The profile of EEG power of Kif5a-conditional KO mice showed a significant power reduction in both rest and locomotive states, suggesting that neuronal network activity is impaired by postnatal loss of KIF5A. All procedures were approved by the Graduate School of Medicine, The University of Tokyo. Because it is known that disturbance of inhibitory synaptic transmission is involved in epileptic seizure generation (Jacob et al., 2008; Rudolph and Möhler, 2004), we speculated that GABAergic synaptic transmission may be impaired in the hippocampus of Kif5a-conditional

KO mice. We performed BMS354825 whole-cell patch-clamp recordings to investigate the miniature Selleck Olaparib inhibitory postsynaptic currents (mIPSCs) in the CA1 region of hippocampal

slices ( Figure 2A). We observed a significantly reduced mean amplitude of mIPSCs in the slices of Kif5a-conditional KO mice compared with those of controls (control, 16.5 ± 0.7 pA; conditional KO, 8.5 ± 0.4 pA). A cumulative probability curve also indicated a leftward shift to smaller amplitudes in Kif5a-conditional KO mice ( Figure 2F). However, the frequency, rise time, and decay time, although slightly reduced, were not statistically different between genotypes ( Figures 2C–2E and 2G). Furthermore, the ratio of evoked (e)IPSC (upward traces in  Figure 2H) to 2-amino-3-(5-methyl-3-oxo-1, 2-oxazol-4-yl) propanoic acid (AMPA)-mediated evoked EPSC (downward traces in Figure 2H) was reduced in Kif5a-conditional KO mouse slices, compared with that in control Metalloexopeptidase mouse slices ( Figure 2H), showing a relative reduction in eIPSC amplitudes in Kif5a-KO neurons. Taken together, these results suggest that GABAA receptor (GABAAR)-mediated synaptic transmission is impaired in the hippocampus of Kif5a-conditional KO mice. Next, to investigate network excitability, we measured

stimulus-evoked population spikes in hippocampal slices of Kif5a-conditional KO and control slices. Epileptiform activities were more frequently observed in Kif5a-conditional KO slices in both standard and Mg2+-depleted artificial cerebrospinal fluid (ACSF), indicating increased excitability in these tissues ( Figures 2J–2O). To investigate the cause of impairment in inhibitory synaptic transmission, we compared cell surface expression of GABAARs in primary hippocampal neurons derived from Kif5a-KO and WT embryos. Most GABAARs at synapses are thought to be composed of two α1, α2, or α3 subunits together with two β2 or β3 subunits and a single γ2 subunit. Using the surface biotinylation method, the cell surface expression level of GABAARβ2 was assessed. In KO neurons, cell surface expression of GABAARβ2 was significantly reduced (52.6% ± 4.9% versus WT), whereas that of GluR2/3 was unchanged ( Figures 3A and 3B).

The early onset ramp we found in the count of cells with significantly different contra versus ipsi memory trial firing rates (orange line, Figure 3C) is paralleled in Figures 4A and 4B by an early onset in population firing rate difference for contra versus ipsi memory trials. Similarly, the late onset ramp in Figure 3C for nonmemory trials is paralleled in Figures 4C and 4D. We then turned to analyzing error trials. The activity on error trials (shaded pink for ipsi-instructed but contra motion, and blue for contra-instructed but ipsi motion; Figures 4A–4D) showed that, on average across selleck chemical the population, cells that fire more on correctly performed

contra-instructed trials also fire more on erroneously performed ipsi-instructed trials; that is, these cells fire more buy MDV3100 on trials where the animal orients contralateral

to the recorded side, regardless of the instruction. Similarly, ipsi preferring cells fire more on trials where the animal orients ipsilaterally, regardless of the instruction. This indicates that the firing rates of FOF cells are better correlated with the subject’s future motor response than with the instructing sensory stimulus. We quantified this observation on a cell-by-cell basis by generating a side-selectivity index (SSI) for each neuron (see Experimental Procedures for details). Positive SSIs mean that a cell fired more on contra-instructed trials. Negative SSIs mean that a cell fired more on ipsi-instructed trials. mafosfamide If cells encode the instruction we would expect SSIcorrect ≈ SSIerror. But if cells encode the direction of the motor response, then we would expect SSIcorrect ≈ −SSIerror. We first

