Prenatal choline supplementation in rats increases the expression of IGF2 and its receptor IGF2R and enhances IGF2-induced acetylcholine release in hippocampus and frontal cortex

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

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Prenatal choline supplementation in rats increases the expression of IGF2 and its receptor IGF2R and enhances IGF2-induced acetylcholine release in hippocampus and frontal cortex Isabella Napoli, Jan Krzysztof Blusztajn, Tiffany J. Mellott⁎ Department of Pathology and Laboratory Medicine, Boston University School of Medicine, 715 Albany Street, Room L810B, Boston, MA 02118, USA

A R T I C LE I N FO

AB S T R A C T

Article history:

Choline is an essential nutrient whose availability during the second half of gestation

Accepted 19 August 2008

produces long-lasting cognitive effects. Rats that obtain supplemental choline during

Available online 28 August 2008

embryonic day (E) 11–17 have enhanced depolarization-evoked acetylcholine (ACh) release from hippocampal slices, whereas choline deficiency during this time reduces this release.

Keywords:

Previously we reported that rats whose mothers consumed a choline-supplemented diet

Acetylcholine

during E11–17 have higher levels of insulin-like growth factor II (IGF2) mRNA and protein in

Choline

the frontal cortex compared to control and prenatally choline-deficient animals. Since IGF2

Nutrition

has been shown to stimulate endogenous ACh release, we measured the release of ACh from

Diet

hippocampal and frontal cortical slices from rats on postnatal day (P) 18, P24, P34 and P80 in

Insulin-like growth factor II

response to a depolarizing concentration of potassium (45 mM or 25 mM) or to IGF2 treatment

Brain

in the absence or presence of a depolarizing concentration of potassium (25 mM). On P18,

Hippocampus

IGF2/depolarization-evoked ACh release from hippocampal slices was enhanced by prenatal

Frontal cortex

choline supplementation. In the frontal cortex on P80, prenatal choline supplementation

Prenatal

dramatically potentiated ACh release induced by depolarization, IGF2 or the combination of

Rat

the two. On P18 and P90 and in both brain regions, IGF2 mRNA and protein levels, as well as

Septum

protein levels of the IGF2 receptor (IGF2R), were higher in prenatally choline-supplemented

Cholinergic

rats. Choline supplementation also increased IGF2R mRNA levels in the septum. In

IGF2R

summary, prenatal choline supplementation produced alterations in IGF2 signaling, via increased levels of IGF2 and IGF2R, which may enhance cholinergic neurotransmission and confer neuroprotection against insult. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Normal development and function of the brain requires the supply of fundamental nutrients, such as choline, during the period of embryonic and fetal growth. Choline is an essential

nutrient that is required for several cellular functions, including growth and maintenance of structural integrity of phospholipid membranes, and it is necessary to establish a pool of acetylcholine (ACh), the neurotransmitter of cholinergic neurons. Maternal dietary choline availability during

⁎ Corresponding author. Fax: +1 617 638 5400. E-mail address: [email protected] (T.J. Mellott). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.08.046

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pregnancy plays a critical role in the organization of the brain and influences the cognitive functions. When rat maternal diets were supplemented with choline during the second half of gestation (embryonic day (E) 11–17), the offspring had improved spatial and temporal memory as well as improved attention compared to control rats at both a young age and in adulthood (Meck et al., 1989; Meck et al., 1988; Meck and Williams, 1997a,b,c; Meck and Williams, 1999; Meck and Williams, 1997a,b,c; Meck and Williams, 1997a,b,c). In contrast, rats whose mothers consumed a diet that was deficient in choline during the E11–17 period had deficits in certain memory tasks (Meck and Williams, 1997a,b,c). The neurochemical mechanisms by which choline supplementation in utero leads to the improvement in memory are not known. However, it has been shown that prenatal choline manipulation modulates ACh synthesis and release in basal forebrain cholinergic neuron (Cermak et al., 1998), which are known to participate in memory processes (Fibiger, 1991). Hippocampi of prenatally choline-supplemented rats had enhanced depolarization-evoked ACh release, whereas ACh synthesis from choline transported by high-affinity choline transporter (CHT) was reduced. In contrast, prenatally choline-deficient rats were characterized as having increased synthesis of ACh following choline uptake by CHT but reduced ACh content relative to the control and prenatally choline-supplemented rats. Prenatally choline-deficient animals were also unable to sustain depolarization-evoked ACh release relative to the choline-supplemented animals (Cermak et al., 1998). Recently, we found that prenatally choline-deficient rats have a higher amount of CHT mRNA in the septum and CHT protein in the hippocampus (Mellott et al., 2007a,b). The augmentation of CHT levels supports the observed increase in ACh synthesis from choline transported by CHT in hippocampal slices from prenatally choline-deficient rats. This pattern of changes suggests that the hippocampus of the prenatally cholinedeficient animals is characterized by fast ACh recycling and efficient choline reutilization for ACh synthesis, presumably to maintain adequate ACh release despite the decrease of the ACh pool, whereas ACh turnover and choline recycling is slower while the evoked release of ACh is high in the prenatally choline-supplemented animals. Recently, we analyzed the effects of prenatal choline availability on gene expression by oligonucleotide microarrays. Insulin-like growth factor 2 (IGF2) was among the genes that were identified as being one whose expression was modified by prenatal choline availability. Its mRNA and protein expression in the frontal cortex was significantly increased by prenatal choline supplementation (Mellott et al., 2007a,b). The insulin-like growth factors (IGFs), their receptors, and binding proteins constitute a family of cellular modulators that play essential roles in the regulation of growth, differentiation and survival, as well as metabolic processes. This family is comprised of three structurally-related peptides: insulin, IGF1, and IGF2. IGF1 stimulates the proliferation of neuron progenitors, induces the differentiation of oligodendrocytes, and increases the survival of neurons and oligodendrocytes in vitro (Barres et al., 1992; D'Mello et al., 1993; Drago et al., 1991; McMorris and Dubois-Dalcq, 1988; Mozell and McMorris, 1991; Pons and Torres-Aleman, 1992; Torres-Aleman et al., 1990; Werther et al., 1993). Although less is known

