ABSTRACT Fetal alcohol spectrum disorder (FASD), the result of fetal alcohol exposure (FAE), affects 2–11% of children worldwide, with no effective treatments. Hippocampus-based learning and

memory deficits are key symptoms of FASD. Our previous studies show hypothyroxinemia and hyperglycemia of the alcohol-consuming pregnant rat, which likely affects fetal neurodevelopment. We

administered vehicle, thyroxine (T4) or metformin to neonatal rats post FAE and rats were tested in the hippocampus-dependent contextual fear-conditioning paradigm in adulthood. Both T4 and

metformin alleviated contextual fear memory deficit induced by FAE, and reversed the hippocampal expression changes in the thyroid hormone-inactivating enzyme, deiodinase-III (_Dio3_) and

imprinting of _Dio3_ and _Igf2_ during development was normalized by both treatments. Administering Dnmt1 inhibitor to control neonates resulted in FAE-like deficits in fear memory and

hippocampal allele-specific expression of _Igf2_, which were reversed by metformin. We propose that neonatal administration of T4 and metformin post FAE affect memory via elevating _Dnmt1_

and consequently normalizing hippocampal _Dio3_ and _Igf2_ expressions in the adult offspring. The present results indicate that T4 and metformin, administered during the neonatal period

that is equivalent to the third trimester of human pregnancy, are potential treatments for FASD and conceivably for other neurodevelopmental disorders with cognitive deficits. SIMILAR

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Despite efforts in awareness and prevention, 1 in 10 pregnant women still reports alcohol consumption.1 As a result, fetal alcohol spectrum disorder (FASD) affects 2–11% of children

worldwide, with increasing prevalence and presumably even more unreported and undiagnosed cases.2 Despite its significance, there are still no validated biological treatments for FASD.3,4

Some preclinical studies suggest that choline administration is beneficial,5,6 while others found it without effect for FASD.7 Current treatments, such as stimulants, antidepressants,

neuroleptics and anti-anxiety drugs, alleviate those FASD symptoms common to many psychiatric disorders but are not specific for FASD.8 Therefore, specific treatments are needed to prevent

or reverse fetal alcohol-induced defects. Hippocampal development is impaired in human FASD,9 consequently some of the most debilitating effects of FASD are on hippocampus-based learning and

memory10 that is mirrored in animal models of fetal alcohol exposure (FAE).11,12 The cause of this cognitive vulnerability is not yet known, but one possible mechanism is via abnormal

thyroid hormone levels during development of the alcohol-exposed fetus.13,14 Excessive alcohol consumption decreases thyroxine (T4) levels,15,16,17 and alcohol use during pregnancy has been

reported with significant changes in thyroid function of neonates.18,19 Preclinical studies demonstrate that maternal alcohol consumption during pregnancy interferes with thyroid hormone

availability or function.11,13,14,20,21,22 Furthermore, clinical or subclinical hypothyroidism of the mother negatively affects neuropsychological development of the child,23,24 and

experimental hypothyroidism in developing rats results in impaired learning.12,21 Sufficient levels of thyroid hormones are essential for normal brain development, and fetus is dependent on

maternal T4 before the adequate functioning of its own thyroid glands.25 Maternal T4 reaching the fetal brain is being deiodinated to the biologically active form of thyroid hormone

(triiodothyronine, T3) in the glia and then transported to the neuron.26 In the neuron, right amount of T3 can regulate the transcription of thyroid hormone-dependent genes. Thus,

ethanol-induced maternal hypothyroxinemia can limit the availability of T3 to the fetal brain and affect the regulation of neurodevelopmental genes. Alternatively, even if necessary amount

of T3 reaches the fetal neurons, it can be excessively metabolized by elevated levels of thyroid hormone-inactivating enzyme, deiodinase-III (_Dio3_) to its inactive form.27 Indeed,

increased hippocampal _Dio3_ expression leads to decreased local T3 levels with subsequent changes in target gene transcription.27 Administration of T4 during gestation normalizes the

increased transcript levels of _Dio3_ in the hippocampus of _in utero_ ethanol-exposed adult offspring.28 Therefore, administering T4 to the ethanol-consuming dams can be effective via

reversing the maternal hypothyroxinemia14 and/or by reducing the expression of _Dio3_ in the fetal and subsequently the adult hippocampus. Both mechanisms can result in alleviation of