calculated the SSI focusing on the delay period of memory trials. We found that, over neurons, SSIcorrect correlates negatively with SSIerror (r = −0.42, p < 10−4), confirming that on memory trials, the delay period firing rates of FOF neurons encode the orienting choice of the rat, not the instruction stimulus. We then repeated this calculation for firing rates over the movement period (from Go signal to 0.5 s after the Go signal), for both memory (SSIcorrect and SSIerror correlation r = −0.59, p < 10−8) and nonmemory (r = −0.78, p < 10−17) trials. These negative correlations indicate that the FOF is again encoding the motor choice of the rat. We summarized the observations from both the delay and movement periods by calculating the SSI for the entire period, from −1.5 s before to 0.5 s after the Go cue. This again resulted in negative SSIcorrect and SSIerror correlations for both memory (r = −0.49, p < 10−5) and nonmemory (r = −0.59, p < 10−8) trials (Figure 4E). Overall, then, the firing rates of FOF neurons encode the orienting choice of the rat, not the instruction stimulus.

The grid location was marked as significant if the spatial Z score exceeded the significance level of 0.05 (corrected for 25 multiple comparisons; see Figure S1A). Spatially significant grid locations for example neurons are marked with “x” or

numerals in Figures 2 and 3. For each spatially significant grid location, we next determined whether the neuron was significantly selective to the composite stimuli at that location. We calculated a Z score for each stimulus: Zshape(x,y,s)=rˆ(x,y,s)−rˆ(x,y,∗)η(x,y,s)×Nj−1. We define a shape selectivity index, SSI(x,y)SSI(x,y), for that spatial location as the maximum of the shape Z   scores: SSI(x,y)=max(Zshape(x,y,s))SSI(x,y)=max(Zshape(x,y,s)). A grid BAY 73-4506 manufacturer location was considered significantly shape selective if the index exceeded the significance level of 0.05 (corrected for 72 × M multiple comparisons, where M was the number ZD1839 clinical trial of significant spatial locations; see Figure S1A). A neuron was considered significantly shape selective if it had at least one spatially significant grid location that was also significantly shape selective. A total of 13 neurons failed this significance test. These neurons had significant spatial RFs, but were not significantly shape selective (Figure 1B). An example of a nonselective neuron is shown in

Figure 2 (example neuron IV). We did not analyze these neurons any further. All subsequent analyses were performed on the remaining 80 neurons. We used the mean responses rˆ(x,y,s)

to generate first three basic response maps: (1) location-specific response maps for the composite stimuli at each location in the 5 × 5 presentation grid (Figures 2B and 3B); (2) average response map, rˆ(∗,∗,s), for the composite stimuli by averaging across spatially significant grid locations; and (3) fine-scale orientation-tuning maps using the same procedure as in (1) for the bar stimuli on the 15 × 15 grid (Figure 3C). For the population analysis, we determined several metrics from the response maps for each neuron: Average shape preference was calculated by first determining the set of composite shapes, sisi, whose firing rate in the average response map, rˆ(∗,∗,si), exceeded 90% of the maximum firing rate. The shape category, cici (0: straight, 1: low curvature, 2: medium curvature, etc.), corresponding to these shapes was weighted and averaged by their firing rates to determine the average shape preference: ∑irˆ(∗,∗,si)ci∑irˆ(∗,∗,si). Local shape preference is same as above but derived from the location-specific response maps. Local preferred shape orientation is the orientation (0°, 22.5°, 45° … 337.5°) of the local preferred shape defined above. We computed the conditional joint distribution of local shape preference and the angular deviation of preferred shape orientation, ΔθprefΔθpref (Figure 4). The computation was conditioned on the shape preference and shape orientation at the maximally responsive location for each neuron.