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about the role of IGF2 in the brain, in vitro studies have shown that IGF2 stimulates proliferation of neuronal and glial cells (Konishi et al., 1994; Lim et al., 1985), promotes survival of several neuronal cell types (Haselbacher et al., 1989; Knusel and Hefti, 1991), regulates the development and turnover of neuromuscular synapses (Ishii, 1989), and potentiates ACh release from hippocampal slices (Kar et al., 1997). IGF2 expression is also reportedly upregulated in response to a penetrating brain injury (Walter et al., 1999). The brains of IGF2 knockout mice (Igf2−/−) did not display any significant differences in morphological structures or in myelination levels compared to control mice (Igf2+/+), suggesting that IGF2 may not be critical for brain development under normal conditions; however, IGF1, IGF2 and insulin receptor binding sites, as well as their response to neurotoxic insult, were altered by the loss of IGF2 (Dikkes et al., 2007). IGF1 and IGF2 are selectively localized in distinct cell types and specific regions of the brain (Kar et al., 1993; Lesniak et al., 1988; Werther et al., 1990) and their physiological responses are presumed to be mediated by specific interactions with cell surface receptors. IGFs bind the tyrosine kinase IGF type 1 receptor (IGF1R), as well as the IGF type 2/mannose-6phosphate receptor (IGF2R or M6PR) that binds IGF2 with higher affinity than IGF1 (Kar et al., 1997). IGF2R is widely distributed in various tissues including the brain. It is a multifunctional single pass transmembrane glycoprotein that mediates the trafficking of lysosomal enzymes and also participates in the degradation of non-glycosylated IGF2 (Hawkes and Kar, 2004). Recently, it was shown that the IGF2R is involved in the regulation of ACh release in response to stimulation by IGF2 in the rat hippocampus and the response is mediated through the activation of protein kinase Cα (Hawkes et al., 2006). Since IGF2 and IGF2R are mediators of endogenous acetylcholine release (Hawkes et al., 2006) and prenatally choline-supplemented rats have increase expression of IGF2 (Mellott et al., 2007a,b), this study was designed to examine the effects of prenatal choline availability on the expression of IGF2 and its receptors, as well as to determine if IGF2-evoked acetylcholine release from frontal cortical and hippocampal slices of these rats is altered. We found that prenatal choline intake altered the mRNA and protein expression of IGF2 and IGF2R in young and adult rats. Moreover, prenatal choline availability affected IGF2-evoked ACh release in hippocampal and frontal cortical slices. The results of this study suggest that the IGF system may help to mediate the differences in cholinergic transmission observed in animals that were exposed to various levels of choline in utero.

2.

Results

2.1.

Hippocampus

Previously, we have shown that prenatal choline availability modulates IGF2 gene expression in the rat frontal cortex (Mellott et al., 2007a,b). Here, we examined the effects of prenatal choline availability on IGF2 and IGF2R mRNA, as well as protein, expression in the hippocampus during early postnatal development (P18) and adulthood (P90). On P18,

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Fig. 1 – IGF2 and IGF2R levels in the hippocampus. RNA from hippocampi of rats was used for RT-PCR of IGF2 (A) and IGF2R (C). IGF2 and IGF2R levels were normalized using β-actin levels and are presented as means ± SEM of eight rats per group. Using a one-way ANOVA, there was a significant effect of diet (p < 0.05) on IGF2 mRNA expression on P18 but not P90. By Tukey's test, the levels of IGF2 mRNA on P18 were significantly higher in prenatally choline-supplemented rats compared to control and choline-deficient rats (p < 0.05 and p < 0.01, respectively). One-way ANOVA revealed a significant effect of diet (p < 0.05) on IGF2R mRNA levels, such that choline-supplemented rats had higher levels of IGF2R mRNA than both control and choline-deficient rats on P18 (p < 0.05 for each) by Tukey's test. Lysates from hippocampi of P18 rats were used for Western blot analysis of IGF2 (B) and IGF2R (D). IGF2 and IGF2R levels are presented as means ± SEM of four rats per group. There were significant differences in IGF2 protein levels between the groups on both P18 and P90 (p < 0.05 and 0.00005, respectively) as determined by one-way ANOVA. Post-hoc analysis revealed that the IGF2 levels in prenatally choline-supplemented rats were significantly higher than control rats on P90 (p < 0.0005) and choline-deficient rats on P18 and P90 (p < 0.05 and p < 0.0005, respectively). IGF2R protein levels were also significantly different between the groups of rats on P18 and P90 (p < 0.05 and p < 0.05, respectively) by one-way ANOVA. On both days, prenatally choline-deficient rats had a lower level of IGF2R than choline-supplemented rats (p < 0.05) as determined by Tukey's test.

the levels of IGF2 mRNA were 2.3-fold and 3.8-fold higher in prenatally choline-supplemented rats than in control and prenatally choline-deficient rats, respectively (Fig. 1A). IGF2 protein levels on P18 were 2.5-fold higher in choline-supplemented than in choline-deficient animals (Fig. 1B). On P90, no statistically significant differences between the three experimental groups were seen in IGF2 mRNA levels (Fig. 1A), although prenatally choline-supplemented animals had a 4fold higher level of IGF2 protein than the other two groups of animals at this age (Fig. 1B). IGF2R mRNA expression was higher in prenatally choline-supplemented animals compared

to control animals on P18, and no significant differences in mRNA levels were observed on P90 (Fig. 1C). On P18 and P90, IGF2R protein levels were 2-fold and 1.8-fold higher, respectively, in prenatally choline-supplemented rats compared to prenatally choline-deficient rats (Fig. 1D). Initial studies measuring ACh levels in the hippocampus were performed to verify and expand upon the results of previous studies that showed higher levels of depolarizationevoked ACh release from the hippocampal slices of prenatally choline-supplemented rats compared to slices from control and prenatally choline-deficient rats (Cermak et al., 1998).

