Fetal Hippocampus Research Articles

An E3 Ubiquitin Ligase, Really Interesting New Gene (RING) Finger 41, Is a Candidate Gene for Anxiety-Like Behavior and β-Carboline-Induced Seizures

Identification of the genes underlying psychiatric illness remains a thorny problem. Previously, Quantitative Trait Loci (QTL) for anxiety-like behaviors and beta-carboline-induced seizure vulnerability have been mapped to the distal portion of mouse chromosome 10, with crosses of A/J and C57BL6 mice. An interval specific congenic strain for this chromosomal 10 region facilitated the genetic dissection of novelty-induced exploratory behaviors. By microarray studies, an unsuspected E3 ubiquitin ligase, Really Interesting New Gene (RING) Finger 41 (Rnf41) was differentially expressed in the region of interest, being upregulated in the hippocampi of B6 compared with A/J as well as congenic A.B6(chr10) versus A/J. By quantitative real-time polymerase chain reaction (qRT-PCR), Rnf41 expression levels were significantly increased 1.5- and 1.3-fold in the hippocampi of C57BL6/J and A.B6(chr10) mice compared with A/J mice, respectively. Protein levels of Rnf41 were increased in hippocampi of B6 mice compared with A/J mice across postnatal development with a 5.5-fold difference at P56. Yeast two-hybrid studies searching for Rnf41 binding partners in fetal hippocampus identified several potential targets. An interaction between Rnf41 and NogoA was validated by glutathionine-S-transferase-Rnf41 pulldown experiments. Re-analysis of a microarray database of human postmortem prefrontal cortex (Brodmann's Area 46/10) found that RNF41 messenger RNA expression levels were reduced significantly in patients with major depression and bipolar disorder compared with unaffected control subjects and confirmed by qRT-PCR. Overall, Rnf41 is nominated as a candidate gene for anxiety-like behaviors, depression, and vulnerability to seizures. The RNF41 and its binding partners suggest molecular pathways underlying behavior, highlighting a potential role for the ubiquitin proteasome system in psychiatric illness.

Dose Response Effects of Dermally applied Diethanolamine on Neurogenesis in Fetal Mouse Hippocampus and Potential Exposure of Humans

Diethanolamine (DEA) is a common ingredient of personal care products. Dermal administration of DEA diminishes hepatic stores of the essential nutrient choline and alters brain development. We previously reported that 80 mg/kg/day of DEA during pregnancy in mice reduced neurogenesis and increased apoptosis in the fetal hippocampus. This study was designed to establish the dose-response relationships for this effect of DEA. Timed-pregnant C57BL/6 mouse dams were dosed dermally from gestation day 7-17 with DEA at 0 (controls), 5, 40, 60, and 80 mg/kg body/day. Fetuses (embryonic day 17 [E17]) from dams treated dermally with 80 mg/kg body/day DEA had decreased neural progenitor cell mitosis at the ventricular surface of the ventricular zone (hippocampus, 54.1 +/- 5.5%; cortex, 58.9 +/- 6.8%; compared to controls; p < 0.01). Also, this dose of DEA to dams increased rates of apoptosis in E17 fetal hippocampus (to 177.2 +/- 21.5% of control; measured using activated caspase-3; p < 0.01). This dose of DEA resulted in accumulation of DEA and its metabolites in liver and in plasma. At doses of DEA less than 80 mg/kg body/day to dams, there were no differences between treated and control groups. In a small group of human subjects, dermal treatment for 1 month with a commercially available skin lotion containing 1.8 mg DEA per gram resulted in detectable plasma concentrations of DEA and dimethyldiethanolamine, but these were far below those concentrations associated with perturbed brain development in the mouse.

Identification of FMRP‐associated mRNAs using yeast three‐hybrid system

Fragile X syndrome, one of the most common forms of inherited mental retardation, results from the absence of the fragile X mental retardation protein (FMRP), which is encoded by the fragile X mental retardation gene 1 (FMR1). FMRP is an RNA-binding protein involved in translational regulation of targeted mRNAs. Identification of targeted mRNAs associated with FMRP is important to understand the function of FMRP and the pathogenic basis of the fragile X syndrome. Employing a yeast three-hybrid system and a human fetal hippocampus cDNA library, we identified 22 candidate target mRNAs, and 18 of them were confirmed to be associated with FMRP in vitro by gel retardation. Some of these mRNAs code for structural proteins, enzymes or proteins involved in cellular processes, especially in the development and function of neural system. To further investigate the role of FMRP in regulating targeted gene expression, we analyzed the expression profile of TXNRD1, one of the candidate mRNAs, after knocking down the expression of endogenous FMRP by siRNA. The results showed that endogenous TXNRD1 translation increased along with depletion of FMRP, which suggested FMRP negatively regulates TXNRD1 translation.