, 2008). Subsequent work showed that blockade of VEGF-A increased leukocyte-endothelial adhesion, which could explain the increase in retinal macrophages following anti-VEGF-A therapy (Walshe et al., 2009). In addition, compensatory elevation in VEGF-A levels following anti-VEGF-A therapy (Willett et al., 2005) might promote inflammatory cell recruitment. These findings raise the following question, the answer to which has important therapeutic implications: What is the ratio of pro- and antiangiogenic macrophages that accumulate after anti-VEGF-A treatment? If the proportion or relative activity of

proangiogenic macrophages increased following anti-VEGF treatments, this finding could explain the tachyphylaxis (desensitization) that occurs with multiple anti-VEGF-A treatments (Forooghian et al., 2009). In that case, the selective inhibition of proangiogenic learn more macrophages would be an appealing adjunct to anti-VEGF therapy. This is in contrast to the

current, Forskolin cost broad-spectrum use of immunosuppression in AMD, which does not target a certain immune cell or subtype. Microglia are another immune cell type that might modulate human CNV pathogenesis. These resident retinal macrophages accumulate in the subretinal space in a CX3CR1-deficient mouse after light-induced and aging models of retinal degeneration; the accumulation of microglia in these mice appears to exacerbate laser-induced CNV ( Combadière et al., 2007). The CX3CR1−/−, CCL2−/−, CCR2−/− mouse models of AMD predominantly exhibit defects in microglia and macrophage function (reviewed in Raoul et al., 2010). When compared to mouse models deficient in either one of these chemokine others receptors, a double-knockout mouse genetically

deficient for both CX3CR1 and CCR2 produced a more penetrant, and earlier-onset, spontaneous phenotype of retinal lesions similar to those seen in dry AMD; also, a subset (15%) of mice spontaneously developed choroidal neovascularization ( Tuo et al., 2007). However, while macrophages accumulate in human CNV membranes, it is not known whether microglia do, too. In the largest histopathologic characterization of microglia in AMD to date, which observed microglia at various stages of AMD pathology, there was a change in microglia morphology, but not number, in AMD compared to nondiseased retinas ( Penfold et al., 1997). The precise functional ramifications of such altered microglia morphology remain to be elucidated. Very recently it has been claimed that many of the early-onset retinal degenerative phenotypes observed in the CX3CR1−/− mice are attributable to the fact that these mice were generated on the C57BL/6N background, which contains the rd8 retinal degeneration mutation of the Crb1 gene ( Mattapallil et al., 2012). However, the CCL2−/− and CCR2−/− mice were generated on the C57BL/6J background, which does not contain the rd8 mutation, and develop retinal findings many months later than rd8 mice ( Ambati et al., 2003b).

When I visited the school last May, I saw that students still performed the routine faithfully but with a different set of calisthenics program. The principal said, exactly like what my principal would, that the morning exercise could help students learn in classroom by following the “Three-Excellence” PF-01367338 price doctrine: excellent health, excellent learner, and excellent citizen. It has been suspected that exercise helps cognitive learning. Physical educators and exercise scientists

alike would like to make the statement intuitively that exercise can facilitate students to learn in all subject areas in schools. Coincidentally with the intuition, emerging research evidence in kinesiology has begun to show that exercise does help improve certain cognitive functions such as reaction time, attention span, or executive functioning (strategies). Learning is defined as a complex, multi-dimensional process resulting in relatively long-term (or permanent) changes in cognitive functioning and behavior. One crucial determinant of learning is long-term memory. It is long-term memory that Labban and Etnier1 were targeting in their research on cognitive impact of acute exercise. The purpose of the study was two-fold: (a) to determine the

“effects of acute aerobic exercise on long-term memory” and (b) to pinpoint “the influence of exposure timing to the to-be-remembered information relative to the exercise bout”. Based on an extensive literature review, the researchers positioned their study ISRIB on a solid theoretical basis. They not only developed their rationale for the study from a comprehensive theoretical articulation, but also used various theories to guide their research decisions on design, grouping, variable selection, treatment spacing, control condition, and experiment protocol. For example, the decision on exercise intensity and duration was based on the relation between cognitive functioning and the degree of dehydration and physiological exhaustion. The detailed attention to theories rendered a design tight Histone demethylase enough for improved internal validity

and reliability of all measures and realistic enough for reasonable generalizability of the results. The study used a randomized, controlled design. The participants were college-age undergraduate students (n = 48). After health and habitual physical activity behavior screening, they were randomly assigned to one of the three experimental conditions: Exercise-Prior – where the participants first exercised 30 min at a moderate-vigorous intensity, then studied two paragraphs of learning material, rested for 30 min while exposed to a “distractor” – procedure to clear working memory about the materials studied, after the rest they took a standardized recall test to determine how much information from the learning material was stored in the long-term memory.