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Hippocampal slices were first incubated in a physiological salt solution (NaPSS), followed by incubation in a solution containing a depolarizing concentration of KCl (45 mM) (KPSS). The amount of ACh released into the medium was measured by HPLC. In NaPSS, hippocampal slices released a small but

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measurable amount of ACh; and there were no differences in basal ACh release between the groups on both P18 and P80 (data not shown). The release of ACh was increased by as much as 2fold over basal release during incubation in KPSS on P18, as well as P80 (Fig. 2A). On P18, potassium-evoked ACh release was

Fig. 2 – Depolarization- and IGF2-evoked ACh release in hippocampal slices. Basal and stimulated ACh release was measured, as described in the experimental procedures, using slices from P18, P24, P34 and P80 hippocampi. Data are reported as means ± SEM of four to six rats per group. (A) A two-way ANOVA for the effects of depolarization and diet on ACh release on P18 revealed a significant effect of depolarization (p < 0.01) and diet (p < 0.05). Tukey's test revealed that prenatal choline supplementation increased depolarization-evoked ACh release compared to control and prenatal choline deficiency (p < 0.05). On P80, a two-way ANOVA for depolarization and diet on ACh release revealed a significant effect of depolarization (p < 0.001), but no statistical significance of diet. (B) A three-way ANOVA for diet, depolarization and IGF2 revealed a significant effect of diet (p < 0.01), depolarization (p < 0.05), and IGF2 (p < 0.001). Prenatal choline supplementation significantly increased ACh release as compared to the control and prenatally choline-deficient group (p < 0.01 and p < 0.005, respectively) by Tukey's test. There was also a significant effect of prenatal choline intake on ACh release co-stimulated by IGF2 and depolarization, such that higher levels of ACh were released from hippocampal slices from prenatally choline-supplemented rats than slices from control or choline-deficient rats (p < 0.05 and p < 0.005, respectively). (C) A three-way ANOVA for the effects of choline, age, and depolarization displayed a significant effect of choline (p < 0.05) and depolarization (p < 0.01) and an interaction between choline and depolarization (p < 0.05). A significant effect of depolarization on ACh release as compared to basal release was only found in the hippocampal slices from prenatally choline-supplemented rats (p < 0.001). Using a Tukey's test, depolarization-evoked ACh release was determined to be significantly higher in choline-supplemented animals compared to choline-deficient animals (p < 0.005). (D) A three-way ANOVA for the effects of choline, age, and IGF2 treatment revealed a significant effect of IGF2 (p < 0.001). (E) A three-way ANOVA for the effects of choline, age, and IGF2/K+ treatment displayed a significant effect of choline (p < 0.005), age (p < 0.005), and IGF2/K+ treatment (p < 0.001) and interaction terms between choline with IGF2/K+ treatment (p < 0.005) and age with IGF2/K+ treatment (p < 0.001). Post-hoc analysis revealed that ACh release in response to IGF2 and depolarization was significantly increased in prenatally choline-supplemented and control rats (p < 0.0005, and p < 0.05, respectively), whereas IGF2-stimulation/ depolarization did not produce significant changes in ACh release compared to basal levels in prenatally choline-deficient rats. IGF2/depolarization-stimulated release of ACh was significantly higher from hippocampal slices from choline-supplemented rats compared to ACh levels produced by slices from control and choline-deficient rats (p < 0.05 and p< 0.001, respectively). Na+, sodium-containing solution (basal release); K+, high potassium-containing solution (25 mM KCl).

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higher in slices from prenatally choline-supplemented rats compared to control and prenatally choline-deficient rats; however, a significant effect of choline was not observed on P80 (Fig. 2A). In addition, we measured ACh release in response to a lower concentration of potassium (25 mM KCl), which would be used in combination with IGF2 treatment of slices. Depolarization-induced ACh release with 25 mM KCl has been shown to be optimal for revealing modulatory increases in release levels by IGF2 and drug-dependent release attenuation (Kar et al., 1997; Kar et al., 1996; Pearce et al., 1991). ACh release in response to 25 mM KCl was lower as compared to the level of ACh released as a result of stimulation by the higher concentration of potassium (Figs. 2A, C). To examine the influence of prenatal choline availability on IGF2-evoked ACh release, hippocampal slices from rats whose mothers were fed either a choline-supplemented, control, or choline-deficient diet were stimulated with IGF2. To determine the dose- and time-dependent effects of IGF2 on ACh release in hippocampus, as well as the frontal cortex, slices were treated with various concentrations of IGF2 (0.05 nM–10 nM) for times ranging from 0 to 15 min. As reported by Kar et al. (1997), IGF2 stimulated ACh release from the slices in the presence of 25 mM KCl in a concentration- and time-dependent manner (data not shown). As the concentration of IGF2 increased, ACh release also increased until peaking at either 0.1 nM for hippocampal slices and 0.5 nM for cortical slices. The effect of IGF2 on ACh release diminished at higher IGF2 concentrations, consistent with the results of Kar et al. (1997). Treatment of slices with the optimal concentrations of IGF2 for different time periods (0– 15 min) produced a proportional increase in ACh release, with the highest release at 15 min (data not shown). On P18, ACh release from hippocampal slices was significantly increased by incubation with a depolarizing concentration of potassium (25 mM), stimulation with IGF2, and the combination of the two (Fig. 2B). IGF2 alone was sufficient to increase the release of ACh from hippocampal slices (1.5- to 2-fold), although there were no significant effects of the prenatal diet on the IGF2-induced ACh release. Treatment with IGF2 also increased the release of ACh in response to depolarization, and this co-stimulation was modulated by prenatal choline status, such that hippocampal slices from prenatally choline-supplemented rats released higher amounts of ACh compared to slices from prenatally cholinedeficient rats (Fig. 2B). Figs. 2C–E show the developmental changes in ACh release in the absence and presence of both IGF2- and potassium-stimulation in the rat hippocampus. Through early postnatal development, prenatal choline supplementation increased the depolarization-evoked ACh release compared to prenatal choline deficiency (Fig. 2C). Although there were no significant differences in IGF2-stimulated ACh release between the groups, the choline-supplemented group tended to release more ACh in response to IGF2 (Fig. 2D). Prenatal diet had a significant effect on ACh release evoked by IGF2 with depolarization, such that the hippocampal slices from cholinesupplemented rats released a higher amount of ACh than the slices from control and choline-deficient animals (Fig. 2E).