Behavior of hippocampal stem/progenitor cells following grafting into the injured aged hippocampus

Multipotent neural stem/progenitor cells (NSCs) from the embryonic hippocampus are potentially useful as donor cells to repopulate the degenerated regions of the aged hippocampus after stroke, epilepsy, or Alzheimer's disease. However, the efficacy of the NSC grafting strategy for repairing the injured aged hippocampus is unknown. To address this issue, we expanded FGF-2-responsive NSCs from the hippocampus of embryonic day 14 green fluorescent protein-expressing transgenic mice as neurospheres in vitro and grafted them into the hippocampus of 24-month-old F344 rats 4 days after CA3 region injury. Engraftment, migration, and neuronal/glial differentiation of cells derived from NSCs were analyzed 1 month after grafting. Differentiation of neurospheres in culture dishes or after placement on organotypic hippocampal slice cultures demonstrated that these cells had the ability to generate considerable numbers of neurons, astrocytes, and oligodendrocytes. Following grafting into the injured aged hippocampus, cells derived from neurospheres survived and dispersed, but exhibited no directed migration into degenerated or intact hippocampal cell layers. Phenotypic analyses of graft-derived cells revealed neuronal differentiation in 3%-5% of cells, astrocytic differentiation in 28% of cells, and oligodendrocytic differentiation in 6%-10% cells. The results demonstrate for the first time that NSCs derived from the fetal hippocampus survive and give rise to all three CNS phenotypes following transplantation into the injured aged hippocampus. However, grafted NSCs do not exhibit directed migration into lesioned areas or widespread neuronal differentiation, suggesting that direct grafting of primitive NSCs is not adequate for repair of the injured aged brain without priming the microenvironment.

Expression of glucocorticoid receptor, mineralocorticoid receptor, and 11β-hydroxysteroid dehydrogenase 1 and 2 in the fetal and postnatal ovine hippocampus: ontogeny and effects of prenatal glucocorticoid exposure

To determine the expression of glucocorticoid metabolizing and action genes in the hippocampus of fetal, neonatal, and adult sheep. Pregnant ewes (or their fetuses) received intramuscular injections of saline or betamethasone (BETA, 0-5 mg/kg) at 104, 111, 118, and/or 125 days of gestation (dG). Hippocampal tissue was collected prior to (75, 84, and 101 dG), during (109 and 116 dG), or after (121, 132, and 146 dG; 6 and 12 postnatal weeks; 3.5 years of age) saline or BETA injections. Hippocampal glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11beta-hydroxysteroid dehydrogenase (11betaHSD)1 and 11betaHSD2 mRNA levels were determined using qRT-PCR. Control animals late in gestation demonstrated a decrease in mRNA encoding GR and 11betaHSD1, whereas 11betaHSD2 was undetectable, consistent with a damping of the negative feedback influence of circulating or locally produced cortisol on the hypothalamic-pituitary-adrenal (HPA) axis. BETA-administration had transient effects on fetal GR and MR, and early in postnatal life (12 weeks of age) 11betaHSD1 mRNA was increased. Hippocampal MR mRNA was elevated in adult offspring exposed to either one or four doses of maternal BETA (P<0.001). Four courses of maternal BETA increased 11betaHSD2 (P<0.05) but not 11betaHSD1 mRNA levels. Late in gestation a reduction in hippocampal GR and 11betaHSD1 mRNA suggests lessening of glucocorticoid negative feedback, facilitating increased preterm HPA activity and parturition. Adult offspring of BETA-treated mothers demonstrated increased MR and 11betaHSD2 mRNA, therefore it appears that exposure of fetus to high levels of synthetic glucocorticoids may have long-lasting effects on the hippocampal expression of HPA-related genes into adulthood.

Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein

To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of GFP. The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP. After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently. We have successfully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies.

