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Sustainable management of landscapes with multiple competing demands such as the Ruaha Landscape is complex due to the diverse preferences and needs of stakeholder groups involved. This study uses conjoint analysis to assess the preferences of representatives from three stakeholder groups-local communities, district government officials, and non-governmental organizations-toward potential solutions of conservation and development tradeoffs facing local communities in the Ruaha Landscape of Tanzania. Results demonstrate that there is little consensus among stakeholders about the best development strategies for the Ruaha region. This analysis suggests a need for incorporating issues deemed important by these various groups into a development strategy that aims to promote conservation of the Ruaha Landscape and improve the livelihood of local communities.

Background: Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [3H]N-methylscopolamine ([3H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca2+]i and [Ca2+]L, respectively) were measured by fluorescent imaging., Results: Activation of M3 muscarinic receptors induced a biphasic increase in [Ca2+]i: an initial, inositol trisphosphate (IP3)-mediated release of Ca2+ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca2+ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca2+]i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca2+]i from 26 to up to 127 nM and decreased [Ca2+]L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca2+ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP., Conclusions: Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca2+]i, [Ca2+]L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.

An experimental and DFT investigation of the mechanism of the coupling of methylisocyanide and C-H activation mediated by the germylene (germanediyl) Ge(Ar(Me6))2 (Ar(Me6) = C6H3-2,6(C6H2-2,4,6-Me3)2) showed that it proceeded by initial MeNC adduct formation followed by an isomerization involving the migratory insertion of the MeNC carbon into the Ge-C ligand bond. Addition of excess MeNC led to sequential insertions of two further MeNC molecules into the Ge-C bond. The insertion of the third MeNC leads to methylisocyanide methyl group C-H activation to afford an azagermacyclopentadienyl species. The X-ray crystal structures of the 1:1 (Ar(Me6))2GeCNMe adduct, the first and final insertion products (Ar(Me6))GeC(NMe)Ar(Me6) and (Ar(Me6))GeC(NHMe)C(NMe)C(Ar(Me6))NMe were obtained. The DFT calculations on the reaction pathways represent the first detailed mechanistic study of isocyanide oligomerization by a p-block element species.

The level and integrity of glutamate transmission during critical periods of postnatal development plays an important role in the refinement of pyramidal neuron dendritic arbor, synaptic plasticity, and cognition. Presently, it is not clear how excitatory transmission via the two predominant isoforms of the vesicular glutamate transporter (VGLUT1 and VGLUT2) participate in this process. To assess a neurodevelopmental role for VGLUT2 in pyramidal neuron maturation, we generated recombinant VGLUT2 knock-out mice and inactivated VGLUT2 throughout development using Emx1-Cre(+/+) knock-in mice. We show that VGLUT2 deficiency in corticolimbic circuits results in reduced evoked glutamate transmission, release probability, and LTD at hippocampal CA3-CA1 synapses during a formative developmental period (postnatal days 11-14). In adults, we find a marked reduction in the amount of dendritic arbor across the span of the dendritic tree of CA1 pyramidal neurons and reduced long-term potentiation and levels of synaptic markers spinophilin and VGLUT1. Loss of dendritic arbor is accompanied by corresponding reductions in the number of dendritic spines, suggesting widespread alterations in synaptic connectivity. Conditional VGLUT2 knock-out mice exhibit increased open-field exploratory activity yet impaired spatial learning and memory, endophenotypes similar to those of NMDA receptor knock-down mice. Remarkably, the impairment in learning can be partially restored by selectively increasing NMDA receptor-mediated glutamate transmission in adult mice by prolonged treatment with d-serine and a d-amino acid oxidase inhibitor. Our data indicate that VGLUT2 expression is pivotal to the proper development of mature pyramidal neuronal architecture and plasticity, and that such glutamatergic deficiency leads to cognitive malfunction as observed in several neurodevelopmental psychiatric disorders.

Previous observational and quasi-experimental studies in sub-Saharan Africa have suggested the effectiveness of youth-targeted HIV prevention interventions using sport as an educational tool. No studies have yet assessed the effect of similar programs in the Caribbean. A quasi-experimental trial was conducted to assess the effectiveness of a sports-based intervention in six migrant settlements in the Puerto Plata Province of the Dominican Republic. A total of 397 structured interviews were conducted with 140 adolescents prior to, immediately following, and four months following 10-hour interventions using the Grassroot Soccer curriculum. Interview responses were coded, aggregated into composite scores, and analyzed using logistic regression, adjusting for baseline differences as well as age, sex, community, and descent. At post-intervention, significant differences were observed between groups in HIV-related knowledge (adjOR = 13.02, 95% CI = 8.26, 20.52), reported attitudes (adjOR = 12.01, 95% CI = 7.61, 18.94), and reported communication (adjOR = 3.13, 95% CI = 1.91, 5.12). These differences remained significant at four-month follow-up, though declines in post-intervention knowledge were observed in the Intervention group while gains in knowledge and reported attitudes were observed in the Control group. Results suggest that this sports-based intervention could play a valuable role in HIV prevention efforts in the Caribbean, particularly those targeting early adolescents. Further evaluation of sports-based interventions should include indicators assessing behavioral and biological outcomes, longer-term follow-up, a larger sample, randomization of study participants, and strenuous efforts to minimize loss-to-follow-up.

Homeostatic scaling of glutamatergic and GABAergic transmission is triggered by prolonged alterations in synaptic neuronal activity. We have previously described a presynaptic mechanism for synaptic homeostasis and plasticity that involves scaling the level of vesicular glutamate (VGLUT1) and gamma-aminobutyric acid (GABA) (VGAT) transporter biosynthesis. These molecular determinants of vesicle filling and quantal size are regulated by neuronal activity in an opposite manner and bi-directionally. Here, we report that a striking induction of VGLUT2 mRNA and synaptic protein is triggered by a prolonged increase in glutamatergic synaptic activity in mature neocortical neuronal networks in vitro together with two determinants of inhibitory synaptic strength, the neuronal activity-regulated pentraxin (Narp), and glutamate decarboxylase (GAD65). Activity-dependent induction of VGLUT2 and Narp exhibits a similar intermediate-early gene response that is blocked by actinomycin D and tetrodotoxin, by inhibitors of ionotropic glutamate receptors and L-type voltage-gated calcium channels, and is dependent on downstream signaling via calmodulin, calcium/calmodulin-dependent protein kinase (CaMK) and extracellular signal-regulated kinase 1/2 (ERK1/2). The co-induction of VGLUT2 and Narp triggered by prolonged gamma-aminobutyric acid type A receptor blockade is independent of brain-derived nerve growth factor and TrkB receptor signaling. VGLUT2 protein induction occurs on a subset of cortically derived synaptic vesicles in excitatory synapses on somata and dendritic processes of multipolar GABAergic interneurons, recognized sites for the clustering of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptors by Narp. We propose that VGLUT2 and Narp induction by excitation-transcription coupling leads to increased glutamatergic transmission at synapses on GABAergic inhibitory feedback neurons as part of a coordinated program of Ca(2+)-signal transcription involved in mechanisms of homeostatic plasticity after prolonged hyperactivity.