2.2.

Frontal cortex

In order to determine if prenatal choline availability also modulates the expression of IGF2 and its receptor in the

frontal cortex, RT-PCR and Western blotting analyses were again performed. On P18, the levels of IGF2 mRNA were not statistically different between the three groups; however, prenatally choline-deficient animals had the least amount of IGF2 mRNA expression compared to both the control and prenatally choline-supplemented group on P90 (Fig. 3A). Choline supplementation tended to increase the levels of IGF2 mRNA expression, however this upregulation of IGF2 expression was more dramatic at the protein level where prenatally choline-supplemented rats had significantly higher levels of IGF2 protein compared to both control and prenatally choline-deficient rats on P18 and P90 (Fig. 3B). The qualitative relationships between the levels of IGF2 mRNA and protein on P90 reported here are consistent with the results previously observed for animals on P34 (Mellott et al., 2007a,b). Fig. 3C shows IGF2R mRNA levels on P18 and P90. On P18, prenatally choline-deficient rats had a slightly reduced amount of IGF2R mRNA compared to the control group. Prenatal choline supplementation tended to increase IGF2R mRNA levels on P90, but this effect was not statistically significant (Fig. 3C). Western blotting analysis of protein lysates obtained from the three experimental groups for IGF2R showed that protein levels of IGF2R were higher in prenatally choline-supplemented rats compared to prenatally choline-deficient rats on P18 and P90 and to control rats on P18 only (Fig. 3D). In order to examine depolarization-evoked ACh release from the rat frontal cortex, the cortical slices were treated as previously described for the hippocampus. Basal levels of ACh released from slices were similar among all ages and groups of animals measured (data not shown). Incubation in a depolarizing medium containing 45 mM KCl induced ACh release from cortical slices on all days examined. On both P18 and P80, the release of ACh was higher in prenatally choline-supplemented rats as compared to control and prenatally cholinedeficient rats (Fig. 4A). Following incubation in a medium containing 45 mM KCl, ACh release increased with age and the difference between prenatal treatment groups also increased such that prenatal choline supplementation produced the highest release of ACh on P80 (Fig. 4A). Specifically, depolarization evoked a 2-fold increase in ACh release from the cortical slices from prenatally choline-supplemented animals on P18, which was augmented to a 5.5-fold induction on P80. The control and prenatally choline-deficient animals also responded to a higher degree to depolarization on P80, but not to the extent of the prenatally choline-supplemented animals. The effects of IGF2 on ACh release in frontal cortical slices were measured on various days as previously described. The most robust differences in ACh release between the groups of rats were observed at P80 (Figs. 4B–E). In the presence of a depolarizing medium containing 25 mM KCl, there was a 4-fold induction of ACh release from slices from prenatally cholinesupplemented animals, which was significantly higher compared to slices from the control and prenatally choline-deficient animals on P80 (Fig. 4B). IGF2 stimulation alone produced a significant 2-fold increase in the release of ACh from slices from prenatally choline-supplemented rats, and the presence of IGF2 with depolarization increased ACh release in prenatally choline-supplemented rats but had little or no effect on slices from the control and prenatally choline-deficient rats. On P80, IGF2/ depolarization-evoked ACh release in prenatally choline-

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Fig. 3 – IGF2 and IGF2R in the frontal cortex. RNA from the frontal cortex of each rat was used for RT-PCR of IGF2 (A) and IGF2R (C). IGF2 and IGF2R levels were normalized using β-actin levels and are presented as means ± SEM of four (P90) or eight (P18) rats per group. Using a one-way ANOVA, there was a significant effect of prenatal choline availability on IGF2 mRNA expression on P90 (p < 0.01) but not P18. The levels of IGF2 mRNA were significantly higher in prenatally choline-supplemented rats compared to prenatally choline-deficient rats on P90 (p < 0.01) as determined by Tukey's test. Control rats also had significantly higher levels of IGF2 mRNA than prenatally choline-deficient rats on P90 (p < 0.05). There was also a significant effect of choline availability on IGF2R mRNA expression on P90 only (p < 0.05) by one-way AVOVA. Cortical lysates of P18 and P90 rats were used for Western blot analysis of IGF2 (B) and IGF2R (D). IGF2 and IGF2R levels are presented as means ± SEM of four rats per group. A one-away ANOVA revealed significant differences in IGF2 protein levels between the groups on both P18 and P90 (p < 0.05 and 0.005, respectively). Specifically, the IGF2 levels in prenatally choline-supplemented rats were significantly higher than control rats on P18 and P90 (p < 0.05 and 0.005, respectively) and choline-deficient rats on P18 and P90 (p < 0.05 and p < 0.001, respectively) as determined by Tukey's test. IGF2R protein levels were also significantly different between the groups of rats on P18 and P90 (p < 0.0005 and p < 0.005, respectively) by one-way ANOVA. Using post-hoc analysis, prenatally choline-deficient rats had a significantly lower level of IGF2R than choline-supplemented animals on both P18 and P90 (p < 0.001 and p < 0.0005, respectively) and control animals on P90 only (p < 0.05). On P18, prenatally choline-supplemented rats also had a significantly higher level of IGF2R than control animals (p < 0.0005).