FAE-caused hippocampus-based cognitive deficits of the adult offspring, as observed.11,21,28 Abnormal thyroid function is often concomitant with glucose metabolic dysfunction.29 Both the

ethanol-consuming dams and their adult offspring are indeed hyperglycemic without any changes in their insulin levels.30,31 This phenomenon suggests insulin resistance, namely an increase in

release of insulin from the pancreas is required to maintain normal plasma glucose levels. As insulin pathway genes, including insulin-like growth factor 2 (_Igf2_), are associated with

hippocampus-based learning and memory processes,32,33,34,35 peripheral and central insulin resistance may also contribute to FAE-induced learning and memory deficits.36 Decreased hippocampal

levels of _Igf2_ is detrimental to cognition.37 Given that FAE leads to decreased _Igf2_ expression during development,38,39 normalizing _Igf2_ expression could reverse FAE-induced

cognitive deficits.40 Metformin, the most widely used insulin-sensitizing drug, is known to affect _Igf2_ expression,41 provide neuroprotection against ethanol-induced neurodegeneration,42

enhance short-term memory43 and spatial memory formation.44 Thus, metformin is a logical choice to explore as a potential treatment to reverse FAE-induced memory deficits. Both _Dio3_ and

_Igf2_ are imprinted genes, known to be preferentially expressed from the paternal allele in the placenta.45,46 However, both of these genes show a preferential maternal expression in the

adult hippocampus.12,47 The reason for this ‘switch’ from paternal to maternal expression is not known, but imprinting in general is regulated by differential DNA methylation of the maternal

and paternal allele.48 Because DNA methylation is altered by FAE,39,49,50,51 the FAE-engendered changes in allelic expression of _Dio3_ (ref.21) and _Igf2_ (ref.38) likely occur via

modifications of DNA methylation in these imprinted loci.52 Methylation maintenance during development by the DNA methyltransferase 1 (Dnmt1)48 and expression regulation by the

enhancer-blocker CCCTC-binding factor (Ctcf)-binding sites are highly analogous in the _Dio3_ and _Igf2_ imprinted regions.53 If FAE affects DNA methylation and imprinting processes of these

genes similarly, the potential beneficial effects of neonatal T4 or metformin treatment could be via reversing the effects of FAE on these imprinted genes and their common regulators. The

primary objective of this study was to identify the effects of neonatal administration of T4 or metformin, respectively, on adult FAE offspring with the goal of finding potential treatment

for FAE-induced cognitive deficits. The desirable time frame of treatment is the period post FAE, and in a neurodevelopmental stage that is equivalent to the third trimester of human

pregnancy. Successful treatment at this period increases our study’s translational value, as some women reduce alcohol consumption toward the end of pregnancy,54 neurodevelopment can still

be affected, and administration of treatments to the women is more feasible than treating the newborns. The secondary objective was to determine whether neonatal treatment with T4 or

metformin would normalize hippocampal expressions of _Dio3_ and _Igf2_. Therefore, we administered T4 and metformin to neonatal rats during postnatal days 1–10 after the maternal consumption

of alcohol ceased, when the offspring have already been exposed to the detrimental effects of alcohol. Both male and female offspring were investigated, as sex differences in the effects of