System A transporters SNAT1 and SNAT2 mediate uptake of neutral alpha-amino acids (e.g. glutamine, alanine, and proline) and are expressed in central neurons. We tested the hypothesis that SNAT2 is required to support neurotransmitter glutamate synthesis by examining spontaneous excitatory activity after inducing or repressing SNAT2 expression for prolonged periods. We stimulated de novo synthesis of SNAT2 mRNA and increased SNAT2 mRNA stability and total SNAT2 protein and functional activity, whereas SNAT1 expression was unaffected. Increased endogenous SNAT2 expression did not affect spontaneous excitatory action-potential frequency over control. Long term glutamine exposure strongly repressed SNAT2 expression but increased excitatory action-potential frequency. Quantal size was not altered following SNAT2 induction or repression. These results suggest that spontaneous glutamatergic transmission in pyramidal neurons does not rely on SNAT2. To our surprise, repression of SNAT2 activity was not limited to System A substrates. Taurine, gamma-aminobutyric acid, and beta-alanine (substrates of the SLC6 gamma-aminobutyric acid transporter family) repressed SNAT2 expression more potently (10x) than did System A substrates; however, the responses to System A substrates were more rapid. Since ATF4 (activating transcription factor 4) and CCAAT/enhancer-binding protein are known to bind to an amino acid response element within the SNAT2 promoter and mediate induction of SNAT2 in peripheral cell lines, we tested whether either factor was similarly induced by amino acid deprivation in neurons. We found that glutamine and taurine repressed the induction of both transcription factors. Our data revealed that SNAT2 expression is constitutively low in neurons under physiological conditions but potently induced, together with the taurine transporter TauT, in response to depletion of neutral amino acids.

One of the goals of birth defects research is to better understand risk or preventive factors for birth defects so that strategies for prevention can be developed. In this article, we have selected four areas of birth defects research that have led to the development of prevention strategies. These areas include rubella virus as a cause of congenital rubella syndrome, folic acid as a preventive factor for neural tube defects, cytomegalovirus infection as a cause of birth defects and developmental disabilities, and alcohol as a cause of fetal alcohol spectrum disorders. For each of these areas, we review key clinical and research findings that led to the identification of the risk or preventive factor, milestones in the development of prevention strategies, and the progress made thus far toward prevention.

In response to bioterrorism threats, anthrax vaccine has been used by the US military and considered for civilian use. Concerns exist about the potential for adverse reproductive health effects among vaccine recipients. This retrospective cohort evaluated birth defects, in relation to maternal anthrax vaccination, among all infants born to US military service women between 1998 and 2004. Department of Defense databases defined maternal vaccination and infant diagnoses; multivariable regression models described potential associations between anthrax vaccination and birth defects in liveborn infants. Among 115,169 infants born to military women during this period, 37,140 were born to women ever vaccinated against anthrax, and 3,465 were born to women vaccinated in the first trimester of pregnancy. Birth defects were slightly more common in first trimester-exposed infants (odds ratio = 1.18, 95% confidence interval: 0.997, 1.41) when compared with infants of women vaccinated outside of the first trimester, but this association was statistically significant only when alternative referent groups were used. Although the small observed association may be unlikely to represent a causal relation between vaccination in early pregnancy and birth defects, this information should be considered when making decisions about administering anthrax vaccine to pregnant women.

Background: There are no large randomized trials of the effect of folic acid dosing regimens on blood folate and homocysteine concentrations., Objective: We aimed to evaluate the changes in folate and homocysteine concentrations in response to different folic acid doses and to withdrawal in young women not exposed to other sources of folic acid., Design: Women (n = 1108) were randomly assigned to 1 of 6 intervention groups for which daily intakes of folic acid for 6 mo were 100 microg 1 time/d, 25 microg 4 times/d, 400 microg 1 time/d, 100 microg 4 times/d, 4000 microg 1 time/d, or 4000 microg 1 time/wk. Plasma and red blood cell folate and homocysteine concentrations were measured at baseline; at 1, 3, and 6 mo; and 3 mo after the discontinuation of folic acid., Results: Folate and homocysteine concentrations were not different at baseline between the groups who had the same daily intake of folic acid as a single dose or multiple doses (P = 0.058). Plasma folate concentrations plateaued at 3 mo with 108% (95% CI: 97.7%, 120%), 259% (95% CI: 240%, 279%), 460% (95% CI: 417%, 503%), and 142% (95% CI: 123%, 162%) observed increases for the folic acid groups receiving 100, 400, and 4000 microg/d and 4000 microg/wk, respectively. The rate of reduction in folate concentrations during the 3 mo after cessation of folic acid was dose-dependent-higher intakes were associated with faster reductions., Conclusions: Changes in folate and homocysteine concentrations were unaffected by different dosing schedules. After folic acid cessation, blood folate declined rapidly, which indicated that the intervention-enhanced folate status was rapidly diminished.

Background: Abnormalities of folate and homocysteine metabolism are associated with a number of pediatric and adult disorders. Folate intake and genetic polymorphisms encoding folate-metabolizing enzymes influence blood folate and homocysteine concentrations, but the effects and interactions of these factors have not been studied on a population-wide basis., Objective: The objective was to assess the prevalence of these genetic polymorphisms and their relation to serum folate and homocysteine concentrations., Design: DNA samples from 6793 participants in the third National Health and Nutrition Examination Survey (NHANES III) during 1991-1994 were genotyped for polymorphisms of genes coding for folate pathway enzymes 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C-->T and 1298A-->C, methionine synthase reductase (MTRR) 66A-->G, and cystathionine-beta-synthase 844ins68. The influence of these genetic variants on serum folate and homocysteine concentrations was analyzed by age, sex, and folate intake in 3 race-ethnicity groups., Results: For all race-ethnicity groups, serum folate and homocysteine concentrations were significantly related to the MTHFR 677C-->T genotype but not to the other polymorphisms. Persons with the MTHFR 677 TT genotype had a 22.1% (95% CI: 14.6%, 28.9%) lower serum folate and a 25.7% (95% CI: 18.6%, 33.2%) higher homocysteine concentration than did persons with the CC genotype. Moderate daily folic acid intake (mean: 150 microg/d; 95% CI: 138, 162) significantly reduced the difference in mean homocysteine concentrations between those with the MTHFR 677 CC and TT genotypes. We found a significant interaction between MTHFR 677C-->T and MTRR 66A-->G on serum homocysteine concentrations among non-Hispanic whites., Conclusions: The MTHFR 677C-->T polymorphism was associated with significant differences in serum folate and homocysteine concentrations in the US population before folic acid fortification. The effect of MTHFR 677C-->T on homocysteine concentrations was reduced by moderate daily folic acid intake.

VGLUT2 is one of three vesicular glutamate transporters that play crucial roles in glutamatergic excitatory neurotransmission. We explored the functional properties of the rat VGLUT2 by heterologous expression of VGLUT2 in Xenopus oocytes. Immunocytochemical analysis indicated that most VGLUT2 protein was expressed in intracellular compartments but that some expression occurred also on the plasma membrane. Functional analysis revealed VGLUT2 to be active in two independent modes, namely, uptake into intracellular organelles and efflux at the plasma membrane. VGLUT-specific transport was identified based on the strong preference for glutamate over aspartate--in contrast to plasma-membrane or mitochondrial glutamate transporters--and sensitivity to known VGLUT blockers. VGLUT2 expression in oocytes (1) stimulated the influx of L-[(3)H]glutamate, but not D-[(3)H]aspartate, into digitonin-permeabilized oocytes and (2) stimulated efflux of L-glutamate, but not L-aspartate, from intact oocytes preinjected with (3)H-labeled amino acids. In the latter assay, cellular efflux of glutamate (which was blocked by rose bengal and trypan blue) may be analogous to vesicular packaging of glutamate. Our data are consistent with VGLUT2-mediated H(+)/L-glutamate antiport, but not antiport with chloride. Expression of mammalian VGLUT1 and VGLUT3 also stimulated L-[(3)H]glutamate efflux from Xenopus oocytes, suggesting that this phenomenon is a general feature of vesicular glutamate transporters. Our findings support the idea that vesicular glutamate transporters, when transiently expressed on the neuronal plasma membrane, may mediate Ca(2+)-independent glutamate leakage in addition to their traditional role of packaging glutamate into synaptic vesicles for Ca(2+)-dependent exocytosis.