supplemented animals was approximately 5-fold higher than basal release and was considerably enhanced compared to levels of release from control (1.6-fold over basal) and prenatally choline-deficient (1.3-fold over basal) animals. Figs. 4C–E show the developmental pattern of ACh release induced by a depolarizing concentration of potassium (25 mM KCl), the presence of IGF2 or both in the rat frontal cortex. Data obtained on P18, P24, P34 and P80 illustrate that the ACh release was modulated by age and prenatal nutrition. Again, cortical slices

obtained from adult rats (P80) had the highest level of ACh release. Depolarization-evoked release of ACh was significantly increased in prenatally choline-supplemented animals compared to control and choline-deficient animals (Fig. 4C). Choline supplementation increased the IGF2-evoked ACh release from cortical slices throughout early postnatal development (Fig. 4D). Cortical slices from control rats did not show a significant response to IGF2 treatment alone until P80. Co-stimulation of frontal cortical slices with IGF2 and depolarization over early

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postnatal development produced the largest effects on ACh release in slices from prenatally choline-supplemented rats and at P80 (Fig. 4E).

2.3.

Septum

In a recent study, cholinergic neurons in the basal forebrain which project to hippocampus and cortex were shown to express IGF2R (Hawkes et al., 2006), and cultured septal neurons have been shown to respond to IGF2 treatment by increasing cholinergic activity (Konishi et al., 1994); therefore, we examined the mRNA expression of the receptor, as well as IGF2 itself, in the septum of prenatally choline-supplemented, control and deficient animals. On P18, IGF2 mRNA levels were

1.6 to 1.9-fold higher in prenatally choline-supplemented rats compared to control and prenatally choline-deficient animals (Fig. 5). Interestingly, IGF2R mRNA levels were 2- to 4-fold higher in prenatally choline-supplemented rats compared to prenatally choline-deficient and control rats, respectively, on both P18 and P90.

3.

Discussion

Prenatal exposure to supplemental choline improves spatial and temporal memory as well as attention in young animals (Meck et al., 1989; Meck et al., 1988; Meck and Williams, 1997a,b, c; Meck and Williams, 1999; Meck and Williams, 1997a,b,c; Meck

Fig. 4 – Depolarization- and IGF2-evoked ACh release in frontal cortical slices. Basal and stimulated ACh release was measured, as described in the experimental procedures, using slices from P18, P24, P34 and P80 frontal cortices. Data are reported as means ± SEM of three to four rats per group. (A) A two-way ANOVA for the effect of depolarization and diet on ACh release on P18 revealed a significant effect of depolarization (p < 0.001) and diet (p < 0.005), as well as a significant interaction between the two (p < 0.005). Using post-hoc analysis, depolarization produced significant increases in the amount of ACh released compared to basal in all three groups (supplemented: p < 0.001; control: p < 0.05; deficient: p < 0.05). Prenatal choline supplementation increased depolarization-evoked ACh release compared to control and prenatal choline deficiency (p < 0.0001 for both). On P80, a two-way ANOVA for depolarization and diet on ACh release again revealed a significant effect of depolarization (p < 0.001) and diet (p < 0.005), as well as a significant interaction between the two (p < 0.005). Depolarization produced significant increases in the amount of ACh released compared to basal in all three groups (supplemented: p < 0.001; control: p < 0.01; deficient: p < 0.05) as determined by Tukey's test. Prenatal choline supplementation increased depolarization-evoked ACh release compared to control and prenatal choline deficiency (p < 0.05 and p < 0.0001, respectively). (B) A three-way ANOVA for diet, depolarization and IGF2 revealed a significant effect of diet (p < 0.001), depolarization (p < 0.001), and IGF2 (p < 0.005). There were also significant interaction terms between choline and depolarization (p < 0.001) and choline and IGF2 (p < 0.005). Tukey's test revealed that prenatal choline supplementation significantly increased ACh release as compared to the control and prenatally choline-deficient group (p < 0.001 and p < 0.001, respectively). There was only a significant effect of depolarization alone, IGF2 stimulation, and co-stimulation by IGF2 and depolarization on ACh release in prenatally choline-supplemented rats (p < 0.001, p < 0.01, and p < 0.0005, respectively). Depolarization with 25 mM KCl lead to a higher level of ACh release in prenatally choline-supplemented rats than in control and choline-deficient rats (p < 0.005 and p < 0.05, respectively). Slices from prenatally choline-supplemented rats released more ACh in response to IGF2 alone compared to slices from choline-deficient rats (p < 0.001). There was significant effect of prenatal choline intake on ACh release co-stimulated by IGF2 and depolarization such that higher levels of ACh was released from slices from prenatally choline-supplemented rats than slices from control or choline-deficient rats (p < 0.05 and p < 0.01, respectively). (C) A three-way ANOVA for the effects of choline, age, and depolarization displayed a significant effect of choline (p < 0.001), age (p < 0.001), and depolarization (p < 0.001) and an interaction terms between choline with depolarization (p < 0.001) and choline with age (p < 0.001), age with depolarization (p < 0.001), and choline with age and depolarization (p < 0.001). Using Tukey's test, a significant effect of depolarization on ACh release as compared to basal release was found in the slices from prenatally choline-supplemented and -deficient rats (p < 0.001 and p < 0.05, respectively), but not in slices from control rats. Depolarization-evoked ACh release was significantly higher in choline-supplemented rats compared to control and choline-deficient rats (p < 0.0005 for both). (D) A three-way ANOVA for the effects of choline, age, and IGF2 treatment revealed a significant effect of choline (p < 0.05), age (p < 0.05), and IGF2 treatment (p < 0.001) and interaction terms between choline with IGF2 treatment (p < 0.05) and age with IGF2 treatment (p < 0.05). Post-hoc analysis revealed that ACh release was significantly evoked by IGF2 treatment in cortical slices from prenatally choline-supplemented rats (p < 0.005), whereas IGF2-stimulation did not produce significant changes in ACh release compared to basal levels in control or prenatally choline-deficient rats. Prenatally choline-supplemented rats had a significantly higher level of IGF2-evoked ACh release compared to levels measured in choline-deficient rats (p < 0.05). (E) A three-way ANOVA for the effects of choline, age, and IGF2/K+ treatment displayed a significant effect of choline (p < 0.001), age (p < 0.001), and IGF2/K+ treatment (p < 0.001) and an interaction terms between choline with IGF2/K+ treatment (p < 0.001), choline with age (p < 0.001), age with IGF2/K+ treatment (p < 0.001), and choline with age and IGF2/K+ treatment (p < 0.001). As determined by Tukey's test, ACh release was significantly stimulated by IGF2/K+ treatment in cortical slices from prenatally choline-supplemented rats (p < 0.0005), whereas IGF2-stimulation/depolarization did not produce significant changes in ACh release compared to basal levels in control or prenatally choline-deficient rats. IGF2/depolarization-stimulated release of ACh was significantly higher from hippocampal slices from choline-supplemented rats compared to ACh levels produced by slices from control and choline-deficient rats (p < 0.0005 and p < 0.0005, respectively). Na+, sodium-containing solution (basal release); K+, high potassium-containing solution (25 mM KCl).