Choline deficiency alters angiogenesis in the fetal brain

Maternal dietary choline intake during pregnancy alters normal fetal neurogenesis and associates with lifelong changes in memory function in the offspring. Mitosis is decreased, while apoptosis is increased, in neural progenitor cells of the hippocampus in the fetuses of rodent dams fed choline deficient (CD) diets. We now report that maternal choline deficiency increases angiogenesis in the fetal hippocampus. Timed pregnant C57BL/6 mice were fed a CD or control (CT) AIN‐76 diet from day 12 to day 17 (E17) of gestation. Fetal brains were studied at day E17. We found increased numbers of endothelial cell‐clusters expressing factor VIII in the fetal hippocampal area in the CD group (68±3 in CD vs. 54±3 in CT, p &lt;0.01). In other experiments, neural progenitor cells from E14 fetal mouse brains were grown in low choline (5 μM) or control (70 μM) for 72 h. In the low choline group we observed increased relative gene expression for vascular endothelial factor (VEGF) (5.03±2.24 in CD vs. 0.52± 0.19 in CT, p&lt;0.001) and angiopoietin 2 (ANGPT2) (4.92±1.7 in CD vs. 1.43±0.6 in CT, p&lt;0.05). We conclude that the changes in neurogenesis are the trigger for altered angiogenesis, probably by increasing VEGF and ANGPT2 signaling. This phenomenon contributes to the effects that maternal intake of choline has on fetal brain development. This research is supported by the grant from NIH‐ AG09525.

Late N‐acetylcysteine treatment prevents the deficits induced in the offspring of dams exposed to an immune stress during gestation

Prenatal infection is a major stressful experience leading to enhanced susceptibility for mental illnesses in humans. We recently reported in rats, that oxidative stress and glutathione (GSH) shortage occurred in fetal male brain after lipopolysaccharide (LPS) to the dams and that these responses might be involved in the neurodevelopmental deficits observed in adolescent offspring. Furthermore, pretreatment with N-acetylcysteine (NAC) before LPS avoided both delayed synaptic plasticity and mnesic performance deficits. Since NAC is one of the few medications permitted in pregnant women, this study evaluated the ability of NAC to serve as a protective therapy even after the LPS challenge. Pregnant rats received a single ip injection of E. coli LPS, two days before delivery, and were given NAC in their tap water after the LPS. GSH was evaluated at the time of its expected drop in the hippocampus of male fetuses, whereas long-term potentiation (LTP) in the CA1 area of the hippocampus and spatial memory in the water-maze were recorded in 28-day-old male offspring. Post-treatment with NAC, four hours after the LPS challenge fully prevented the drop in the GSH hippocampal content. LTP, as well as spatial learning were completely protected. NAC administration at delivery also partially restored the LTP whereas post-treatment two days later was inefficient. Another set of dams were supplemented with alpha-tocopherol prior to LPS exposure, enhancing the alpha-tocopherol levels in fetal hippocampus. This treatment did not prevent the LPS-induced synaptic plasticity impairment. These results point to fetal hippocampal GSH as a major target of the detrimental effects of in utero LPS challenge. The therapeutic window of NAC extends up to birth, suggesting that this drug might be clinically useful even after an immuno-inflammatory episode.

Development of an artificial neuronal network with post-mitotic rat fetal hippocampal cells by polyethylenimine

The selection of appropriate surface materials that promote cellular adhesion and growth is an important consideration when designing a simplified neuronal network in vitro. In the past, extracellular matrix proteins such as laminin (LN) or positively charged substances such as poly- l-lysine (PLL) have been used. In this study, we examined the ability of another positively charged polymer, polyethyleneimine (PEI), to promote neuronal adhesion, growth and the formation of a functional neuronal network in vitro. PEI, PLL and LN were used to produce grid-shape patterns on glass coverslips by micro-contact printing. Post-mitotic neurons from the rat fetal hippocampus were cultured on the different polymers and the viability and morphology of these neurons under serum-free culture conditions were observed using fluorescent microscopy and atomic force microscopy (AFM). We show that neurons cultured on the PEI- and PLL-coated surfaces adhered to and extended neurites along the grid-shape patterns, whereas neurons cultured on the LN-coated coverslips clustered into clumps of cells. In addition, we found that the neurons on the PEI and PLL-coated grids survived for more than 2 weeks in serum-free conditions, whereas most neurons cultured on the LN-coated grids died after 1 week. Using AFM, we observed some neurosynapse-like structures near the neuronal soma on PEI-coated coverslips. These findings indicate that PEI is a suitable surface for establishing a functional neuronal network in vitro.