Wasting of lean tissue as a consequence of metabolic acidosis is a serious problem in patients with chronic renal failure. A possible contributor is inhibition by low pH of the System A (SNAT2) transporter, which carries the amino acid L-glutamine (L-Gln) into muscle cells. The aim of this study was to determine the effect of selective SNAT2 inhibition on intracellular amino acid profiles and amino acid-dependent signaling through mammalian target of rapamycin in L6 skeletal muscle cells. Inhibition of SNAT2 with the selective competitive substrate methylaminoisobutyrate, metabolic acidosis (pH 7.1), or silencing SNAT2 expression with small interfering RNA all depleted intracellular L-Gln. SNAT2 inhibition also indirectly depleted other amino acids whose intracellular concentrations are maintained by the L-Gln gradient across the plasma membrane, notably the anabolic amino acid L-leucine. Consequently, SNAT2 inhibition strongly impaired signaling through mammalian target of rapamycin to ribosomal protein S6 kinase, ribosomal protein S6, and 4E-BP1, leading to impairment of protein synthesis comparable with that induced by rapamycin. It is concluded that even though SNAT2 is only one of several L-Gln transporters in muscle, it may determine intracellular anabolic amino acid levels, regulating the amino acid signaling that affects protein mass, nucleotide/nucleic acid metabolism, and cell growth.

Background: Neural tube defects are serious birth defects of the brain and spinal cord. Up to 70% of neural tube defects can be prevented by the consumption of folic acid by women before and early during pregnancy., Objective: The objective was to examine folic acid intake in women of childbearing age in the United States., Design: We analyzed nutrient intake data reported by 1685 nonpregnant women aged 15-49 y who participated in the National Health and Nutritional Examination Survey, 2001-2002., Results: The adjusted geometric mean consumption of folic acid from fortified foods was 128 microg/d (95% CI: 123, 134 microg/d) in nonpregnant women. Eight percent (95% CI: 5.8%, 11.0%) of nonpregnant women reported consuming >or=400 microg folic acid/d from fortified foods. This proportion was lower among non-Hispanic black women (5.0%) than among non-Hispanic white (8.9%) or Hispanic (6.8%) women. A smaller percentage of non-Hispanic black (19.1%) and Hispanic (21%) women than of non-Hispanic white women (40.5%) consumed >or=400 microg folic acid from supplements, fortified foods, or both, in addition to food folate, as recommended by the Institute of Medicine to reduce the frequency of neural tube defects., Conclusions: Most nonpregnant women of childbearing age in the United States reported consuming less than the recommended amount of folic acid. The proportion with low daily folic acid intake was significantly higher in non-Hispanic black and Hispanic women than in non-Hispanic white women. At the present level of folic acid fortification, most women need to take a folic acid-containing dietary supplement to achieve the Institute of Medicine recommendation.

Dendritic development is essential for the establishment of a functional nervous system. Among factors that control dendritic development, brain-derived neurotrophic factor (BDNF) has been shown to regulate dendritic length and complexity of cortical neurons. However, the cellular and molecular mechanisms that underlie these effects remain poorly understood. In this study, we examined the role of amino acid transport in mediating the effects of BDNF on dendritic development. We show that BDNF increases System A amino acid transport in cortical neurons by selective up-regulation of the sodium-coupled neutral amino acid transporter (SNAT)1. Up-regulation of SNAT1 expression and System A activity is required for the effects of BDNF on dendritic growth and branching of cortical neurons. Further analysis revealed that induction of SNAT1 expression and System A activity by BDNF is necessary in particular to enhance synthesis of tissue-type plasminogen activator, a protein that we demonstrate to be essential for the effects of BDNF on cortical dendritic morphology. Together, these data reveal that stimulation of neuronal differentiation by BDNF requires the up-regulation of SNAT1 expression and System A amino acid transport to meet the increased metabolic demand associated with the enhancement of dendritic growth and branching.

The neutral amino acid transporters SNAT1-3 and ASCT1 play critical roles in the recycling of glutamine, and subsequently glutamate, via the glutamine-glutamate cycle. Hypoxia-ischemia was induced in rat pups using the Rice-Vannucci model. Brains were harvested at 1 h, 24 h and 7 days after ischemia. The expression of NAATs was evaluated using immunoblotting, real-time PCR, and immunohistochemistry. Results were compared with age-matched controls and shams. SNAT1 mRNA decreased at 1 h after injury in both hemispheres when compared with the control animals and correlated with a decrease in protein expression at 24 h in the hippocampus and cortex. SNAT1 protein expression increased globally at 7 days after injury and specifically in the hippocampus. Finally, SNAT2 and 3 demonstrated subtle changes in various brain regions after injury. These data suggest that neutral amino acid transporters remain largely intact after hypoxia-ischemia., (Copyright (c) 2006 S. Karger AG, Basel.)

1. Selective protein-protein interactions between neurotransmitter transporters and their synaptic targets play important roles in regulating chemical neurotransmission. We screened a yeast two-hybrid library with bait containing the C-terminal amino acids of VGLUT1 and obtained clones that encode endophilin 1 and endophilin 3, proteins considered to play an integral role in glutamatergic vesicle formation. 2. Using a modified yeast plasmid vector to enable more cost-effective screens, we analyzed the selectivity and specificity of this interaction. Endophilins 1 and 3 selectively recognize only VGLUT1 as the C-terminus of VGLUT2 and VGLUT3 do not interact with either endophilin isoform. We mutagenized four conserved stretches of primary sequence in VGLUT1 that includes two polyproline motifs (Pro1, PPAPPP, and Pro2, PPRPPPP), found only in VGLUT1, and two conserved stretches (SEEK, SYGAT), found also in VGLUT2 and VGLUT3. The absence of the VGLUT conserved regions does not affect VGLUT1-endophilin association. Of the two polyproline stretches, only one (Pro2) is required for binding specificity to both endophilin 1 and endophilin 3. 3. We also show that endophilin 1 and endophilin 3 co-localize with VGLUT1 in synaptic terminals of differentiated rat neocortical neurons in primary culture. These results indicate that VGLUT1 and both endophilins are enriched in a class of excitatory synaptic terminals in cortical neurons and there, may interact to play an important role affecting the vesicular sequestration and synaptic release of glutamate.