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and Williams, 1997a,b,c). Moreover, prenatal and early postnatal choline supplementation has been shown to protect against age-induced impairments in performance on hippocampallydependent tasks (McCann et al., 2006; Meck et al., 1989; Meck et al., 1988; Meck and Williams, 2003; Meck et al., 2008). In the current study, we explored the possibility that some of these actions of prenatal choline supplementation may be related to changes in the cholinergic system because ACh released from the long axon basal forebrain cholinergic neurons that project to the hippocampus and cerebral cortex regulate multiple processes including attention, learning and memory (Sarter and Parikh, 2005). We found that ACh release was indeed significantly higher in hippocampal and cortical slices obtained from prenatally choline-supplemented rats as compared to controls and prenatally choline-deficient animals. These findings extend our previous observations in similar studies performed on hippocampal slices of juvenile rats (Cermak et al., 1998). Here we also show a dramatic enhancing effect of prenatal choline supplementation on ACh release in the cortex. Moreover, the latter is seen in adult animals (P80) suggesting that it is longlasting and likely to mediate some of the behavioral effects of prenatal choline exposure. Another remarkable effect of modulation of prenatal availability of choline is the altered developmental pattern of expression of multiple genes observed with the use of microarrays in the hippocampus and cerebral cortex (Mellott et al., 2007a,b). One of the genes upregulated by prenatal choline supplementation in brain is IGF2. Interestingly, IGF2 promotes the differentiation of basal forebrain cholinergic neurons in culture (Konishi et al., 1994) and, as noted above, increases ACh release from the hippocampus and frontal

131

cortex (Kar et al., 1997). We previously reported that cortical expression of IGF2 mRNA was increased by more than 2-fold on P34 in the prenatally choline-supplemented rats as compared to the two other dietary groups. The differences in IGF2 mRNA expression among the groups of rats were also observed on P25 at the protein level, specifically 2.5-fold higher in prenatally choline-supplemented rats than in control rats and 9-fold higher than in prenatally choline-deficient rats. We now confirm and extend these findings to adult rats. Moreover, we report a striking upregulation of IGF2 protein expression in the hippocampus by prenatal choline supplementation (4-fold over the control and prenatally choline-deficient groups), indicating that high prenatal choline intake increases IGF2 levels in adult rats in both brain regions. In this study we also explored the possible relationships between the IGF2 system (i.e. IGF2 and IGF2R) and ACh release. Consistent with previous studies by Kar et al. (1997), we found that IGF2, in combination with depolarization, evoked ACh release in the hippocampus. Moreover, we observed that, in cortical and hippocampal slices from young rats, IGF2 alone evoked ACh release. The latter action of IGF2 was seen most dramatically in cortical slices obtained from prenatally choline-supplemented rats and roughly correlated with the enhanced expression of IGF2R protein seen in these animals. In the hippocampus, the largest difference in IGF2R protein expression between the groups of animals, observed at age P18, corresponds with the most significant differences observed in IGF2-stimulated ACh release. This is particularly interesting since IGF2R has been reported to mediate the release of endogenous ACh following stimulation with IGF2 (Hawkes et al., 2006) It was also shown that the cholinergic neurons in the basal forebrain (septum

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Fig. 5 – IGF2 and IGF2R levels in the septum. Septal RNAs of P18 and P90 rats were used for RT-PCR of IGF2 (A) and IGF2R (B). IGF2 and IGF2R levels were normalized using β-actin levels and are presented as means ± SEM of four rats per group. Using a one-way ANOVA, there was a significant effect of choline availability on IGF2 expression on P18 (p < 0.005). IGF2 mRNA levels were significantly higher in prenatally choline-supplemented rats compared to control and prenatally choline-deficient rats on P18 (p < 0.05 and p < 0.005, respectively) by Tukey's test. One-way ANOVA revealed a significant effect of prenatal choline availability on IGF2R mRNA expression on P18 and P90 (p < 0.01 and p < 0.005, respectively). Using post-hoc analysis, the levels of IGF2R mRNA were significantly higher in prenatally choline-supplemented rats compared to control (P18: p < 0.01 and P90: p < 0.005) and prenatally choline-deficient rats (P18: p < 0.05 and P90: p < 0.05).