FAE have been reported both in human55 and animal studies.56,57 We hypothesized that altered maternal thyroid and glucose functions by alcohol consumption generates an adverse intrauterine

environment for the development of the offspring. We further hypothesized that these adverse effects can be reversed by neonatal T4 and metformin administration within a specific

developmental window. MATERIALS AND METHODS Please see Supplementary Materials and Methods for a more detailed description of the methods. ANIMALS All animal procedures were approved by the

Northwestern University Animal Care and Use Committee. All rats were housed in controlled environment and received water _ad libitum_. For the _ex vivo_ and _in vivo_ treatment studies,

maternal diets and animal procedures were performed as described previously.58 Briefly, adult female Sprague–Dawley (S) rats (Harlan, Indianapolis, IN, USA) were mated with adult Brown

Norway (B) males (Charles River, Wilmington, MA, USA). We chose to study the S by B (SB) offspring because of their vulnerability to FAE.12,27 This cross also allows studying allele-specific

expression of imprinted genes. Pregnant females received control (C, _ad libitum_ standard lab chow) or pair-fed (PF) and ethanol (E) liquid diets (Lieber-DeCarli ’82; Bio-Serv, Frenchtown,

NJ, USA) during gestation days (G) 8–21. The E diet contained 5% ethanol (w/v, 35% ethanol-derived calories). PF dams received the same amount of isocaloric liquid diet as the paired E

dams. Regular laboratory chow was provided _ad libitum_ to all pregnant rats on G21 and their offspring. Neonates from each prenatal diet group (C, PF and E) and each litter received T4

(0.05 μg g−1 per day; Sigma, St. Louis, MO, USA)59 or metformin (200 μg g−1 per day; Sigma),60 or distilled water as vehicle by intraperitoneal injection in a volume of 10 μl g−1 for 10

days, postnatal days 1–10. Two different cohorts of animals were used in the T4 and metformin studies with their own matched vehicle littermates. The two sets of vehicle cohorts were

statistically the same and therefore combined throughout the study. The thyroxin dose administered to neonates restored the attenuated thyroid-stimulating hormone levels in the adult FAE

offspring without altering thyroid-stimulating hormone levels of the control animals (Supplementary Figure 1). The dose of metformin has been chosen from published data,60 and the human

equivalent of this dose is 2400 mg per 75 kg per day,61 which is within the recommended dose range. In two additional studies, postnatal day 1 pups of C animals received vehicle,

5-aza-2′-deoxycytidine (5-Aza; 1 μg g−1 per day; Sigma)62 or 5-Aza and metformin (5-Aza+Met; 1 and 200 μg g−1 per day, respectively) by intraperitoneal injection for 10 days. The number of

animals in each experimental condition is provided in the figure legends. PRIMARY HIPPOCAMPAL CULTURE Primary hippocampal cultures were prepared from embryonic day 18 fetuses of dams on C,

PF or E diets (_n_=3 dams per diet) as described previously.58 Hippocampal neurons were plated at a density of 8 × 105 cells per 60 mm dish and cultured for 10 days. Metformin (final

concentration of 0.4 mm in sterile water) was administered every 48 h.63 All cultures were incubated at 37 °C with 5% CO2 and processed for RNA isolation. BEHAVIORAL TESTING

_Context-dependent fear conditioning_ One to two male and female adult offspring per litter were tested as described previously.21 Rats were placed into the automated fear-conditioning

apparatus (TSE, Bad Homburg, Germany) for 3 min followed by three mild shocks (0.8 mA, 1 s each, 60 s between each shock). Twenty-four hours later, the animals were placed into the same

chamber for 3 min without shock exposure, and their freezing behavior was measured. Rats that did not respond to the initial shock were excluded from the study. Two weeks later, adult

offspring were killed between 10:00 and 12:00 h. RNA ISOLATION AND QUANTITATIVE REAL-TIME PCR Whole hippocampi were dissected as described64 and collected directly into RNAlater reagent

(Ambion, Austin, TX, USA). RNA was isolated by using Direct-zol RNA MiniPrep (Zymo Research, Orange, CA, USA) and reverse transcription was performed using SuperScript VILO Master Mix

(Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR was conducted as described previously58 with 5 ng cDNA, specific primer pairs (Supplementary Table 1) and SYBR Green Master Mix

(Applied Biosystems, Foster City, CA, USA) using the ABI 7900HT cycler. PYROSEQUENCING We used the identified single-nucleotide polymorphisms in the _Dio3_ exon12 and in the 3′-untranslated

region of _Igf2_ (ref.47) between S and B strains to track allele-specific expression. PCR was conducted using a forward primer and biotinylated reverse primer flanking the single-nucleotide

polymorphisms. Purification of biotinylated PCR products and pyrosequencing were performed by EpigenDx (Hopkinton, MA, USA) using a sequencing forward primer. STATISTICAL ANALYSIS _Ex vivo_

data were analyzed by two-way analysis of variance (ANOVA; prenatal diet and _ex vivo_ treatment). All _in vivo_ data containing prenatal diet were analyzed by three-way ANOVA (prenatal

diet, neonatal drug treatment and sex). Significant ANOVA results were followed by Bonferroni _post hoc_ tests corrected for multiple comparisons. In the 5-Aza studies, data were analyzed by

two-way ANOVA (neonatal drug treatment and sex). As there were no differences in sex effects, male and female data of the 5-Aza studies were combined and analyzed by unpaired two-tailed

Student’s _t_-test. All statistical analyses were performed using Systat (Chicago, IL, USA) and GraphPad Prism 7 (La Jolla, CA, USA) programs. RESULTS Please see Table 1 for the summary of

the different neonatal treatments’ effects on the measured consequences of FAE. NEONATAL T4 TREATMENT RESTORES FAE-INDUCED HIPPOCAMPAL FEAR MEMORY DEFICIT AND _DIO3_ EXPRESSION FAE resulted

in a fear memory deficit in male and female adults, measured as decreased freezing duration in the contextual fear-conditioning test (F(2,110)=5.75, _P_<0.01, sex × diet, F(2,110)=2.78,

not significant; Figure 1a). Neonatal T4 treatment, administered after FAE, alleviated this deficit without affecting the memory of C and PF offspring (diet × drug, F(2,110)=3.53,

_P_<0.05). Expression of hippocampal _Dio3_ was increased in the hippocampus of male and female E offspring (diet, F(2,57)=10.89, _P_<0.01; Figure 1b). Neonatal T4 administration

reduced _Dio3_ expression at large, even more so in the E offspring to the levels of T4-treated controls (drug, F(1,57)=117.37, _P_<0.01; diet × drug, F(2,57)=4.92, _P_<0.01). Body

weights of all groups of animals at weaning and in adulthood are shown in Supplementary Tables 2 and 3. METFORMIN AFFECTS FAE-INDUCED GENE EXPRESSION CHANGES IN PRIMARY HIPPOCAMPAL CULTURE

As metformin has never been used in FAE studies, its efficacy was evaluated first in our _ex vivo_ primary culture model.53 Metformin is known to affect insulin pathway genes and its effect

on the FAE-induced changes of these genes were examined.58 The _ex vivo_ model consisted of our routine FAE ethanol administration _in vivo_ and subsequent evaluation of gene expression

after 10 days of metformin or vehicle treatment of the fetal hippocampal culture.58 Among the previously validated insulin pathway genes, FAE effects persisted after 10 days in culture for

transcript levels of insulin receptor (_Insr_; F(2,33)=3.74, _P_<0.05), _Igf2_ (F(2,35)=7.07, _P_<0.01) and growth factor receptor bound protein 10 (_Grb10_; F(2,37)=3.58, _P_<0.05)

(Figures 2a–c). Metformin treatment reversed the FAE-induced decrease in the expression of _Insr_ and _Igf2_ (_Insr_: diet × drug, F(2,33)=3.08, _P_=0.05; _Igf2_: drug F(1,35)=16.5,

_P_<0.01; diet × drug F(2,35)=9.33, _P_<0.01) (Figures 2a and b). Metformin also normalized the FAE-induced increase in _Grb10_ levels (drug, F(1,37)=19.76, _P_<0.01; Figure 2c).

