Effect of marginal vitamin A deficiency during pregnancy on retinoic acid receptors and N-methyl-D-aspartate receptor expression in the offspring of rats

This study examined whether pregnancy-related marginal vitamin A deficiency (MVAD) influences postnatal development of retinoic acid receptors (RARs) and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1) in hippocampus of rat pups. Sixteen female rats were randomized equally into control and MVAD groups. Dams and pups were fed with either a normal control diet or one deficient in vitamin A. Eight female pups in each group were killed at 1 day, 2 weeks, 4 weeks and 8 weeks after birth, respectively. Serum retinol levels were monitored. The messenger RNA (mRNA) and protein expressions and subcellular localization of RARα, RARβ and NR1 in postnatal hippocampus were detected. At 1 day, 2 weeks and 8 weeks after birth, serum retinol levels in the MVAD group were significantly lower than those in the control group. Results of Morris water maze test at 7 weeks of age showed that spatial learning and memory in the MVAD group were affected. Vitamin A deficiency resulted in decreased mRNA levels of RARα, RARβ and NR1 (P<.05). The protein level of RARα and NR1 in the MVAD group was lower than that of the control group (P<.05). There was no significant difference in RARβ between the groups (P>.05). A mass of RARα and NR1 colocalized in hippocampal cell cytoplasm on postnatal day 1. Our data suggested that vitamin A deficiency in pregnancy may affect the postnatal expression of RARα and NR1, affecting learning and memory function in the hippocampus and synaptic plasticity of the calcium signaling pathway.     

Retinoic acid restores adult hippocampal neurogenesis and reverses spatial memory deficit in vitamin A deprived rats

A dysfunction of retinoid hippocampal signaling pathway has been involved in the appearance of affective and cognitive disorders. However, the underlying neurobiological mechanisms remain unknown. Hippocampal granule neurons are generated throughout life and are involved in emotion and memory. Here, we investigated the effects of vitamin A deficiency (VAD) on neurogenesis and memory and the ability of retinoic acid (RA) treatment to prevent VAD-induced impairments. Adult retinoid-deficient rats were generated by a vitamin A-free diet from weaning in order to allow a normal development. The effects of VAD and/or RA administration were examined on hippocampal neurogenesis, retinoid target genes such as neurotrophin receptors and spatial reference memory measured in the water maze. Long-term VAD decreased neurogenesis and led to memory deficits. More importantly, these effects were reversed by 4 weeks of RA treatment. These beneficial effects may be in part related to an up-regulation of retinoid-mediated molecular events, such as the expression of the neurotrophin receptor TrkA. We have demonstrated for the first time that the effect of vitamin A deficient diet on the level of hippoccampal neurogenesis is reversible and that RA treatment is important for the maintenance of the hippocampal plasticity and function.     

A Mid-Life Vitamin A Supplementation Prevents Age-Related Spatial Memory Deficits and Hippocampal Neurogenesis Alterations through CRABP-I

Age-related memory decline including spatial reference memory is considered to begin at middle-age and coincides with reduced adult hippocampal neurogenesis. Moreover, a dysfunction of vitamin A hippocampal signalling pathway has been involved in the appearance of age-related memory deficits but also in adult hippocampal neurogenesis alterations. The present study aims at testing the hypothesis that a mid-life vitamin A supplementation would be a successful strategy to prevent age-related memory deficits. Thus, middle-aged Wistar rats were submitted to a vitamin A enriched diet and were tested 4 months later in a spatial memory task. In order to better understand the potential mechanisms mediating the effects of vitamin A supplementation on hippocampal functions, we studied different aspects of hippocampal adult neurogenesis and evaluated hippocampal CRABP-I expression, known to modulate differentiation processes. Here, we show that vitamin A supplementation from middle-age enhances spatial memory and improves the dendritic arborisation of newborn immature neurons probably resulting in a better survival and neuronal differentiation in aged rats. Moreover, our results suggest that hippocampal CRABP-I expression which controls the intracellular availability of retinoic acid (RA), may be an important regulator of neuronal differentiation processes in the aged hippocampus. Thus, vitamin A supplementation from middle-age could be a good strategy to maintain hippocampal plasticity and functions.     