We studied the distribution and cellular localization of Na(+)-coupled neutral amino acid transporter 2, a member of the system A family of amino acid transporters, in the rat and human cerebral cortex using immunocytochemical methods. Na(+)-coupled neutral amino acid transporter 2-positive neurons were pyramidal and non-pyramidal, and Na(+)-coupled neutral amino acid transporter 2/GABA double-labeling studies revealed that Na(+)-coupled neutral amino acid transporter 2 was highly expressed by GABAergic neurons. Double-labeling studies with the synaptophysin indicated that rare axon terminals express Na(+)-coupled neutral amino acid transporter 2. Na(+)-coupled neutral amino acid transporter 2-immunoreactivity was also found in astrocytes, leptomeninges, ependymal cells and choroid plexus. Electron microscopy showed robust Na(+)-coupled neutral amino acid transporter 2-immunoreactivity in the somato-dendritic compartment of neurons and in glial processes, but, as in the case of double-labeling studies, failed to reveal Na(+)-coupled neutral amino acid transporter 2-immunoreactivity in terminals. To rule out the possibility that the absence of Na(+)-coupled neutral amino acid transporter 1- and Na(+)-coupled neutral amino acid transporter 2-positive terminals was due to insufficient antigen detection, we evaluated Na(+)-coupled neutral amino acid transporter 1/synaptophysin and Na(+)-coupled neutral amino acid transporter 2/synaptophysin coexpression using non-standard immunocytochemical procedures and found that Na(+)-coupled neutral amino acid transporter 1 and Na(+)-coupled neutral amino acid transporter 2+ terminals were rare in all conditions. These findings indicate that Na(+)-coupled neutral amino acid transporter 1 and Na(+)-coupled neutral amino acid transporter 2 are virtually absent in cortical terminals, and suggest that they do not contribute significantly to replenishing the Glu and GABA transmitter pools through the glutamate-glutamine cycle. The strong expression of Na(+)-coupled neutral amino acid transporter 2 in the somato-dendritic compartment and in non-neuronal elements that are integral parts of the blood-brain and brain-cerebrospinal fluid barrier suggests that Na(+)-coupled neutral amino acid transporter 2 plays a role in regulating the levels of Gln and other amino acids in the metabolic compartment of cortical neurons.

The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements.

Objective: The United States food supply has been fortified with folic acid since 1998. Information about folate levels early in pregnancy before the fortification is limited. This study examined the associations between serum folate at first prenatal visit and maternal race/ethnicity, age, vitamin use, and body mass index., Study Design: This cross-sectional study assessed serum folate levels among 9421 women who entered prenatal care in 1999 and 2000 in southern California. Information on race/ethnicity, vitamin use, weight, height, and age was obtained from surveys and birth certificates., Results: After adjustment for vitamin use, the strongest predictor of serum folate level, being in the lowest folate quartile (< or = 16 ng/mL) was related independently to being of black, Hispanic, or Asian/Pacific Islander race/ethnicity, being younger age, and being overweight or obese., Conclusion: After food fortification with folic acid, differences in serum folate values in pregnant women by maternal race/ethnicity, age, and body mass index persisted.

Homeostatic control of pyramidal neuron firing rate involves a functional balance of feedforward excitation and feedback inhibition in neocortical circuits. Here, we reveal a dynamic scaling in vesicular excitatory (vesicular glutamate transporters VGLUT1 and VGLUT2) and inhibitory (vesicular inhibitory amino acid transporter VIAAT) transporter mRNA and synaptic protein expression in rat neocortical neuronal cultures, using a well established in vitro protocol to induce homeostatic plasticity. During the second and third week of synaptic differentiation, the predominant vesicular transporters expressed in neocortical neurons, VGLUT1 and VIAAT, are both dramatically upregulated. In mature cultures, VGLUT1 and VIAAT exhibit bidirectional and opposite regulation by prolonged activity changes. Endogenous coregulation during development and homeostatic scaling of the expression of the transporters in functionally differentiated cultures may serve to control vesicular glutamate and GABA filling and adjust functional presynaptic excitatory/inhibitory balance. Unexpectedly, hyperexcitation in differentiated cultures triggers a striking increase in VGLUT2 mRNA and synaptic protein, whereas decreased excitation reduces levels. VGLUT2 mRNA and protein are expressed in subsets of VGLUT1-encoded neocortical neurons that we identify in primary cultures and in neocortex in situ and in vivo. After prolonged hyperexcitation, downregulation of VGLUT1/synaptophysin intensity ratios at most synapses is observed, whereas a subset of VGLUT1-containing boutons selectively increase the expression of VGLUT2. Bidirectional and opposite regulation of VGLUT1 and VGLUT2 by activity may serve as positive or negative feedback regulators for cortical synaptic transmission. Intracortical VGLUT1/VGLUT2 coexpressing neurons have the capacity to independently modulate the level of expression of either transporter at discrete synapses and therefore may serve as a plastic interface between subcortical thalamic input (VGLUT2) and cortical output (VGLUT1) neurons.

A fundamental question in synaptic physiology is whether the unitary strength of a synapse can be regulated by presynaptic characteristics and, if so, what those characteristics might be. Here, we characterize a newly proposed mechanism for altering the strength of glutamatergic synapses based on the recently identified vesicular glutamate transporter VGLUT1. We provide direct evidence that filling in isolated synaptic vesicles is subject to a dynamic equilibrium that is determined by both the concentration of available glutamate and the number of vesicular transporters participating in loading. We observe that changing the number of vesicular transporters expressed at hippocampal excitatory synapses results in enhanced evoked and miniature responses and verify biophysically that these changes correspond to an increase in the amount of glutamate released per vesicle into the synaptic cleft. In addition, we find that this modulation of synaptic strength by vesicular transporter expression is endogenously regulated, both across development to coincide with a maturational increase in vesicle cycling and quantal amplitude and by excitatory and inhibitory receptor activation in mature neurons to provide an activity-dependent scaling of quantal size via a presynaptic mechanism. Together, these findings underscore that vesicular transporter expression is used endogenously to directly regulate the extent of glutamate release, providing a concise presynaptic mechanism for controlling the quantal efficacy of excitatory transmission during synaptic refinement and plasticity.

System A is a highly regulated, Na+-dependent transporter that accepts neutral amino acids containing short, polar side chains. System A plays an important role during rat development as decreased pup weights are observed in dams infused during gestation with a non-metabolizable System A substrate. Given the potential importance of SNAT1 during development in the rat brain, we examined whether SNAT1 would be present at an earlier gestation during organogenesis in multiple organs by immunohistochemistry and immunoblotting. SNAT1 protein was observed in the developing lungs, intestines, kidneys, heart, pancreas, and skeletal muscle of rats at prenatal days 14, 17, 19, 21, and postnatal day 2 rats. SNAT1 protein expression decreased in the liver and intestine shortly after birth and as the rat matured. SNAT1 expression was constant throughout development in the lungs and kidney and increased in the heart from prenatal day 19 to postnatal day 2. Highest levels of expression in older animals were seen in organs undergoing rapid cell division.

The study evaluated the sex distribution of major isolated malformations and common trisomies among a large and geographically varied sample. Eighteen registries from 24 countries contributed cases, which were centrally reviewed and classified in three clinical types as isolated, associated, or syndromic. We selected cases of 26 major defects (n = 108,534); trisomy 21, 18, and 13 (n = 30,114); other syndromes (n = 2,898); and multiple congenital anomalies (n = 24,197), for a total of 165,743 cases. We observed a significant deviation of sex distribution (compared to a sex ratio of 1.06 or male proportion of 51.4%) for 24 of the 29 groups (a male excess in 16, a female excess in 8), and in 8 of such groups these estimates varied significantly across registries. A male excess was noted for two left obstructive cardiac defects (hypoplastic left heart and coarctation of the aorta) and a female excess for all the main types of neural tube defects. A male excess was seen for omphalocele but not gastroschisis. For neural tube defects the female excess tended to be stronger in areas with historically high prevalence for these defects. For 15 of the 26 birth defects the sex distribution differed among isolated, associated, and syndromic cases. Some of these epidemiologic commonalities are consistent with known or putative developmental processes. Further, the geographic variation for some defects may reflect local prevalence rates and risk factors. Finally, the findings underscore the need for clinical classification (e.g., into isolated, multiple, syndromes) in studies of birth defects.