Repeated administrations of adrenocorticotropic hormone during late gestation in pigs: Maternal cortisol response and effects on fetal HPA axis and brain neurotransmitter systems

The present study examined the effects of repeated adrenocorticotropic hormone (ACTH) administrations to sows during late gestation on hypothalamic-pituitary-adrenocortical (HPA) axis and brain neurotransmitter systems in their fetuses. ACTH (100 IU per animal, Synacthen Depot, n=6) or saline (n=5) was administered intramuscularly to sows every 2nd day from gestational day (GD) 85 to GD 101. Blood samples were taken from sows repeatedly within 12h after ACTH application on GD 85 and GD 101. On GD 105, fetuses were recovered under general anaesthesia for the collection of blood and brain samples. Plasma cortisol concentrations in sows increased significantly within 2h after ACTH application and returned to control levels after 10h post-application, showing a similar response at the beginning and at the end of the 16-day stimulation period. On GD 101, a significant increase of plasma glucose and insulin concentrations was found in sows after administration of ACTH and after a following feeding time. Number and body weight of fetuses were not affected by the maternal ACTH treatment. Cortisol concentrations in the umbilical vein were significantly decreased in fetuses from ACTH sows and a similar trend was observed in the umbilical artery and in the vena cava cranialis. Glucocorticoid receptor (GR) binding in hippocampus and hypothalamus did not differ between treatments. However, in hippocampus, serotonergic activity was increased in fetuses from ACTH-treated mothers as shown by significantly elevated 5-hydroxytryptamine (5-HT) levels. In conclusion, repeated administrations of ACTH during late gestation resulted in a reproducible cortisol response of sows and reduced cortisol concentrations in the fetal umbilical vein after the treatment period. Although the number of sows used in this experiment was low and differences between treatments were limited these findings indicate that excessive glucocorticoid exposure during gestation alters serotonergic activity in hippocampus of fetuses and may affect the emotional reactivity later in life.

A yeast model of Down syndrome

The recent discovery that cellular proliferation was reduced in aneuploid haploid yeast supports a long-standing argument that the developmental neurophenotype of Down syndrome is not uniquely a result of the effects of increased gene dosage. Instead, some phenotypic outcomes appear to resemble those caused by disrupted cellular homeostasis induced by aneuploidy. Decreased cellular proliferation has been identified in the cerebellum and hippocampus of Down syndrome mouse models and in the post-mortem hippocampus and germinal matrix of Down syndrome fetuses. Consistent with predominantly stochastic gene expression and increased energy demands induced by aneuploidy, the “buffering” canalization processes in Down syndrome would be reduced thereby giving rise to increased variance to less stable developmental pathways such as proliferation. The nature and extent of phenotypes due to reduced canalization would depend on the tissue; which is also a question for future research to address. A conceptual model is presented here to demonstrate the nature of influences affecting phenotypes. Ultimately, in Down syndrome, exigent periods of neurodevelopment increasingly appear to reflect the burden of disrupted homeostasis.

Effects of repeated prenatal glucocorticoid exposure on long‐term potentiation in the juvenile guinea‐pig hippocampus

Synthetic glucocorticoids (sGCs) are routinely used to treat women at risk of preterm labour to promote fetal lung maturation. There is now strong evidence that exposure to excess glucocorticoid during periods of rapid brain development has permanent consequences for endocrine function and behaviour in the offspring. Prenatal exposure to sGC alters the expression of N-methyl-D-aspartate receptor (NMDA-R) subunits in the fetal and neonatal hippocampus. Given the integral role of the NMDA-R in synaptic plasticity, we hypothesized that prenatal sGC exposure will have effects on hippocampal long-term potentiation (LTP) after birth. Further, this may occur in either the presence or absence of elevated cortisol concentrations, in vitro. Pregnant guinea-pigs were injected with betamethasone (Beta, 1 mg kg(-1)) or vehicle on gestational days (gd) 40, 41, 50, 51, 60 and 61 (term approximately 70 days), a regimen comparable to that given to pregnant women. On postnatal day 21, LTP was examined at Schaffer collateral synapses in the CA1 region of hippocampal slices prepared from juvenile animals exposed to betamethasone or vehicle, in utero. Subsequently, the acute glucocorticoid receptor (GR)- and mineralocorticoid receptor (MR)-dependent effects of cortisol (0.1-10 microM; bath applied 30 min before LTP induction) were examined. There was no effect of prenatal sGC treatment on LTP under basal conditions. The application of 10 microM cortisol depressed excitatory synaptic transmission in all treatment groups regardless of sex. Similarly, LTP was depressed by 10 microM cortisol in all groups, with the exception of Beta-exposed females, in which LTP was unaltered. Hippocampal MR and GR protein levels were increased in Beta-exposed females, but not in any other prenatal treatment group. This study reveals sex-specific effects of prenatal exposure to sGC on LTP in the presence of elevated cortisol, a situation that would occur in vivo during stress.