Cloning of cDNAs encoding retinoic acid receptors RAR gamma 1, RAR gamma 2, and a new splicing variant, RAR gamma 3, from Aambystoma mexicanum and characterization of their expression during early development

To analyze retinoic acid (RA) receptor (RAR) expression during early development in the urodele embryo, we have isolated cDNAs for four members of the axolotl (Ambystoma mexicanum) RAR family, namely RAR alpha (NR1B1), aRAR gamma 1 (NR1B3a), aRAR gamma 2 (NR1B3b), and a new splicing variant of aRAR gamma 2, aRAR gamma 3 (NR1B3c), which contains an insertion of five hydrophobic amino acids in the C-terminal region of the DNA binding domain. The temporal expression pattern of the RAR gamma isoforms was established by RT-PCR using total RNA from embryos of different stages. The expression of aRAR gamma 2 coincides with neurulation and is enhanced in the extremities of the embryo's anteroposterior axis. The aRAR gamma 3 is specifically expressed during gastrulation and early neurulation, whereas aRAR gamma 1 is expressed later during organogenesis. Global aRAR gamma 2 mRNA levels, as well as their spatio-temporal expression pattern in the neurula, were not affected by treatment with RA. These results show that several RARs are expressed in the axolotl embryo during early development, and reveal the existence of a new RAR gamma variant.     

Regulation of N-methyl-D-aspartate receptor subunit expression in the fetal guinea pig brain

N-methyl-d-aspartate receptors (NMDARs) are critical for neuronal maturation and synaptic formation as well as for the onset of long-term potentiation, a process critical to learning and memory in postnatal life. In the current study, we demonstrated that NMDAR subunits undergo spatial, temporal, and sex-specific regulation. During development, we observed increasing NR1 and NR2A expression at the same time as levels of NR2B subunits decreased in the hippocampus and cortex in the fetal guinea pig. We have also shown that glucocorticoids can modulate fetal NMDAR subunit expression in a sex-specific fashion. This is clinically important because synthetic glucocorticoids are administered to pregnant women at risk of preterm labor. Repeated exposure to exogenous glucocorticoids caused a dose-dependent decrease in NR1 mRNA levels and increased NR2A mRNA expression in the female hippocampus at Gestational Day 62. There are significant changes in NMDAR subunit expression in late gestation. It is possible that these alter NMDA-dependent signaling at this time. Prenatal exposure to exogenous glucocorticoids modifies the trajectory of NMDAR subunit expression in females but not in males.     

Prenatal stress in rat causes long-term spatial memory deficit and hippocampus MRI abnormality: differential effects of postweaning enriched environment

Prenatal stress (PS) can cause long-term hippocampus alternations in structure and plasticity in adult offspring. Enriched environment (EE) has an effect in rescuing a variety of neurological disorders. Pregnant dams were left undisturbed (prenatal control, PC) or restrained 6h per day from days 14 to 21 (prenatal stress, PS). Control and prenatal stressed offspring rats were subjected to a standard rearing environment (SE) or an EE on postnatal days 22-120 (PC/SE PC/EE, PS/SE, and PS/EE; n=5, each group). At ～4 months of age, all rats underwent Morris water maze test and brain MRI examination. Hippocampi were then dissected for biochemical analyses, including, Western blot for NMDA receptor (NR) subunits and synaptophysin and RT-PCR forβ1 integrin and tissue-plasminogen activator (t-PA). MRI showed all 5 rats in the PS/SE group and 5 in the PS/EE group exhibited increased signals in bilateral hippocampus and increased T2 time in the PS/SE group. Exposure to EE treatment on postnatal days 22-120 counteracted the deficit in spatial memory and increased NR1 protein expression, but it did not affect the rate of high signals and increased T2 time, decreased NR2, synaptophysin, β1 integrin and t-PA mRNA expressions in PS adult offspring. The results of this study indicate PS in rats causes long-term spatial memory deficits and gross hippocampus pathology. Postnatal EE treatment has differential benefits in terms of spatial learning, signaling molecules, and gross hippocampus pathology.     