Objectives: To evaluate the effectiveness of policies and recommendations on folic acid aimed at reducing the occurrence of neural tube defects., Design: Retrospective cohort study of births monitored by birth defect registries., Setting: 13 birth defects registries monitoring rates of neural tube defects from 1988 to 1998 in Norway, Finland, Northern Netherlands, England and Wales, Ireland, France (Paris, Strasbourg, and Central East), Hungary, Italy (Emilia Romagna and Campania), Portugal, and Israel. Cases of neural tube defects were ascertained among liveborn infants, stillbirths, and pregnancy terminations (where legal). Policies and recommendations were ascertained by interview and literature review., Main Outcome Measures: Incidences and trends in rates of neural tube defects before and after 1992 (the year of the first recommendations) and before and after the year of local recommendations (when applicable)., Results: The issuing of recommendations on folic acid was followed by no detectable improvement in the trends of incidence of neural tube defects., Conclusions: Recommendations alone did not seem to influence trends in neural tube defects up to six years after the confirmation of the effectiveness of folic acid in clinical trials. New cases of neural tube defects preventable by folic acid continue to accumulate. A reasonable strategy would be to quickly integrate food fortification with fuller implementation of recommendations on supplements.

In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.

Background: Since fortification of cereal grain products with synthetic folic acid (FA) became mandatory in January 1998, women in the United States who have become pregnant have been exposed to a higher level of FA than women who became pregnant previously. Some studies have suggested that increased FA consumption might increase the risk of multiple gestation pregnancies., Methods: Women who had a live birth in Kaiser Foundation Health Plan hospitals from January 1, 1994 through December 31, 2000; all multiple births; and the use of ovulation-inducing drugs were ascertained from electronic databases. Medical records of a sample of women with multiple births who did not use ovulation-inducing drugs were reviewed to determine whether they used assisted reproductive technology. Exposure to FA-fortified foods was based on date of delivery., Results: The rate of multiple births increased from 13.6 to 14.8 per 1000 live births from 1994 through 2000. The percentage of women who had a multiple birth and who filled a prescription for an ovulation-inducing drug in the 12 months before delivery increased from a low of 6.6% in 1994 to a high of 14.9% in 2000. After excluding women using ovulation-inducing drugs, the increased rate of multiple births was no longer observed., Conclusions: While the rates of multiple births have increased since FA fortification became mandatory, this increase can be explained by the increased use of ovulation-inducing drugs. Our findings show no relationship between food fortification with FA and the rates of multiple births in this large, managed health care population.

Previous studies have shown that the vesicular monoamine transporter 2 (VMAT2) is localized to both large dense core vesicles and synaptic vesicles in vivo. However, when exogenously expressed in PC12 cells, VMAT2 localizes only to large dense core vesicles. This distribution is similar to that of the endogenous vesicular monoamine transporter 1 (VMAT1) in PC12 cells. When VMAT2 was expressed in a protein kinase A (PKA)-deficient PC12 cell line it localized to synaptic-like microvesicles. Expression of recombinant VMAT1 in the same cell line showed a heterogeneous distribution to both large dense core vesicles and synaptic-like microvesicles. Coexpression of the PKA catalytic subunit partially restored trafficking of both VMAT2 and VMAT1 to large dense core vesicles; treatment of wild-type PC12 cells with the PKA inhibitor H89 increased VMAT2 on synaptic-like microvesicles. The VMAT1 and VMAT2 in large dense core vesicles exhibit a larger molecular size than those located on synaptic-like microvesicles. This difference is due to differential N-linked glycosylation. In vitro phosphorylation experiments show that PKA does not phosphorylate VMAT2. A chimera containing the VMAT2 cytoplasmic C-terminus fused to vesicular acetylcholine transporter (VAChT) shows mislocalization to synaptic-like microvesicles and VAChT-like glycosylation in the PKA-deficient cell line. However, coexpression with PKA changes the chimera's trafficking to large dense core vesicles and increases the molecular size. These results suggest that protein kinase A affects the formation and/or composition of VMAT trafficking complexes.

SNAT1 mediates glutamine (Gln) influx into neurons and is believed to replenish the transmitters pools of glutamate (Glu) and gamma-aminobutyric acid (GABA). We investigated its distribution and cellular localization in the cerebral cortex and neighboring regions of rats and humans using light and electron microscopic immunocytochemical methods with specific antibodies. In the first somatic sensory cortex of rats and in areas 9, 10, 21 and 46 of the human cortex, numerous SNAT1-positive (+) cells were present in the cortical parenchyma and in the white matter; >95% of SNAT1+ cells were neurons, but some were astrocytes. Most SNAT1+ cells were pyramidal neurons, but numerous non-pyramidal neurons were also observed: SNAT1/GABA double-labeling studies showed that SNAT1 is expressed in all GABA+ neurons. SNAT1/synaptophysin studies showed that <0.1% of all synaptophysin+ puncta coexpressed SNAT1. SNAT1 immunoreactivity (ir) was also in leptomeninges, ependymal cells and choroid plexus. Electron microscopic studies showed that neuronal SNAT1 ir was almost exclusively observed in perikarya and dendritic profiles. SNAT1 ir was also in distal astrocytic processes, including end feet profiles, and in leptomeninges. These findings suggest that the major function of SNAT1 is not to replenish the transmitter pools of Glu and GABA.

Recent reports suggest that women carrying certain polymorphisms of folate genes associated with suboptimal folate status might be at increased risk for having a child with Down syndrome or other autosomal trisomies, and hypothesized that maternal use of multivitamin supplements might reduce such risk. To evaluate this hypothesis, we examined data from a population-based case-control study, and contrasted cases of Down syndrome, trisomy 18, and trisomy 13, with unaffected controls. Periconceptional multivitamin use, compared to no such use, was associated with an odds ratio (OR) of 0.9 (95% confidence interval [CI], 0.6-1.3) for having a pregnancy affected by an autosomal trisomy. The OR was 0.8 (95% CI, 0.5-1.3) for Down syndrome and 1.4 (95% CI, 0.5-3.6) for trisomies 13 and 18, with little variation by maternal race or age. Periconceptional multivitamin use was not associated with a major reduction in the risk for common autosomal trisomies.

