et al., 2000; Figure S1A). The two eGFP-like mutations simplify the excitation spectra of super ecliptic pHluorin to a single peak (∼490 nm) and produce a brighter and more photo-stable FP that retains PARP inhibitor review already the basic-shifted pH/fluorescence intensity profile (Sankaranarayanan et al., 2000). The FP voltage sensors containing either super ecliptic pHluorin or eGFP do not produce substantial ΔF with depolarizing steps (Figure 1B). However, introducing the A227D mutation dramatically increased the response magnitude of the super ecliptic pHluorin containing sensor (Figure 1B). In contrast, introducing the A227D mutation did not increase the response magnitude of the sensor

containing eGFP (Figure 1B). Cells expressing probes containing super ecliptic pHluorin A227D were brighter than ones expressing ecliptic pHluorin A227D (3460 ± 609 AU, n = 12 cells versus 373 ± 40 AU, n = 11 cells, respectively); however, the bleach rates were not significantly different (−4.8% ± 0.8% versus −6.4% ± 0.7% over 2 s of laser illumination). We conclude from these results that the mutations found in ecliptic pHluorin are required for the A227D mutation to confer its effect. However, the dual peak excitation spectrum of ecliptic pHluorin is not required for the enhanced response. The FP voltage sensor containing the super ecliptic pHluorin A227D was named ArcLight. The A227D mutation did not alter the level of expression of the probes at the plasma membrane or the basal cellular fluorescence level in HEK293 cells (data not shown).

Athletic and PE program questionnaires were pre-tested for comprehension and face validity with school personnel from five non-study schools and modified based on their feedback. The primary outcome measure for this analysis was sports team participation in high school. We assessed this by asking high school students, signaling pathway “In the past 12 months, on how many sports teams did you play?” Responses were dichotomized into a dummy variable (i.e., 0 = 0 sports teams, 1 = 1 or more sports teams). Sports offered per 100 students. Based on information from the

New Hampshire Interscholastic Athletic Association 24 and school websites, we created a comprehensive list of interscholastic and intramural sports and asked school personnel to indicate which sports their school offered for boys and girls. We used these responses to create a sex-specific index reflecting the number of sports available per 100 students for each school (i.e., by sex: ((number of interscholastic + intramural sports offered)/number of students × 100)). We used this metric to standardize sport availability across schools of different sizes. We dichotomized responses into two groups: less than one sport per 100 students and at least one sport per

100 students. Percent of unrestricted sports. We defined unrestricted sports as sports in which there RG7204 order was no limit to the number of students who could participate. Because intramural sports are unlimited by design, we counted all the intramural sport opportunities as unrestricted. For interscholastic sports, we determined whether or not participation was limited by asking school personnel many to “list any (interscholastic) sport for which participation is limited (i.e., the number of players per sport is capped)” and to specify whether boys and/or girls teams were limited. We then calculated the percent of unrestricted sports separately (i.e., by sex: (number of unrestricted

sports offered/(number of unrestricted + restricted sports offered) × 100)). We categorized responses into three groups of unrestricted sports based on the distribution of the data: less than 85%, 85%–99.9%, and 100%. We measured and adjusted for a number of potential confounders. Adolescent-level covariates measured concurrent with the outcome included sex, grade in school, ethnicity, and overweight/obese status. Overweight/obese status was based on self-reported height and weight and sex-specific Centers for Disease Control and Prevention BMI-for-age growth charts.25 and 26 We also adjusted for sports participation at baseline, when adolescents were in elementary school. This was assessed by asking, “In the past year, did you participate in any of the following things? Team sports (yes/no)?”27 Parent/household level covariates measured through parent surveys included education, income, and single vs. two parent household. School and school-town covariates included school enrollment, town median household income, and school town population.