Micro-RNA speciation in fetal, adult and Alzheimer's disease hippocampus

Micro-RNAs constitute a family of small noncoding ribonucleic acids that are posttranscriptional regulators of messenger RNA activity. Although micro-RNAs are known to be dynamically regulated during neural development, the role of micro-RNAs in brain aging and neurodegeneration is not known. This study examined micro-RNA abundance in the hippocampal region of fetal, adult and Alzheimer's disease brain. The data indicate that micro-RNAs encoding miR-9, miR-124a, miR-125b, miR-128, miR-132 and miR-219 are abundantly represented in fetal hippocampus, are differentially regulated in aged brain, and an alteration in specific micro-RNA complexity occurs in Alzheimer hippocampus. These data are consistent with the idea that altered micro-RNA-mediated processing of messenger RNA populations may contribute to atypical mRNA abundance and neural dysfunction in Alzheimer's disease brain.

Neurodevelopmental damage after prenatal infection: Role of oxidative stress in the fetal brain

Prenatal infection is a major risk responsible for the occurrence of psychiatric conditions in infants. Mimicking maternal infection by exposing pregnant rodents to bacterial endotoxin lipopolysaccharide (LPS) also leads to major brain disorders in the offspring. The mechanisms of LPS action remain, however, unknown. Here, we show that LPS injection during pregnancy in rats, 2 days before delivery, triggered an oxidative stress in the hippocampus of male fetuses, evidenced by a rapid rise in protein carbonylation and by decreases in α-tocopherol levels and in the ratio of reduced/oxidized forms of glutathione (GSH/GSSG). Neither protein carbonylation increase nor decreases in α-tocopherol levels and GSH/GSSG ratio were observed in female fetuses. NMDA synaptic currents and long-term potentiation in CA1, as well as spatial recognition in the water maze, were also impaired in male but not in female 28-day-old offspring. Pretreatment with the antioxidant N-acetylcysteine prevented the LPS-induced changes in the biochemical markers of oxidative stress in male fetuses, and the delayed detrimental effects in male 28-day-old offspring, completely restoring both long-term potentiation in the hippocampus and spatial recognition performance. Oxidative stress in the hippocampus of male fetuses may thus participate in the neurodevelopmental damage induced by a prenatal LPS challenge.

The Effects of Maternal Betamethasone Administration on the Intrauterine Growth-Restricted Fetus

Intrauterine growth restriction (IUGR) is associated with altered fetal cardiovascular function to ensure adequate perfusion of essential organs. IUGR fetuses are at risk of preterm delivery and so are likely to receive antenatal glucocorticoids to promote lung maturation. Because glucocorticoids alter vascular tone, we questioned whether such treatment may induce fetal cardiovascular alterations. Using pregnant sheep carrying twins, we induced IUGR at approximately 0.7 gestation by single umbilical artery ligation in one twin, using the other twin as a control. In each fetus, we monitored carotid blood flow and arterial blood gases. We administered 11.4 mg betamethasone (n = 5) or vehicle (n = 4) to the ewe on d 5 (BM1) and 6 (BM2) postsurgery. On d 7, fetal brains were collected for immunohistochemistry. In control fetuses, carotid blood flow decreased 3.5 h post-BM1 by 24% (P < 0.001), returning to baseline at 5.5 h. In IUGR fetuses, carotid flow decreased 2.5 h post-BM1 by 27% and then increased by 25% over baseline, peaking at 11 h (P < 0.001). Compared to control + saline, we observed a significant increase in oxidative damage (4-hydroxynonenal-positive cells) in the fetal hippocampus and subcallosal area of all treatment groups (IUGR + BM > IUGR + saline = control + BM). There was a significant correlation between carotid blood flow reperfusion after betamethasone and the number of 4-hydroxynonenal-positive cells in the cortex and hippocampus. These data suggest that antenatal betamethasone may induce brain injury in the IUGR fetus but not in the normally grown fetus.