Randomized trials, supported by many observational studies, have shown that periconceptional use of folic acid, alone or in multivitamin supplements, is effective for the primary prevention of neural tube defects (NTDs). Whether this is true also for other congenital anomalies is a complex issue and the focus of this review. It is useful to consider the evidence not only for specific birth defects separately but, importantly, also for all birth defects combined. For the latter, the Hungarian randomized clinical trial indicated, for periconceptional multivitamin use, a reduction in the risk for all birth defects (odds ratio (OR) = 0.53, 95% confidence interval (CI) = 0.35-0.70), even after excluding NTDs (OR = 0.53, 95% CI = 0.38-0.75). The Atlanta population-based case-control study, the only large observational study to date on all major birth defects, also found a significant risk reduction for all birth defects (OR = 0.80, 95% CI = 0.69-0.93) even after excluding NTDs (OR = 0.84, 95% CI = 0.72-0.97). These and other studies also evaluated specific anomalies, including those of the heart, limb, and urinary tract, as well as orofacial clefts, omphalocele, and imperforate anus. For cardiovascular anomalies, two studies were negative, whereas three, including the randomized clinical trial, suggest a possible 25-50% overall risk reduction, more marked for some conotruncal and septal defects. For orofacial clefts, six of seven case-control studies suggest an apparent reduced risk, which could vary by cleft type and perhaps, according to some investigators, by pill dosage. For limb deficiencies, three case-control studies and the randomized trial estimated approximately a 50% reduced risk. For urinary tract defects, three case-control studies and the randomized trial reported reduced risks, as did one study of nonsyndromic omphalocele. All these studies examined multivitamin supplement use. With respect to folic acid alone, a reduced rate of imperforate anus was observed among folic acid users in China. We discuss key gaps in knowledge, possible avenues for future research, and counseling issues for families concerned about occurrence or recurrence of these birth defects., (Copyright 2004 Wiley-Liss, Inc.)

The sodium-coupled neutral amino acid transporters (SNAT) of the SLC38 gene family resemble the classically-described System A and System N transport activities in terms of their functional properties and patterns of regulation. Transport of small, aliphatic amino acids by System A subtypes (SNAT1, SNAT2, and SNAT4) is rheogenic and pH sensitive. The System N subtypes SNAT3 and SNAT5 also countertransport H(+), which may be key to their operation in reverse, and have narrower substrate profiles than do the System A subtypes. Glutamine emerges as a favored substrate throughout the family, except for SNAT4. The SLC38 transporters undoubtedly play many physiological roles including the transfer of glutamine from astrocyte to neuron in the CNS, ammonia detoxification and gluconeogenesis in the liver, and the renal response to acidosis. Probing their regulation has revealed additional roles, and recent work has considered SLC38 transporters as therapeutic targets in neoplasia.

The prescription of known teratogenic medications requires a careful balance between allowing women access to medications that they might need and avoiding unnecessary exposure to these medications during pregnancy because of their devastating fetal effects. Isotretinoin, a potent human teratogen, is of particular concern because of its widespread use among reproductive-aged women and the dramatic increase in use from 1992 through 2000. A revised risk management system was implemented in 2002 because of concerns about the continued occurrence of isotretinoin-exposed pregnancies. However, the recent approval of three generic versions of isotretinoin in the US has further complicated risk management and raises concerns that use might increase further if the lower cost of generics serves to increase accessibility. There are now four separate isotretinoin risk management systems in the US, each with its own distinct packaging, though the requirements for and substance of each are identical. Some additional concrete steps could be taken to minimise any unnecessary use of isotretinoin and help allow an adequate assessment of the current risk management systems. In addition to being familiar with and following all aspects of the current risk management system, physicians could choose to limit the use of isotretinoin to those who meet the labelled indications in order to reduce the number of exposed pregnancies. All four companies currently marketing isotretinoin in the US could jointly and voluntarily establish a consolidated, mandatory registration and follow-up of all women of reproductive potential who receive an isotretinoin prescription. Mandatory registration has many challenges, but it could allow a clear accounting of the total number of women for whom follow-up information is and is not available. Although the companies cannot be legally compelled to use a consolidated approach, the use of a single registry for the originator's product and all generic brands would allow identification of duplicates and also avoid the confusion that is introduced by providing materials that not only look different, but also have different addresses, contact information and names for participation in follow-up surveys. This is particularly important because women might take more than one version of isotretinoin during a single course of therapy or might receive a different programme's materials from their doctor than from the pharmacy. Though the introduction of generic versions of isotretinoin further complicates risk management, the companies marketing isotretinoin have an opportunity to work together to demonstrate their commitment to both limit the occurrence of exposed pregnancies and conduct a meaningful evaluation of the occurrence of pregnancies exposed to isotretinoin.

Presynaptic P2X(7) receptors are thought to play a role in the modulation of transmitter release and have been localised to terminals with the location and morphology typical of excitatory boutons. To test the hypothesis that this receptor is preferentially associated with excitatory terminals we combined immunohistochemistry for the P2X(7) receptor subunit (P2X(7)R) with that for two vesicular glutamate transporters (VGLUT1 and VGLUT2) in the rat CNS. This confirmed that P2X(7)R immunoreactivity (IR) is present in glutamatergic terminals; however, whether it was co-localised with VGLUT1-IR or VGLUT2-IR depended on the CNS region examined. In the spinal cord, P2X(7)R-IR co-localised with VGLUT2-IR. In the brainstem, co-localisation of P2X(7)R-IR with VGLUT2-IR was widespread, but co-localisation with VGLUT1-IR was seen only in the external cuneate nucleus and spinocerebellar tract region of the ventral medulla. In the cerebellum, P2X(7)R-IR co-localised with both VGLUT1 and VGLUT2-IR in the granular layer. In the hippocampus it was co-localised only with VGLUT1-IR, including in the polymorphic layer of the dentate gyrus and the substantia radiatum of the CA3 region. In other forebrain areas, P2X(7)R-IR co-localised with VGLUT1-IR throughout the amygdala, caudate putamen, striatum, reticular thalamic nucleus and cortex and with VGLUT2-IR in the dorsal lateral geniculate nucleus, amygdala and hypothalamus. Dual labelling studies performed using markers for cholinergic, monoaminergic, GABAergic and glycinergic terminals indicated that in certain brainstem and spinal cord nuclei the P2X(7)R is also expressed by subpopulations of cholinergic and GABAergic/glycinergic terminals. These data support our previous hypothesis that the P2X(7)R may play a role in modulating glutamate release in functionally different systems throughout the CNS but further suggest a role in modulating release of inhibitory transmitters in some regions.

Background: Neural tube defect (NTD) rates can be lowered by increased consumption of folic acid (FA) by women before and during early pregnancy. The crude proportion of reproductive-aged women taking FA supplements has been used to predict a decline of the NTD rate in the general population. In this study we examine the potential error in using the crude proportion to predict NTD risk reduction, and offer an alternative method., Methods: The crude proportion measures the number of women taking FA. It ignores the substantial variability by maternal age in the probability of giving birth. Age-specific fertility rates (ASFRs) reflect the probability that a woman in a specific age group will give birth in a given year. In this study, we show how to calculate a proportion weighted by ASFRs to predict a decline in the NTD rate, and to assess the effectiveness of FA consumption in preventing NTDs., Results: Our results show that a crude proportion of 50% of women (15-49 years old) taking FA is associated with a range of 24-77% in weighted proportions. Assuming a 40% risk reduction from taking 400 microg of FA daily, the expected NTD rate decline could vary from 9.6% to 30.6%, depending on the age distribution of women taking FA., Conclusions: The ASFR-weighted proportion estimates the proportion of babies born to women taking FA, as opposed to the crude proportion of women taking FA. We recommend using the ASFR-weighted proportion to predict an NTD rate decline and measure the success of FA education campaigns. We found that when women in high-fertility age groups increased their FA consumption, the decline in the NTD rate was greater than when women in low-fertility age groups did so. Our findings suggest that the more efficient approach to NTD prevention is to focus on women with a higher probability of giving birth. For example, by focusing on <50% of women of childbearing age (20-34 years), as much as 76% of the maximum NTD rate reduction can be achieved.