, 2012, Soc. Neurosci., abstract). Therefore, the Shox2+ dI5 INs and/or the V2d neurons are likely responsible for decreased locomotor LY294002 datasheet frequency seen in this study. Another hallmark of vertebrate excitatory rhythm generating neurons is their recurrent connectivity (Li et al., 2006 and Parker and Grillner, 2000). Although connectivity was seen among Shox2 INs, it was sparse and we cannot ascribe this connectivity directly to Shox2+ non-V2a INs. It is notable that synaptic connectivity was not observed in previous studies of V2a neurons in the rodent spinal cord in Chx10-GFP

mice (Dougherty and Kiehn, 2010a and Zhong et al., 2010), nor has it been seen among excitatory Hb9 neurons (Wilson et al., 2005 and Hinckley and Ziskind-Conhaim, 2006). The rostrocaudal distribution of rhythmicity found in Shox2 INs may match with the subsets of Shox2 INs having a role in rhythm generation. Thus, the rhythm generating capability in the spinal cord is distributed (Kiehn and Kjaerulff, 1998) throughout the lumbar cord but with a rostral (L1–L3) dominance (Cazalets, 2005 and Kiehn and Kjaerulff,

1998). Notably, this rostral-caudal difference in rhythmicity was not seen in V2a neurons, as Chx10-GFP rhythmic neurons were equally distributed along the lumbar spinal cord (Dougherty and Kiehn, 2010a, Dougherty and Kiehn, 2010b and Zhong et al., 2010). Could there be an alternate explanation for the decrease in frequency observed in this study? Shox2 neurons could provide drive to the rhythm generating neurons—in which case a reduction in the glutamatergic drive AZD2281 order to rhythm generating neurons would account for the decrease in locomotor frequency. Metalloexopeptidase We do not favor this possibility, since the majority of Shox2 neurons, particularly in more rostral segments, are rhythmically active during locomotion, thereby placing them either as part of the rhythm generator or downstream from it. If Shox2 neurons provide tonic drive to rhythm generating neurons, they would have to be located locally as Shox2-halorhodopisin experiments involved application

of yellow light to an area of approximately three lumbar segments—with a consequent reduction in locomotor frequency. Another possibility is that the non-V2a Shox2 neurons are not rhythm generating but the effect seen is due to a nonspecific decrease in the number of excitatory neurons required for rhythm generation. Essentially when a critical mass of excitatory cells is eliminated, the frequency will drop. However, the Chx10 neurons outnumber the Shox2 neurons by at least 20%–25%. Therefore, if the critical excitatory cell mass hypothesis was correct, we would expect there to be a pronounced reduction in frequency in Chx10DTA experiments (that Crone et al., 2008 did not see), an intermediate reduction in the Shox2-Chx10DTA experiments (that we did not see), and a reduction in the frequency in Isl1-vGlut2Δ/Δ experiments (which Bui et al., 2012, Soc. Neurosci., abstract did not see).

In GluK3, this glycine residue is replaced by D759, producing unique rapid desensitization, whereas in GluK4 and GluK5, the asparagine substitution at this position

would likely not destabilize the LBD dimer interface, imparting different gating properties to these receptors. CT99021 clinical trial Zinc can modulate excitatory synaptic transmission through multiple mechanisms, which are not all well described (Paoletti et al., 2009). Although the inhibitory regulation of postsynaptic glutamate receptors, principally NMDARs, appears as a primary function of synaptic zinc, other potential roles in the regulation of synaptic transmission have also been proposed by Paoletti et al. (2009). Technical limitations have yet precluded a direct measurement of zinc in the synaptic cleft. However, the peak concentration was initially estimated to be in the order of 100 μM (Vogt et al., 2000). This value

Epigenetic inhibitor cost is well within the range of efficacy for the allosteric potentiation of GluK3 by zinc but may be overestimated and may depend on experimental conditions (Paoletti et al., 2009). Moreover, simultaneous application of zinc and glutamate does not potentiate GluK3-mediated currents (data not shown), which likely excludes an effect of zinc during low-frequency stimulation. However, high-frequency trains of synaptic stimulation are thought to trigger a substantial increase in extracellular zinc, and the accumulated zinc could potentiate presynaptic GluK2/GluK3 receptors present at hippocampal mossy fiber synapses (Pinheiro et al., 2007). Interestingly, it has been