and the nucleus basalis) that were identified by their expression of vesicular ACh transporter, the protein responsible for packaging ACh into synaptic vesicles, co-express IGF2R (Hawkes et al., 2006). We found that the septum of prenatally choline-supplemented animals contained higher amounts of IGF2R mRNA than the septum from the other groups of rats. The inconsistencies between the mRNA and protein levels of IGF2R in both the hippocampus and frontal cortex may be due to the alterations in the levels of IGF2R protein in the processes

projecting from neurons that reside in the basal forebrain and express different levels of IGF2R mRNA. Thus, it is possible that the cholinergic neurons of the basal forebrain of prenatally choline-supplemented animals express higher levels of IGF2R, which is transported down the axon to their terminals in the cortex and hippocampus and renders these terminals more responsive to stimulation by IGF2 and increases the amount of ACh released. IGF2R is also responsible for sequestering IGF2 thus preventing it from interacting with other receptors (mainly IGF1R) and targeting IGF2 for degradation (Hawkes et al., 2007). In addition to being expressed in the basal forebrain cholinergic neurons, IGF2R is found in many classes of neurons in brain (Hawkes et al., 2006). Thus, one would anticipate that regulation of IGF2R expression by prenatal availability of choline would result in modulation of IGF2 signaling in multiple neurons and may include both potentiation of signaling, as reported here for the prenatally choline-supplemented animals, and, possibly downregulation of IGF2 action in neurons whose IGF2R mediates predominantly IGF2 clearance. Typically, IGF2 is viewed as neurotrophic or neuroprotective factor due to its ability to stimulate proliferation of neuronal and glial cells and to increase survival of various neuronal cell types (Haselbacher et al., 1989; Knusel and Hefti, 1991; Konishi et al., 1994; Lim et al., 1985). Increased levels of IGF2 in prenatally choline-supplemented animals may not only enhance cholinergic neurotransmission in these animals but also protect animals against the detrimental effects of aging and brain injury or insult. Prenatal and early postnatal choline supplementation has been shown to protect against age-induced impairments in performance on hippocampallydependent tasks (McCann et al., 2006; Meck et al., 1989; Meck et al., 1988; Meck and Williams, 2003). In addition, perinatal choline supplementation has been shown to reduce the severity of behavioral deficits in animals prenatally exposed to alcohol (Thomas et al., 2007; Thomas et al., 2004; Thomas et al., 2000) and to improve motor defects in a mouse model of Rett syndrome (Nag and Berger-Sweeney, 2007). Dietary choline supplementation has also been shown to protect rats from seizure-induced spatial memory retention deficits (Holmes et al., 2002; Yang et al., 2000). Recently, we determined that, in prenatally choline-supplemented rats, the damaging effects of the neuroexcitotoxic agent kainic acid (KA) on the hippocampus were reduced, possibly due to enhanced pre-seizure levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and IGF1 (WongGoodrich et al., 2008). However, perhaps in contrast to our findings, Dikkes et al. (Dikkes et al., 2007) reported that Igf2−/− mice were more resistant to the effects of KA compared to Igf2+/+ mice, suggesting that IGF2 expression in the adult brain increases an animal's susceptibility to epilepsy and neurodegeneration. Dikkes et al. (2007) also suggested that the absence of IGF2 during development may alter the circuitry of the brain (i.e. an increase in inhibition) that reduces the KA-induced insult to the hippocampus. Our data showing elevated amounts of other trophic and survival factors such as BDNF, NGF, and IGF1 (Glenn et al., 2007; Sandstrom et al., 2002; Williams et al., 1998; Wong-Goodrich et al., 2008) in prenatally choline-supplemented animals point to the possibility that these factors may counteract any negative effects of increased

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levels of IGF2. Moreover, as noted above the high levels of IGF2R in the prenatally choline-supplemented rats provide a potential mechanism whereby IGF2 may be sequestered thus maintaining a balance of IGF2 signaling. Among its biochemical functions, choline acts as a donor of methyl groups that can be utilized for DNA and histone methylation – the salient processes in epigenetic modulation of gene expression (Jaenisch and Bird, 2003) – and we and others have previously provided evidence consistent with the notion that the molecular mechanisms of the effects of prenatal choline intake on development may include altered global- and gene-specific (including Igf2, Dnmt1) DNA methylation in brain and liver (Kovacheva et al., 2007; Niculescu et al., 2006). Importantly the expression of both IGF2 and IGF2R is regulated by the methylation of DNA within the regulatory regions of these genes as well as by methylation of histones and it will be important to test the hypothesis that the observed changes in the expression of these genes seen in postnatal brain governed by prenatal choline availability are related to these epigenetic mechanisms.

4.

Experimental procedures

4.1.

Animal subjects

neostigmine, 10 mM HEPES, pH 7.4) and incubated for 15 min at 37 °C. In addition, slices were treated for 15 min in NaPSS in the absence or presence of IGF2, followed by a 15 min incubation in 25 mM KPSS (containing 125 mM NaCl, 25 mM KCl, 1 mM CaCl2, 0.75 mM MgCl2, 10 mM glucose, 15 μM neostigmine, 10 mM HEPES, pH 7.4) in the absence or presence of IGF2 (0.1 nM for hippocampal slices; 0.5 nM, for cortical slices). After the incubations, the media were collected, centrifuged to remove debris, vacuum dried, and reconstituted in 300 μl of water. Acetylcholine and choline were quantified by high-performance liquid chromatography (HPLC) using an Acetylcholine/Choline analytical column and immobilized enzyme reactor from BAS (West Lafayette, IN). The conditions of the HPLC were as follows: 1.5 ml min-1 flow rate, mobile phase (22 mM phosphate buffer, pH 8.5) containing 0.005% (v/ v) ProClin reagent (BAS).

4.3.

4.2.

Acetylcholine release

On postnatal days 18, 24, 34, and 80, four to six offspring (one per dam) per experimental group were anesthetized and the brains were dissected. Frontal cortices (without the olfactory bulb) and hippocampi were removed rapidly on ice. Hippocampi and frontal cortices were cut into 400 μm transverse slices with a McIlwain tissue chopper. The slices were then incubated at 37 °C for 1 h in a physiological salt solution (NaPSS) (in mM: 145 NaCl, 5 KCl, 1 CaCl2, 0.75 MgCl2, 10 glucose, 15 μM neostigmine, 10 HEPES, pH 7.4), while changing the medium every 15 min. The slices (2–3 slices per well) were transferred to cell strainers and incubated at 37 °C for a 15 min period in NaPSS. The strainers were then transferred into depolarizing solution (KPSS) (containing 105 mM NaCl, 45 mM KCl, 1 mM CaCl2, 0.75 mM MgCl2, 10 mM glucose, 15 μM

Protein assay

Protein content was determined using the bicinchoninic acid assay with bovine serum albumin as standard according to Smith et al. (1985).