Since January 2000 the Chilean Ministry of Health has required the fortification of wheat flour with folic acid (FA) at a concentration of 2.2 mg FA/kg in order to reduce the risk of neural tube defects (NTD) in newborns. This policy was expected to result in a mean additional intake of approximately 400 microg FA/d. We assessed the effectiveness of the FA flour fortification program on bread folate content and on blood folate concentration in women of childbearing age in Santiago, Chile. The prefortification folate status of 751 healthy women of reproductive age was assessed. The folate content of 100 bread samples bought at retail bakeries was measured, average wheat flour consumption was estimated and postfortification FA dietary intake was calculated. The effect of flour fortification on blood folate concentration in this group of women (n = 605) was evaluated in a follow-up study. Blood folate concentrations of the 605 women in the follow-up group increased (P < 0.0001) following fortification. Before fortification the mean serum and red blood cell folate concentrations were 9.7 +/- 4.3 and 290 +/- 102 nmol/L, respectively, compared with 37.2 +/- 9.5 and 707 +/- 179 nmol/L postfortification, respectively. The mean FA content of bread was 2020 +/- 940 micro g/kg. The median FA intake of the group evaluated postfortification was 427 microg/d (95% CI 409-445) based on an estimated intake of 219 g/d (95% CI 201-229) of wheat flour, mainly as bread. Fortification of wheat flour substantially improved folate status in a population of women of reproductive age in Chile. The effect of the FA fortification program on the occurrence of NTD is currently being assessed.

Congenital heart defects are among the most common congenital anomalies and are the leading cause of infant death due to congenital anomalies. Except for a few known measures, effective primary prevention is not yet feasible for most heart anomalies. Recent reports have associated the use of multivitamin supplements around the time of conception and during early pregnancy with a reduced risk for heart defects in the offspring. We review and discuss the evidence and suggest a framework for further investigation in this area.

Cortical regions of the brain stand out for their high content in synaptic zinc, which may thus be involved in synaptic function. The relative number, chemical nature and transmitter receptor profile of synapses that sequester vesicular zinc are largely unknown. To address this, we combined pre-embedding zinc histochemistry and post-embedding immunogold electron microscopy in rat hippocampus. All giant mossy fibre (MF) terminals in the CA3 region and approximately 45% of boutons making axospinous synapses in stratum radiatum in CA1 contained synaptic vesicles that stained for zinc. Both types of zinc-positive boutons selectively expressed the vesicular zinc transporter ZnT-3. Zinc-positive boutons further immunoreacted to the vesicular glutamate transporter VGLUT-1, but not to the transmitter gamma-aminobutyric acid. Most dendritic spines in CA1 immunoreacted to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunits GluR1-3 (approximately 80%) and to N-methyl-D-aspartate receptor (NMDAR) subunits NR1 + NR2A/B (approximately 90%). Synapses made by zinc-positive boutons contained 40% less AMPAR particles than those made by zinc-negative boutons, whereas NMDAR counts were similar. Further analysis indicated that this was due to the reduced synaptic expression of both GluR1 and GluR2 subunits. Hence, the levels of postsynaptic AMPARs may vary according to the presence of vesicular zinc in excitatory afferents to CA1. Zinc-positive and zinc-negative synapses may represent two glutamatergic subpopulations with distinct synaptic signalling.

The glutamine-glutamate/GABA cycle is critical for the developing brain as glutamatergic neurotransmission is important for neuronal survival and drives synaptogenesis and activity-dependent synaptic plasticity. GABAergic transmission may be essential for the formation of neural circuits. Recently a cDNA encoding a brain-enriched System A transporter (SAT1/ATA1), has been identified which may provide glutamine to neurons for the biosynthesis of neurotransmitters glutamate and gamma-aminobutyric acid (GABA). In this study, we have examined the developmental expression pattern of SAT1/ATA1 protein in rat brain by immunohistochemistry. We find that SAT1/ATA1 was present in the developing rat brain at all gestational ages examined including prenatal days 17 and 19 and postnatal days 2, 10, 14, and adult. SAT1/ATA1 immunoreactivity was seen in the neocortex, hippocampus, and neuroepithelium at the earliest time point examined, prenatal day 17. SAT1/ATA1 was prominent in the striatum, the hippocampus and the cortex in the postnatal animals. In adults, SAT1/ATA1 was limited to the cell body region while in developing animals SAT1/ATA1 protein was found in neuronal processes. These results contribute to our understanding of the relationship between the cycling of glutamate and glutamine between astrocytes and glia and the pathophysiological conditions that occur in hypoxic ischemic encephalopathy.

We describe a unique birth defects surveillance system in the People's Republic of China. The system was instituted in March 1992 as a component of an evaluation of the effectiveness of a public health campaign using periconceptional folic acid supplementation to prevent neural tube defects, and currently surveys birth cohorts of approximately 150 000 infants per year. Local health care providers collect information in the form of detailed written descriptions and photographs of affected infants. The system allows for detection of birth defects at the local level with later definitive classification and coding; however, information is limited to structural anomalies that are visible on physical examination. This birth defects surveillance system provides an extensive database of infants with major and minor external structural anomalies, including the unique feature of a photographic record for most cases. These data can be used for aetiological studies, descriptive epidemiology and identification of unusual trends.

Background: Periconceptional folic acid use reduces the risk of neural tube defects and possibly other birth defects. The effectiveness of two interventions to increase the use of multivitamins among women of childbearing ages was evaluated., Methods: Quasi-experimental interrupted time series design with a nonequivalent control group. Participants included female members of Kaiser Foundation Health Plan aged 18 to 39 years residing in the three geographic service areas of California under study from 1998 through 2000. The central component of the direct mail/pharmacy information intervention was the mailing of "starter kits" of 100 multivitamins, while the provider education intervention used primary care providers to deliver the study message. Main outcomes included the use of multivitamins containing folic acid at least four times per week ("regularly"), intention to use multivitamins regularly, and knowledge and attitudes about multivitamins. Outcomes were measured via telephone interviews of nonpregnant women of childbearing age., Results: A total of 3438 women were interviewed. There was a small but significant increase in the percentage of women using multivitamins in the direct mail/pharmacy information intervention group at the beginning of the intervention period (p =0.006), but this increase was not sustained after the interventions ended. No other significant change was observed., Conclusions: Despite our ability to reach many women of childbearing age with multiple messages about regularly using multivitamins, only a small temporary increase was found in the percentage of women using multivitamins who received the messages in the mail. Other interventions and further evaluation of the impact of food fortification with folic acid should be considered.