shown that vesicular zinc is required for presynaptic LTP at hippocampal mossy fiber synapses by a yet undisclosed mechanism (Pan et al., 2011). This result can be correlated with the fact that mossy fiber LTP is absent in GluK3−/− mice (Pinheiro et al., 2007). The positive allosteric modulation of these presynaptic GluK2/GluK3 receptors may impart increased sensitivity to glutamate and prolonged channel opening, inducing a possible increase Bay 11-7085 in presynaptic Ca2+ influx. Hence, allosteric modulation of presynaptic GluK3 receptors may be one of the mechanisms by which zinc promotes presynaptic long-term potentiation. In conclusion, we have identified zinc as a positive allosteric modulator of presynaptic KARs, with a potential role in synaptic plasticity. Our structure-function analyses lend further support to the notion that the stability of the LBD dimer interface is essential for dictating the desensitization properties of KARs. Our data help explain the fast desensitization properties of GluK3 as compared to GluK2 and pinpoint a single amino acid residue in the upper lobe of the clamshell of the GluK3 LBD, D759, as responsible for the specific properties of GluK3.

RU486 pretreatment prevented the increased ethanol self-administration induced by nicotine pretreatment (Figure 5D). The mean ethanol intake for the group pretreated with RU486 and nicotine (0.74 ± 0.06 g/kg/session, n = 12) was significantly lower than the nicotine pretreatment alone (0.97 g/kg; n = 17) (p < INCB018424 solubility dmso 0.01) and nearly identical to the saline pretreatment control (Figure 5D, dashed line). Thus, nicotine required the activation

of stress hormone receptors to enhance subsequent ethanol self-administration. Because local infusions of RU486 into the VTA prevented the inhibition of ethanol-induced DA release, we hypothesized that stress hormone action altered ethanol-induced GABA transmission onto DA neurons. Therefore, we pretreated rats with RU486 prior to nicotine pretreatment and measured ethanol-induced sIPSCs 15 hr later. The nicotine-pretreatment potentiation of the sIPSC frequency by ethanol was prevented by RU486 pretreatment

(Figures 6A and 6B) (p < 0.05). The average sIPSC frequency BMN 673 nmr induced by ethanol (relative to basal) was 187% ± 12% after nicotine pretreatment (Figure 6B, red bar) and 118% ± 8% after RU486 and nicotine pretreatment (Figure 6B, blue bar). In addition, the enhanced paired-pulse depression after the nicotine pretreatment (78.6% ± 3.4%; see Figure 4D) was also prevented by RU486 (92.1% ± 3.0%; n = 15; data not shown) (p < 0.05). These results and others indicate that stress hormone receptor activation in response to nicotine pretreatment altered ethanol-induced GABA network activity in the VTA. Acute pretreatment with nicotine

induced a long-lasting attenuation of ethanol-induced DA signals within the mesoaccumbens pathway. The decreased ethanol-induced DA signals were due to an increase in GABAergic inhibition of DA neurons and a consequent decrease in VTA DA neuron firing. These nicotine-induced neuroadaptations PDK4 required a stress hormone signal that acted significantly within the VTA. Concomitant with these physiological changes, we also show that increases in ethanol self-administration induced by nicotine were prevented by RU486, a glucocorticoid/progesterone receptor antagonist (Cadepond et al., 1997). In addition to other interactions with ethanol (Al-Rejaie and Dar, 2006, Collins et al., 1996, Gulick and Gould, 2008 and López-Moreno et al., 2008), nicotine exposure influences subsequent ethanol consumption and abuse (Barrett et al., 2006, Grant, 1998, Lê et al., 2003, Morgen et al., 2008 and Smith et al., 1999). Although the development of drug abuse involves the mesolimbic DA system, there are little mechanistic data indicating how nicotine influences DA responses to ethanol. Our results suggest that nicotine acts through stress hormone signaling pathways in the VTA to enhance subsequent ethanol-induced GABAergic drive onto DA neurons, thereby decreasing ethanol-induced DA signals.