Interleukin-1β Released by gp120 Drives Neural Death through Tyrosine Phosphorylation and Trafficking of NMDA Receptors

Interleukin-1beta is a proinflammatory cytokine implicated under pathological conditions involving NMDA receptor activation, including the AIDS dementia complex (HAD). No information is available on the molecular mechanisms recruited by native interleukin-1beta produced under this type of condition. Using a sandwich co-culture of primary hippocampal neurons and glia, we investigated whether native interleukin-1beta released by HIV-gp120-activated glia (i) affects NMDAR functions and (ii) the relevance on neuronal spine density and survival, two specific traits of HAD. Increased phosphorylation of NR2B Tyr-1472 was observed after 24 h of exposure of neurons to 600 pm gp120. This effect occurred only when neurons were treated in the presence of glial cells and was abolished by the interleukin-1 receptor antagonist (IL-1ra). Gp120-induced phosphorylation of NR2B resulted in a sustained elevation of intracellular Ca(2+) in neurons and in a significant increase of NR2B binding to PSD95. Increased intracellular Ca(2+) was prevented by 10 mum ifenprodil, that selectively inhibits receptors containing the NR2B, by interleukin-1ra and by Ca-pYEEIE, a Src family SH2 inhibitor peptide. These last two inhibitors, prevented also NR2B binding to PSD95. Finally, gp120 reduced by 35% of the total PSD95 positive spine density after 48 h of treatment and induced by 30% of the neuronal death. Again, both of these effects were blocked by Ca-pYEEIE. Altogether, our data show that gp120 releasing interleukin-1beta from glia increases tyrosine phosphorylation of NMDAR. Thus, tyrosine phosphorylation may contribute to the sensitization of the receptor increasing its function and synaptic localization. Both of these effects are relevant for neurodegeneration.

Chronic Prenatal Ethanol Exposure Increases Glucocorticoid‐Induced Glutamate Release in the Hippocampus of the Near‐Term Foetal Guinea Pig

Exposure to high cortisol concentration can injure the developing brain, possibly via an excitotoxic mechanism involving glutamate (Glu). The present study tested the hypothesis that chronic prenatal ethanol exposure (CPEE) activates the foetal hypothalamic-pituitary-adrenal axis to produce high cortisol exposure in the foetal compartment and alters sensitivity to glucocorticoid-induced Glu release in the foetal hippocampus. Pregnant guinea pigs received daily oral administration of ethanol (4 g/kg maternal body weight/day) or isocaloric-sucrose/pair-feeding from gestational day (GD) 2 until GD 63 (term, approximately GD 68) at which time they were euthanised, 1 h after their final treatment. Adrenocorticotrophic hormone (ACTH) and cortisol concentrations were determined in foetal plasma. Basal and electrically stimulated Glu and gamma-aminobutyric acid (GABA) efflux in the presence or absence of dexamethasone (DEX), a selective glucocorticoid-receptor agonist, were determined ex vivo in foetal hippocampal slices. Glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and N-methyl-D-aspartate (NMDA) receptor NR1 subunit mRNA expression were determined in situ in the hippocampus and dentate gyrus. In the near-term foetus, CPEE increased foetal plasma ACTH and cortisol concentrations. Electrically stimulated glutamate, but not GABA, release was increased in CPEE foetal hippocampal slices. Low DEX concentration (0.3 microM) decreased stimulated glutamate, but not GABA, release in both CPEE and control foetal hippocampal slices. High DEX concentration (3.0 microM) increased basal release of Glu, but not GABA, in CPEE foetal hippocampal slices. GR, but not MR, mRNA expression was elevated in the hippocampus and dentate gyrus, whereas NR1 mRNA expression was increased in the CA1 and CA3 fields of the foetal hippocampus. These data demonstrate that CPEE increases high glucocorticoid concentration-induced Glu release in the foetal hippocampus, presumably as a consequence of increased GR expression. These effects of CPEE, coupled with increased glutamate release and increased NMDA receptor expression, may predispose the near-term foetal hippocampus to GR and Glu-NMDA receptor-mediated neurodevelopmental toxicity.