Vitamin A and Feeding Statuses Modulate the Insulin-Regulated Gene Expression in Zucker Lean and Fatty Primary Rat Hepatocytes

Unattended hepatic insulin resistance predisposes individuals to dyslipidemia, type 2 diabetes and many other metabolic complications. The mechanism of hepatic insulin resistance at the gene expression level remains unrevealed. To examine the effects of vitamin A (VA), total energy intake and feeding conditions on the insulin-regulated gene expression in primary hepatocytes of Zucker lean (ZL) and fatty (ZF) rats, we analyze the expression levels of hepatic model genes in response to the treatments of insulin and retinoic acid (RA). We report that the insulin- and RA-regulated glucokinase, sterol regulatory element-binding protein-1c and cytosolic form of phosphoenolpyruvate carboxykinase expressions are impaired in hepatocytes of ZF rats fed chow or a VA sufficient (VAS) diet ad libitum. The impairments are partially corrected when ZF rats are fed a VA deficient (VAD) diet ad libitum or pair-fed a VAS diet to the intake of their VAD counterparts in non-fasting conditions. Interestingly in the pair-fed ZL and ZF rats, transient overeating on the last day of pair-feeding regimen changes the expression levels of some VA catabolic genes, and impairs the insulin- and RA-regulated gene expression in hepatocytes. These results demonstrate that VA and feeding statuses modulate the hepatic insulin sensitivity at the gene expression level.     

Rapid and Transient Learning-Associated Increase in NMDA NR1 Subunit in the Rat Hippocampus

Several lines of evidence indicate that glutamate NMDA receptors are critically involved in long-term potentiation (LTP) and in certain forms of learning. It was previously demonstrated that memory formation of an inhibitory avoidance task in chick is specifically associated with an increase in the density of NMDA receptor in selected brain regions. Here we report on the effect of a one trial inhibitory avoidance training in rats, a hippocampal-dependent learning task, on the levels of different subunits of the glutamate NMDA receptor in synaptic plasma membranes (SPM) isolated from the hippocampus. Training rats on a one trial inhibitory avoidance task results in a rapid, transient and selective increase (+33 %, p < 0.05) in NMDA NR1 subunit expression in hippocampal SPM of rats sacrificed 30 min posttraining. No changes were observed at 0 or 120 min after training or in shocked animals in comparison to naive control rats. In addition, no training-associated increase in the levels of NMDA NR2A and NR2B or AMPA GluR 2/3 subunits was observed at any timepoint tested. In conclusion, the present findings support the hypothesis that alterations in expression of synaptic NMDA NR1 subunits in the hippocampus are specifically associated with memory formation of an inhibitory avoidance task and strongly suggest that hippocampal NMDA receptors are crucially involved in the neural mechanisms underlying certain forms of learning.These authors contributed equally to this work     

microRNA-124 attenuates isoflurane-induced neurological deficits in neonatal rats via binding to EGR1

Isoflurane anesthesia induces neuroapoptosis in the development of the brain. In this study, neonatal rats and hippocampal neurons were subjected to isoflurane exposure, in which the effect of miR-124 on the neurological deficits induced by isoflurane was evaluated. Isoflurane anesthesia models were induced in neonatal SD rats aged 7 days and then treated with miR-124 agomir, miR-124 antagomir, or LV-CMV-early growth response 1 (EGR1) plasmids. Then, the spatial learning and memory ability of rats were evaluated by Morris water maze. Furthermore, primary hippocampal neurons cultured 7 days were also exposed to isoflurane and transfected with miR-124 agomir, miR-124 antagomir, or LV-CMV-EGR1 plasmids. The targeting relationship of miR-124 and EGR1 was verified by the dual-luciferase reporter gene assay. To identify the effect of miR-124 on neuron activities, the viability and apoptosis of hippocampal neurons were assessed. In response to isoflurane exposure, miR-124 expression was reduced and EGR1 expression was increased in the hippocampal tissues and neurons. The isoflurane anesthesia damaged rats' spatial learning and memory ability, and reduced viability, and promoted apoptosis of hippocampal neurons. EGR1 was targeted and negatively regulated by miR-124. The treatment of miR-124 agomir improved rats' spatial learning and memory ability and notably increased hippocampal neuron viability and resistance to apoptosis, corresponding to an increased brain-derived neurotrophic factor (BDNF) expression, inhibited expression of proapoptotic factors (cleaved-Caspase-3 and Bax), and enhanced the expression of antiapoptotic factor (Bcl-2). Upregulated miR-124 inhibited the expression of EGR1, by which mechanism miR-124 reduced the neurological deficits induced by isoflurane in neonatal rats through inhibiting apoptosis of hippocampal neurons.     