EFFECTS OF NEONATAL METFORMIN TREATMENT ON FAE-INDUCED FEAR MEMORY DEFICIT AND HIPPOCAMPAL _IGF2_ EXPRESSION Neonatal metformin treatment tended to alleviate the FAE-induced fear memory

deficit in both male and female adult offspring (diet, F(2,93)=6.37, _P_<0.01; sex × diet, F(2,93)=1.09, not significant; drug, F(1,93)=3.56, _P_=0.06; Figure 3a). Hippocampal transcript

levels of _Insr_ were not different between the prenatal diet groups (data not shown). However, metformin not only reversed the FAE-induced decrease but also enhanced the expression of

_Igf2_ in the adult male hippocampus (diet, F(2,71)=5.04, _P_<0.01; sex, F(1,71)=10.41, _P_<0.01; drug, F(1,71)=22.14, _P_<0.01; diet × drug, F(2,71)=11.32, _P_<0.01; sex × diet

× drug, F(2,71)=6.36, _P_<0.01). In females, the FAE- and PF-induced decrease in _Igf2_ expression were normalized, but not enhanced by metformin (Figure 3b). Hippocampal _Grb10_

transcript levels of E male and female adult offspring were normalized by neonatal metformin (diet, F(2,55)=9.78, _P_<0.01; sex, F(1,55)=2.15, not significant; drug, F(1,55)=16.81,

_P_<0.01; diet × drug, F(2,55)=7.63, _P_<0.01; Figure 3c). Body weights of all groups of animals at weaning and in adulthood are shown in Supplementary Tables 4 and 5. TREATMENT

SPECIFICITY IN HIPPOCAMPAL ALLELE-SPECIFIC AND TOTAL EXPRESSION OF _DIO3_ AND _IGF2_ Determining the parental contribution to the expression of the imprinted _Dio3_ and _Igf2_ genes was

possible due to single-nucleotide polymorphisms previously identified between the S and the B strains.12,47 We were not able to track allele-specific expression for _Grb10_ because there are

no single-nucleotide polymorphisms between the S and B cDNAs. Allele-specific expression of hippocampal _Dio3_ was affected by FAE and neonatal T4 or metformin treatments in a sex- and

treatment-specific manner. Decreased maternal allelic expression of hippocampal _Dio3_ tended to be normalized by T4 in the adult FAE male (diet, F(2,65)=4.07, _P_<0.05; drug,

F(1,65)=3.08, _P_=0.08; diet × sex × drug, F(2,65)=2.63, _P_=0.08; Figure 4a). In contrast, the decreased maternal allelic expression of hippocampal _Dio3_ was not normalized by metformin in

the adult FAE male hippocampus (Figure 4a). There were no effects of FAE, T4 or metformin on _Dio3_ expression in the females (Supplementary Figure 2a). Allelic expression of hippocampal

_Igf2_ also showed sex differences in response to FAE and was affected by both treatments. Maternal contribution to _Igf2_ expression was significantly lower in the adult PF and FAE male

offspring compared to controls (diet, F(2,69)=23.76, _P_<0.01; sex × diet, F(2,69)=49.40, _P_<0.01; Figure 4b). Both T4 and metformin treatment reversed the FAE-induced decrease (T4:

drug, F(1,67)=133.68, _P_<0.01; sex × drug, F(1,67)=4.85, _P_<0.05; metformin: drug, F(1,69)=32.44, _P_<0.01; sex × drug, F(1,69)=30.45, _P_<0.01). However, only the neonatal T4

treatment restored the decrease in maternal allelic _Igf2_ expression in the adult male PF hippocampus (Figure 4b). There were no effects of FAE or treatments on _Igf2_ expression in the

females (Supplementary Figure 2b). To interpret the parental dosage effects (allelic expression) in the light of total expression, a schematic representation of the maternal and the paternal

allelic contributions to the total hippocampal expression of _Dio3_ and _Igf2_ is shown in Figures 4c and d, respectively (drawn not to scale). The effects of T4 and metformin on the