Glutamine, the preferred precursor for neurotransmitter glutamate and GABA, is likely to be the principal substrate for the neuronal System A transporter SNAT1 in vivo. We explored the functional properties of SNAT1 (the product of the rat Slc38a1 gene) by measuring radiotracer uptake and currents associated with SNAT1 expression in Xenopus oocytes and determined the neuronal-phenotypic and cellular distribution of SNAT1 by confocal laser-scanning microscopy alongside other markers. We found that SNAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine (K0.5 approximately 0.3 mm), alanine, and the System A-specific analogue 2-(methylamino)isobutyrate. Amino acid transport is driven by the Na+ electrochemical gradient. The voltage-dependent binding of Na+ precedes that of the amino acid in a simultaneous transport mechanism. Li+ (but not H+) can substitute for Na+ but results in reduced Vmax. In the absence of amino acid, SNAT1 mediates Na+-dependent presteady-state currents (Qmax approximately 9 nC) and a nonsaturable cation leak with selectivity Na+, Li+ >> H+, K+. Simultaneous flux and current measurements indicate coupling stoichiometry of 1 Na+ per 1 amino acid. SNAT1 protein was detected in somata and proximal dendrites but not nerve terminals of glutamatergic and GABAergic neurons throughout the adult CNS. We did not detect SNAT1 expression in astrocytes but detected its expression on the luminal membranes of the ependyma. The functional properties and cellular distribution of SNAT1 support a primary role for SNAT1 in glutamine transport serving the glutamate/GABA-glutamine cycle in central neurons. Localization of SNAT1 to certain dopaminergic neurons of the substantia nigra and cholinergic motoneurons suggests that SNAT1 may play additional specialized roles, providing metabolic fuel (via alpha-ketoglutarate) or precursors (cysteine, glycine) for glutathione synthesis.

Background: Folic acid intake is the most important predictor of blood folate concentrations among nonpregnant women, but the reporting of folic acid-containing supplement use is subject to error., Objective: We assessed the effect of reporting error of supplement use on blood folate concentrations., Design: Data from the third National Health and Nutrition Examination Survey were analyzed. Respondents to that survey were asked twice about supplement use: ie, during the household interview, to recall use in the previous month, and during the physical examination, to recall use in the previous 24 h. To examine the effect of error reporting, we classified women (aged 15-44 y) into 5 groups according to supplement use in the previous month (nonusers, those ingesting < 400 micro g/d, and those ingesting >or= 400 micro g/d) and in the 24 h before the physical examination (yes or no). We expected nonappreciable differences in red blood cell (RBC) folate concentration by status of 24-h recall within the same category of previous-month use because RBC folate reflects long-term average consumption. We calculated covariate-adjusted means of serum and RBC folate concentrations., Results: Among women who reported average daily use of >or= 400 micro g folic acid in the previous month, the adjusted mean RBC folate was 436.5 nmol/L (95% CI: 406.7, 466.3 nmol/L) in those who did not take the supplement in the previous 24 h and 519.7 nmol/L (95% CI: 496.2, 543.2 nmol/L) in those who did do so (P < 0.01). This significant difference indicates apparently erroneous reporting of supplement use in the previous month by some participants., Conclusion: The effect of reporting error on blood folate concentrations is important in interpreting survey results, evaluating health education campaigns, and identifying populations needing special education programs.

We have cloned and functionally characterized a third isoform of a vesicular glutamate transporter (VGLUT3) expressed on synaptic vesicles that identifies a distinct glutamatergic system in the brain that is partly and selectively promiscuous with cholinergic and serotoninergic transmission. Transport activity was specific for glutamate, was H(+)-dependent, was stimulated by Cl(-) ion, and was inhibited by Rose Bengal and trypan blue. Northern analysis revealed higher mRNA levels in early postnatal development than in adult brain. Restricted patterns of mRNA expression were observed in presumed interneurons in cortex and hippocampus, and projection systems were observed in the lateral and ventrolateral hypothalamic nuclei, limbic system, and brainstem. Double in situ hybridization histochemistry for vesicular acetylcholine transporter identified VGLUT3 neurons in the striatum as cholinergic interneurons, whereas VGLUT3 mRNA and protein were absent from all other cholinergic cell groups. In the brainstem VGLUT3 mRNA was concentrated in mesopontine raphé nuclei. VGLUT3 immunoreactivity was present throughout the brain in a diffuse system of thick and thin beaded varicose fibers much less abundant than, and strictly separated from, VGLUT1 or VGLUT2 synapses. Co-existence of VGLUT3 in VMAT2-positive and tyrosine hydroxylase -negative varicosities only in the cerebral cortex and hippocampus and in subsets of tryptophan hydroxylase-positive cell bodies and processes in differentiating primary raphé neurons in vitro indicates selective and target-specific expression of the glutamatergic/serotoninergic synaptic phenotype.

In September 1992, the U.S. Public Health Service (USPHS) recommended that all women capable of becoming pregnant should consume 400 microg of folic acid/day on an ongoing basis to reduce their risk for having a pregnancy affected by spina bifida and anencephaly (i.e., neural tube defects [NTDs]). The recommendation was preceded a year earlier by a CDC recommendation for women at high risk (i.e., those women who have had an earlier pregnancy affected by an NTD). The 1991 CDC recommendation stated that women at high risk should plan subsequent pregnancies and consume 4,000 microg/day of folic acid from the time they begin trying to become pregnant through the first trimester of pregnancy to reduce their risk. The 1992 USPHS recommendation specified that women at high risk should follow the general population recommendation for consumption of 400 microg/day when not trying to become pregnant.

Background: Multivitamin use has been associated with lower risks for some birth defects. We evaluated whether multivitamin use modified birth defect risks associated with febrile illness, a common and possibly teratogenic exposure., Methods: From the population-based Atlanta Birth Defects Case-Control Study (1968-1980) we selected seven defects (neural tube defects, cleft lip and palate, cardiac outflow tract defects, ventricular septal defects, atrial septal defects, omphalocele, and limb deficiencies) because of their inverse relation with multivitamin supplement use documented in previous analyses. We defined four exposure categories from combinations of multivitamin use (periconceptional use compared with no use) and febrile illness (early pregnancy compared with no illness). The reference category was no multivitamin use and no illness., Results: Febrile illness with no multivitamin use was associated with generally increased risk for the seven defects and the combined group (odds ratio = 2.1, 1.7, 1.5, 1.9, 2.9, 4.4, 3.3, and 2.3, respectively). With multivitamin use, however, the risk estimates associated with febrile illness were generally lower (odds ratio = 0.6, 1.1, 0.0, 1.5, 0.0, 0.8, 0.0, and 0.8, respectively). Some of the associated 95% confidence intervals included one., Conclusions: The pattern of findings suggests that multivitamin use might decrease the risk associated with febrile illness.

Objective: We evaluated the association between mothers' use of multivitamin supplements and their infants' risk for omphalocele, a congenital anomaly of the abdominal wall. Omphalocele can occur in certain multiple congenital anomaly patterns with neural tube defects, for which a protective effect of multivitamins with folic acid has been demonstrated., Methods: We used data from a population-based case-control study of infants born from 1968-1980 to mothers residing in metropolitan Atlanta. Case-infants with nonsyndromic omphalocele (n = 72) were actively ascertained from multiple sources. Control-infants (n = 3029), without birth defects, were selected from birth certificates by stratified random sampling., Results: Compared with no use in the periconceptional period, periconceptional use of multivitamin supplements (regular use from 3 months before pregnancy through the first trimester of pregnancy) was associated with an odds ratio for nonsyndromic omphalocele of 0.4 (95% confidence interval [CI]: 0.2-1.0). For the subset comprising omphalocele alone or with selected midline defects (neural tube defects, hypospadias, and bladder/cloacal exstrophy), the odds ratio was 0.3 (95% CI: 0.1-0.9). These estimates were similar when the reference group also included women who began using multivitamins late in pregnancy (during the second or third month of pregnancy). The small number of participants limited the precision of subgroup analyses and translated into wide confidence intervals that included unity., Conclusions: Periconceptional multivitamin use was associated with a 60% reduction in the risk for nonsyndromic omphalocele. These findings await replication from additional studies to confirm the findings, generate more precise estimates, and detail possible mechanisms of actions.