We present here a method for broadly characterizing single cells at the molecular level beyond the more common morphological and transmitter/receptor classifications. The RNA from defined single cells is amplified by microinjecting primer, nucleotides, and enzyme into acutely dissociated cells from a defined region of rat brain. Further processing yields amplified antisense RNA. A second round of amplification results in >106-fold amplification of the original starting material, which is adequate for analysis-e.g., use as a probe, making of cDNA libraries, etc. We demonstrate this method by constructing expression profiles of single live cells from rat hippocampus. This profiling suggests that cells that appear to be morphologically similar may show marked differences in patterns of expression. In addition, we characterize several mRNAs from a single cell, some of which were previously undescribed, perhaps due to "rarity" when averaged over many cell types. Electrophysiological analysis coupled with molecular biology within the same cell will facilitate a better understanding of how changes at the molecular level are manifested in functional properties. This approach should be applicable to a wide variety of studies, including development, mutant models, aging, and neurodegenerative disease.

Neural tube closure takes place during early embryogenesis and requires interactions between genetic and environmental factors. Failure of neural tube closure is a common congenital malformation that results in morbidity and mortality. A major clinical achievement has been the use of periconceptional folic acid supplements, which prevents ~50-75% of cases of neural tube defects. However, the mechanism underlying the beneficial effects of folic acid is far from clear. Biochemical, genetic and epidemiological observations have led to the development of the methylation hypothesis, which suggests that folic acid prevents neural tube defects by stimulating cellular methylation reactions. Exploring the methylation hypothesis could direct us towards additional strategies to prevent neural tube defects.Neural tube defects (NTDs) are complex congenital malformations of the CNS. Anencephaly and spina bifida are the most common and severe forms of NTD, with a prevalence of about 1 in 1,000 births, although this varies throughout the world 1 . Infants with anencephaly are stillborn or die shortly after birth, whereas infants with spina bifida can survive but are likely to have severe lifelong disabilities and are at risk of psychosocial maladjustment. The estimated lifetime medical costs are high; for example, in California, USA, the costs for children born with spina bifida exceed US$81 million per year 2 .It has been known for more than 20 years that women can reduce their risk of having an NTDaffected pregnancy by taking folic acid supplements. However, the underlying mechanisms byCorrespondence to H.J.B. H.Blom@cukz.umcn.nl. Competing interests statementThe authors declare no competing financial interests. DATABASES NIH Public Access Author ManuscriptNat Rev Neurosci. Author manuscript; available in PMC 2010 November 2. Neurulation and neural tube defectsNeurulation has long fascinated scientists, as evidenced by a devoted literature that extends back hundreds of years. The process of neurulation occurs during early embryogenesis, starting with the formation of the neural plate from specialized ectodermal cells and culminating in the closure of the neural tube (FIG. 1), the precursor of both the CNS and most of the PNS. Considered most simply, the neural plate develops bilateral neural folds, which elevate and fuse at the midline to create the neural tube. Subsequent development, together with vesiculation within the tube, gives rise to the brain and spinal cord. Further details of this complex process are beyond the scope of this review (for reviews, see REFS 4,5 ).Neurulation seems to be highly complex and tightly regulated. The development and closure of the neural tube is usually completed by 28 days post-conception. This means that by the time a woman finds out that she is pregnant, the neural tube is almost completely closed. For the neural tube to close properly, genes that regulate various morphogenetic activities must function cooperatively. These activities include programmed cell death, neural crest...

Prevention or Repair Neural tube defects, such as spina bifida, remain remarkably common, despite widespread efforts to prevent them through supplementing maternal diets with folic acid. Surgery early in development has seen some success, but problems often remain. Wallingford et al. ( 10.1126/science.1222002 ) review normal and abnormal neural tube development and suggest that discovering the genetic risk factors for these serious birth defects could provide ways to prevent and treat neural tube defects.

Periconceptional folic acid supplementation reduces the occurrence of several human congenital malformations, including craniofacial, heart and neural tube defects. Although the underlying mechanism is unknown, there may be a maternal-to-fetal folate-transport defect or an inherent fetal biochemical disorder that is neutralized by supplementation. Previous experiments have identified a folate-binding protein (Folbp1) that functions as a membrane receptor to mediate the high-affinity internalization and delivery of folate to the cytoplasm of the cell. In vitro, this receptor facilitates the accumulation of cellular folate a thousand-fold relative to the media, suggesting that it may be essential in cytoplasmic folate delivery in vivo. The importance of an adequate intracellular folate pool for normal embryogenesis has long been recognized in humans and experimental animals. To determine whether Folbp1 is involved in maternal-to-fetal folate transport, we inactivated Folbp1 in mice. We also produced mice lacking Folbp2, another member of the folate receptor family that is GPI anchored but binds folate poorly. Folbp2-/- embryos developed normally, but Folbp1-/- embryos had severe morphogenetic abnormalities and died in utero by embryonic day (E) 10. Supplementing pregnant Folbp1+/- dams with folinic acid reversed this phenotype in nullizygous pups. Our results suggest that Folbp1 has a critical role in folate homeostasis during development, and that functional defects in the human homologue (FOLR1) of Folbp1 may contribute to similar defects in humans.