Diethanolamine alters neurogenesis and induces apoptosis in fetal mouse hippocampus

Diethanolamine (DEA) is present in many consumer products such as shampoo. Dermal administration of DEA diminishes hepatic stores of the essential nutrient choline, and we previously reported that dietary choline deficiency during pregnancy reduces neurogenesis and increases apoptosis in the hippocampus of fetal rats and mice. Therefore, DEA could also alter brain development. Timed-pregnant C57BL/6 mice were dosed dermally from gestation day 7 through 17 with DEA at 0, 20, 80, 160, 320, and 640 mg/kg body/day. At doses of DEA > 80 mg/kg body/day, we observed decreased litter size. In fetuses (embryonic day 17) collected from dams treated dermally with 80 mg/kg body/day DEA, we observed decreased neural progenitor cell mitosis at the ventricular surface of the ventricular zone of the hippocampus [to 56+/-14% (se) histone 3 (H3) phosphorylation as compared to controls; P < 0.01]. We also observed increased apoptosis in fetal hippocampus (to 170+/-10% of control measured using TUNEL and to 178+/-7% of control measured using activated caspase 3; P < 0.01). Thus, maternal exposure to DEA reduces the number of neural progenitor cells in hippocampus by two mechanisms, and this could permanently alter memory function in offspring of mothers exposed to this common ingredient of shampoos and soaps.

Analysis of human hippocampus gangliosides by fully-automated chip-based nanoelectrospray tandem mass spectrometry

Modern microfluidic devices are currently introduced in electrospray (ESI) mass spectrometry (MS), tending to substitute the classical capillary-based ESI infusion. Automated systems using the combination of robotized sample handling and chip-based ESI are significantly increasing the analysis reproducibility, precision, throughput, and efficiency. In the last couple of years our group developed the chip-based ESI–MS approach for glycomics in biomedical research and applied it for oligosaccharide, glycopeptide and ganglioside investigation. Here we report upon the optimization and application of this modern technique for the analysis of differential ganglioside expression patterns in human fetal and adult hippocampus. By this methodology, ganglioside species exhibiting high degree of heterogeneity in the ceramide motifs and biologically-relevant modifications could be identified in human hippocampus. The ultra-high reproducibility of the experiments uniquely provided by the chip-ESI approach allowed for a reliable MS-based ganglioside comparative assay. Moreover, the particular feature of chip ESI-tandem MS to provide structural information at high sensitivity was useful for detailed characterization of hippocampus-associated species. The experimental data presented in this study indicate the benefits of microfluidic/MS for determination of the topospecific brain ganglioside composition and development-related changes in their expression, which might be of high value in clinical investigation and for studies related to ganglioside-based therapy of central nervous system diseases.

Subcellular Targeting of RGS9-2 Is Controlled by Multiple Molecular Determinants on Its Membrane Anchor, R7BP

RGS9-2, a member of the R7 regulators of G protein signaling (RGS) protein family of neuronal RGS, is a critical regulator of G protein signaling. In striatal neurons, RGS9-2 is tightly associated with a novel palmitoylated protein, R7BP (R7 family binding protein). Here we report that R7BP acts to target the localization of RGS9-2 to the plasma membrane. Examination of the subcellular distribution in native striatal neurons revealed that both R7BP and RGS9-2 are almost entirely associated with the neuronal membranes. In addition to the plasma membrane, a large portion of RGS9-2 was found in the neuronal specializations, the postsynaptic densities, where it forms complexes with R7BP and its constitutive partner Gbeta5. Using site-directed mutagenesis we found that the molecular determinants that specify the subcellular targeting of RGS9-2.Gbeta5.R7BP complex are contained within the 21 C-terminal amino acids of R7BP. This function of the C terminus was found to require the synergistic contributions of its two distinct elements, a polybasic motif and palmitoylated cysteines, which when combined are sufficient for directing the intracellular localization of the constituent protein. In differentiated neurons, the C-terminal targeting motif of R7BP was found to be essential for mediating its postsynaptic localization. In addition to the plasma membrane targeting elements, we identified two functional nuclear localization sequences that can mediate the import of R7BP into the nucleus upon depalmitoylation. These findings provide a mechanism for the subcellular targeting of RGS9-2 in neurons.