Memory-improving effect of formulation-MSS by activation of hippocampal MAPK/ERK signaling pathway in rats

MSS, a comprising mixture of maesil (Prunus mume Sieb. et Zucc) concentrate, disodium succinate and Span80 (3.6:4.6 :1 ratio) showed a significant improvement of memory when daily administered (460 mg/kg day, p.o.) into the normal rats for 3 weeks. During the spatial learning of 4 days in Morris water maze test, both working memory and short-term working memory index were significantly increased when compared to untreated controls. We investigated a molecular signal transduction mechanism of MSS on the behaviors of spatial learning and memory. MSS treatment increased hippocampal mRNA levels of NR2B and TrkB without changes of NR1, NR2A, ERK1, ERK2 and CREB. However, the protein levels of pERK/ERK and pCREB/CREB were all significantly increased to 1.5+/-0.17 times. These results suggest that the improving effect of spatial memory for MSS is linked to MAPK/ERK signaling pathway that ends up in the phosphorylation of CREB through TrkB and/or NR2B of NMDA receptor.     

Effects of isoflurane exposure during pregnancy on postnatal memory and learning in offspring rats

Emerging evidence has demonstrated that exposure to anesthetics early in life caused neurohistopathologic changes and persistent behavioral impairments. In this study, a maternal fetal rat model was developed to study the effects of isoflurane exposure during pregnancy on postnatal memory and learning in the offspring. Pregnant rats at gestational day 14 were either exposed to 1.3% isoflurane in a humidified 100% oxygen carrier gas or simply humidified 100% oxygen without any inhalational anesthetic for 2 h every day before delivery. Four weeks later, spatial learning and memory of the offspring were examined using the Morris Water Maze. The expression levels of GAP-43 and NPY in the hippocampal CA1 region of the pups were determined by immunohistochemistry and RT-PCR. Simultaneously, the ultrastructure changes in synapse of the hippocampus were also observed by transmission electron microscopy (TEM). Isoflurane exposure during pregnancy impaired postnatal spatial memory and learning in the offspring as shown by the longer escape latency and the fewer original platform crossings in the Morris Water Maze test. The number and optical densities of GAP-43 and NPY positive cells, as well as the levels of GAP-43 and NPY mRNA, decreased significantly in the hippocampus of isoflurane-exposed pups. Furthermore, TEM studies showed remarkable changes in synaptic ultrastructure of hippocampus. These results indicate that isoflurane exposure during pregnancy could cause postnatal spatial memory and learning impairments in offspring rats, which may be partially explained by the down-regulation of GAP-43 and NPY in the hippocampal area.     

Impaired long-term memory and long-term potentiation in N-type Ca2+ channel-deficient mice

Voltage-dependent N-type Ca(2+) channels, along with the P/Q-type, have a crucial role in controlling the release of neurotransmitters or neuromodulators at presynaptic terminals. However, their role in hippocampus-dependent learning and memory has never been examined. Here, we investigated hippocampus-dependent learning and memory and synaptic plasticity at hippocampal CA3-CA1 synapses in mice deficient for the alpha(1B) subunit of N-type Ca(2+) channels. The mutant mice exhibited impaired learning and memory in the Morris water maze and the social transmission of food preference tasks. In particular, long-term memory was impaired in the mutant mice. Interestingly, among activity-dependent long-lasting synaptic changes, theta burst- or 200-Hz-stimulation-induced long-term potentiation (LTP) was decreased in the mutant, compared with the wild-type mice. This type of LTP is known to require brain-derived neurotrophic factor (BDNF). It was found that both BDNF-induced potentiation of field excitatory postsynaptic potentials and facilitation of the frequency of miniature excitatory postsynaptic currents (mEPSCs) were reduced in the mutant. Taken together, these results demonstrate that N-type Ca(2+) channels are required for hippocampus-dependent learning and memory, and certain forms of LTP.     