4.4.

Pregnant Sprague–Dawley CD strain rats were obtained from Charles River Laboratories and were housed in individual cages with a 12 h light/dark cycle. The rats were divided into three groups (4–6 animals per group): Choline-supplemented, control, and choline-deficient. During days 11–17 of pregnancy, the rats were fed a modified AIN 76A diet that was either choline-deficient (0 mmol kg-1 choline), a control (7.9 mmol kg-1 choline), or a choline-supplemented diet (35.6 mmol kg-1 choline) (Dyets Inc.). Following embryonic day 17, all rats were returned to a control diet. Offspring also consumed a control diet once weaned at a postnatal age of 22 days. No significant differences in the amounts of diet or water consumed by the different groups were observed (data not shown). Litter sizes ranged from 9–14 and no significant differences in the number of offspring per group were observed. For all experiments equal numbers of both male and female offspring were used.

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Reverse transcriptase PCR

On postnatal days 18 and 90, four to six offspring (one pre dam) per experimental group were anesthetized and the brains were dissected. The frontal cortex (without the olfactory bulb) and hippocampus were removed rapidly on ice. The brain tissues were immediately homogenized in cold guanidine isothiocyanate solution, frozen on dry ice, and stored at −80 °C. Total RNA was extracted from tissues by phenol and chloroform method (Chomczynski and Sacchi, 1987; Sambrook et al., 1989) and precipitated. RNA was resuspended and its quantity was determined using Quant-iT™ RiboGreen® RNA assay kit (Molecular Probes) and the Victor3 multi-label plate reader (PerkinElmer Life Sciences). Hippocampal and frontal cortex RNA were used for reverse transcriptase (RT)PCR using Superscript One-Step RT-PCR with Platinum Taq (Invitrogen). First strand cDNA synthesis was performed with either 10 ng (used for β-actin and IGF2R) or 40 ng (used for IGF2) of total RNA, oligo dT primers and reverse transcriptase at 48 °C (45 min). Primers used for PCR include β-actin (Forward: CAC AGC TGA GAG GGA AAT C, Reverse: TCA GCA ATG CCT GGG TAC), IGF2 (Forward: GAA ACT ATG GGT AGG AAG TGG TCC, Reverse: CCC GTT ACA TAG GGA ATG GGA), IGF2R (Forward: AAG GTG TTG CTT GAA TCG GC, Reverse: CCC TTA CAA AAC GCA AAG CG). PCR was performed using Platinum Taq DNA polymerase with a denaturing step for 2 min at 94 °C, followed by 32–40 cycles (40 cycles for IGF2, 32 cycles for IGF2R, 32 cycles for β-actin) of 1 min at 94 °C, 1 min at 55 °C and 2 min at 72 °C, and terminated by an elongation step at 72 °C for 7 min. PCR products were separated on a 10% TBE polyacrylamide gel and stained with ethidium bromide. PCR products were then visualized with the Kodak Image Station 440 (Rochester, NY). As expected, the PCR products from the amplification of IGF2 were present at 251 bp, of IGF2R at 406 bp, and of β-actin at 327 bp. Product intensities were quantified using Kodak software. PCR product levels were normalized with β-actin values.

134 4.5.

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Western blot analysis

Hippocampi and frontal cortices were dissected as described before, frozen on dry ice, and stored at −80 °C. For Western blot analysis, whole tissue extracts were prepared by adding lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.02% (m/v) sodium azide, 1% (v/v) Nonidet NP-40, 0.1% (m/v) SDS, 10% (v/v) glycerol, 25 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM AEBSF, 1 μg ml-1 leupeptin, 2 μg ml-1 pepstatin) to a frozen sample, gently sonicating, incubating for 15 min on ice, and briefly centrifuging to clear. The extracts were normalized for total protein and subjected to SDS-PAGE. After transfer of protein to an Immoblin P membrane (Millipore), the membrane was blocked with 5% nonfat dry milk in 1× Tris-buffered saline (TBS) containing 0.1% Tween 20 for 1 h and then probed with primary antibody overnight. The primary antibodies used included a monoclonal β-actin antibody (1:5000) (Sigma), a monoclonal IGF2 antibody (1:500) (Upstate), and a monoclonal IGF2R antibody (1:750) (BD Biosciences). The antibody/antigen complexes on the blots were recognized with anti-rabbit (1:5000) or anti-mouse (1:2000) IgG peroxidase conjugate and were visualized using the chemiluminescence method (SuperSignal West Femto Maximum Sensitivity Substrate) on the Kodak Image Station 440. IGF2 appeared as a 12 kDa band; IGF2R, a 273 kDa band; and β-actin, a 42 kDa band. Band intensities were quantified using Kodak software. Protein levels were normalized with β-actin values. For immunoprecipitation, tissue lysates were prepared as described above and incubated overnight with polyclonal antiIGF2 antibody (Santa Cruz Biotechnology Inc.) (2 μg per 500 μg of extract) and protein A Sepharose (3.5 mg per sample) in a rotary mixer at 4 °C. Samples were briefly centrifuged and washed three times. The pellet was resuspended in lysis buffer and proteins were electrophoretically separated by SDS/ PAGE.

4.6.

Statistical analysis

Data were analyzed using an analysis of variance (ANOVA). If significant differences were found, data were further analyzed by Tukey's multiple comparison test. Analyses were performed with the statistical program SYSTAT (SPSS Inc., Chicago, IL).

Acknowledgments We would like to thank Martina Brock, Vesela Kovacheva and Kei Satoh for their help with experiments performed in this project. These studies were supported by a grant from the National Institute on Aging AG009525.

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