, 2000 and Leech et al., 1999), we aim to determine the relationship between prenatal cannabis use and early indications of childhood attention problems and aggressive behavior. It is important to investigate early childhood behavior, because it has been shown that childhood www.selleckchem.com/products/SB-431542.html behavior disturbances may be predictive for psychopathology in adulthood (Caspi, 2000). We did this using a well-validated

instrument in a general population birth cohort of children at 18 months of age. This study was conducted within the Generation R Study, a population based birth cohort in Rotterdam, the Netherlands (Jaddoe et al., 2008 and Jaddoe et al., 2010). More information on the Generation R Study, including eligibility, recruitment, and enrollment can be found in the Supplemental buy Olaparib material. The study was conducted in accordance with the guidelines proposed in the World Medical Association Declaration of Helsinki, and was approved by the Medical Ethics Committee of the Erasmus Medical Centre, Rotterdam. Written informed consent was obtained from all participating parents and anonymity was guaranteed. Information on prenatal substance use was available for 5512 children. Information on child behavioral problems at 18 months was available in 4077 children (74.0% of 5512). These children form the study population for the analyses. Tobacco, alcohol and substance use were measured using a self-report

questionnaire given to both parents during the first trimester of pregnancy. More information on these questionnaires is provided in the Supplemental material. The agreement between maternal self-report and urinalyses

was good (Yule’s Y = 0.77) and has been described previously ( El Marroun et al., 2011). The self-reported prevalence was in agreement with national numbers in the same period ( Rodenburg et al., 2007). The pregnant mothers were also asked about the father’s Rolziracetam cannabis use. We used maternal report of paternal cannabis use only when the fathers did not complete the questionnaire (26%). Maternal report of paternal cannabis use was highly correlated to paternal self-reported cannabis use (r = 0.83 p < 0.001). In order to assess the gestational influence of cannabis, we categorized intrauterine exposure into four non-overlapping groups, according to cannabis and/or tobacco use. 1. Cannabis exposure in pregnancy (n = 88), mostly with co-use of tobacco during pregnancy (84.5%), The Child Behavior Checklist for toddlers (CBCL 1½–5 years) was used to acquire a standardized maternal report of children’s problem behaviors. We focused on three specific syndrome scales: Anxious/Depressed, Attention Problems and Aggressive Behavior. Each item is scored 0 = not true, 1 = somewhat or sometimes true and 2 = very true or often true, based on the preceding two months. Good reliability and validity have been reported for the CBCL (Achenbach and Rescorla, 2000). We used both continuous scores for the CBCL and dichotomous cut-off scores reflecting clinical cases.

While universal equitable coverage would reduce disparities, an alternative would be to target accelerated introduction or expanded coverage of high-risk children, based on geography or other population characteristics. The cost-effectiveness and impact estimates in Table 4 and Fig. 2 and Fig. 4 can be interpreted as the incremental cost-effectiveness of introducing the vaccine into higher risk populations first. The results GSK1349572 research buy suggest that it would be most cost-effective to target these children first. Although few countries are considering sub-national introduction, this could be done to target high-risk regions. In order to be most effective, these regions would also need to have adequate levels of vaccine

coverage. Geographic targeting could also focus on more remote areas

where access to timely treatment of diarrhea is lower. For other infections with clear geographic hotspots (e.g., malaria and soil transmitted helminthes) this is a clear strategy for improving value for money [30] and [31]. Although it can be more difficult to target children based on socio-economic characteristics, there are examples of programs Selleck Cisplatin designed to do this, such as conditional cash transfer programs that target low-income communities and households [32] and [33]. A related approach would be to target based on other risk factors such as nutritional status by coordinating with maternal and newborn nutrition programs. These targeting strategies would increase the likelihood that investments go disproportionately to the areas the or children where they provide the greatest value for money. While these targeting strategies would create challenges, the level of potential benefit (a 38% increase in mortality reduction) is too great to ignore. The current study is a preliminary assessment of the distributional effects and, as such, it has a number of limitations. First, no systematic data are available for directly estimating rotavirus mortality or burden by wealth quintile or sub-national

regions. As a result, we aggregated data on post-neonatal infant mortality and low weight-for-age as a proxy measure. It is important to note that there is variability in estimated mortality disparities, depending on which proxy measure is used. For example, in Table 3 post-neonatal mortality is highest in the second poorest quintile, rather than the poorest. This may be the product of higher neonatal mortality among the poorest, differences in reporting biases or other factors. This suggests that better proxy measures, at the level of quintiles or individuals could provide more accurate estimates of disparities. In addition, the analysis only explores one dimension of equity at a time (either socio-economic status or geographic location) without exploring the interaction between them or whether other factors such as maternal education may explain both reduced vaccination and increased mortality risk.