Maternal zinc deficiency reduces NMDA receptor expression in neonatal rat brain, which persists into early adulthood

Prenatal and early postnatal zinc deficiency impairs learning and memory and these deficits persist into adulthood. A key modulator in this process may be the NMDA receptor; however, effects of zinc deficiency on the regulation of NMDA receptor activity are not well understood. Female Sprague-Dawley rats were fed diets containing 7 (zinc deficient, ZD), 10 (marginally zinc deficient, MZD) or 25 (control) mg Zn/g diet preconception through postnatal day (PN) 20, at which time pups were weaned onto their maternal or control diet. Regulation of NMDA receptor expression was examined at PN2, PN11, and PN65. At PN2, expression of whole brain NMDA receptor subunits NR1, NR2A, and NR2B was lower in pups from dams fed ZD and MZD compared to controls, as analyzed using relative RT-PCR and immunoblotting. At PN11, whole brain and hippocampi NR1, NR2A, NR2B and PSA-NCAM (polysialic acid-neural cell adhesion molecule) expression and the number of PSA-NCAM immunoreactive cells were lower in pups from dams fed ZD compared to controls. Whole brain brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) concentrations were lower in pups from dams fed ZD or both low zinc diets, respectively. Whole brain NR1 expression remained lower in previously zinc-deficient rats at PN65. These data indicate potential mechanisms through which developmental zinc deficiency can impair learning and memory later in life.     

Prenatal exposure to interleukin-6 results in inflammatory neurodegeneration in hippocampus with NMDA/GABA(A) dysregulation and impaired spatial learning

During pregnancy, infection or immune responses induce cytokine release, which might influence fetal neurodevelopment, leading to neurodegenerative disease in adulthood. Because the hippocampus is a key area for learning and memory, we evaluated 4- and 24-wk-old rats for the effects of early and late prenatal exposure to interleukin-6 (IL-6) on hippocampal morphology, expression of mRNA for IL-6, the gamma-aminobutyric acid receptor (GABA(Aalpha5)), the NR1 subunit of the N-methyl-D-aspartate receptor, and glial fibrillary acidic protein (GFAP), caspase-3 protein and mRNA levels, and learning abilities. Late exposure increased serum IL-6 and hippocampal expression of IL-6 mRNA at 4 and 24 wk. All adult rats showed neuronal loss in the hilus and astrogliosis; males had losses mainly in the CA2 and CA3 regions, and females in CA1. Expression of GABA(Aalpha5), NR1, and GFAP mRNA increased in late-exposed males and females at 4 and 24 wk. mRNA and protein levels of the apoptosis marker caspase-3 were increased in all late-exposed rats except males at 4 wk. Evaluation of hippocampus-dependent working memory in the Morris water maze at 20 wk of age showed increases in escape latency and time spent near the pool wall in all IL-6 adult rats, especially females. These findings suggest that fetal IL-6 exposure, especially in late pregnancy, leads to increased IL-6 levels in the circulation and hippocampus, abnormalities of hippocampal structural and morphology, and decreased learning during adulthood.     

NMDA receptor ablation on parvalbumin-positive interneurons impairs hippocampal synchrony, spatial representations, and working memory

Activity of parvalbumin-positive hippocampal interneurons is critical for network synchronization but the receptors involved therein have remained largely unknown. Here we report network and behavioral deficits in mice with selective ablation of NMDA receptors in parvalbumin-positive interneurons (NR1(PVCre-/-)). Recordings of local field potentials and unitary neuronal activity in the hippocampal CA1 area revealed altered theta oscillations (5-10 Hz) in freely behaving NR1(PVCre-/-) mice. Moreover, in contrast to controls, in NR1(PVCre-/-) mice the remaining theta rhythm was abolished by the administration of atropine. Gamma oscillations (35-85 Hz) were increased and less modulated by the concurrent theta rhythm in the mutant. Positional firing of pyramidal cells in NR1(PVCre-/-) mice was less spatially and temporally precise. Finally, NR1(PVCre-/-) mice exhibited impaired spatial working as well as spatial short- and long-term recognition memory but showed no deficits in open field exploratory activity and spatial reference learning.