FAE-induced changes in the hippocampal total expression of _Dio3_ and _Igf2_ have been shown on Figures 1b and 3b, respectively. If an overlapping mechanism is responsible for these

treatment effects, T4 and metformin would also alter FAE-induced changes in _Igf2_ and _Dio3_, respectively. Indeed, the FAE-induced increase in the total expression of _Dio3_ was normalized

by metformin in the hippocampus of adult male, while the correction by metformin was not complete in the female (sex, F(1,62)=27.53, _P_<0.01; diet × drug, F(2,62)=4.32, _P_<0.05; sex

× drug, F(1,62)=9.61, _P_<0.01; diet × sex × drug, F(2,62)=3.00, _P_=0.05; E vehicle vs E metformin _P_<0.01; Figure 4e). Decreased transcript levels of _Igf2_ in the E and PF

hippocampus were restored to control levels by neonatal T4 (drug, F(1,63)=9.20, _P_<0.01; diet × drug, F(2,63)=3.42, _P_<0.05) in both sexes equally (Figure 4f). These latter effects

parallel those of T4 on _Dio3_ expression (Figure 1b). DNMT1 AS POTENTIAL MEDIATOR OF FAE EFFECTS Dnmt1 is involved in learning and memory processes,65 known to be affected by FAE66 and it

is the primary regulator of DNA methylation maintenance relevant to allele-specific imprinting processes.48 Transcript levels of _Dnmt1_ were decreased in the adult hippocampus by FAE (diet,

F(2,69)=15.21, _P_<0.01) and normalized by both treatments with no sex effects (T4: drug, F(1,63)=13.22, _P_<0.01; diet × drug, F(2,63)=3.13, _P_<0.05; metformin: diet × drug,

F(2,69)=9.37, _P_<0.01; Figure 5a). _Ctcf_, one of the main regulator of allele-specific _Igf2_ expression,67 showed increased hippocampal expression after FAE (diet, F(2,52)=20.92,

_P_<0.01). Neonatal T4 did not reverse this effect, but rather it increased _Ctcf_ expression equally in all prenatal treatment groups (T4: drug, F(1,45)=197.39, _P_<0.01; diet × drug,

F(2,45)=31.49, _P_<0.01; Figure 5b). In contrast, neonatal metformin reversed the FAE-induced increase in _Ctcf_ expression (metformin: drug, F(1,52)=4.35, _P_<0.05; diet × drug,

F(2,52)=5.54, _P_<0.01; Figure 5b). As a proof of concept for the role of _Dnmt1_ in FAE-induced memory deficit, 5-Aza, a Dnmt inhibitor that preferentially inhibits Dnmt1 (refs66,68) was

administered to control neonates between postnatal days 1 and 10. As expected, 5-Aza treatment reduced _Dnmt1_ expression in the adult hippocampus (drug, F(1,25)=24.46, _P_<0.01; Figure 

5c). Neonatal 5-Aza treatment mimicked the effects of FAE on fear memory in both male and female adults (drug, F(1,25)=7.19, _P_<0.05; Figure 5d). Total and maternal allelic expression of

hippocampal _Igf2_ were decreased by 5-Aza, the latter with a male-specificity similar to the FAE effects (allelic expression in males, F(1,13)=12.59, _P_<0.01; total expression in males

and females, F(1,24)=72.26, _P_<0.01; Figures 5e and f). No effect of 5-Aza treatment has been seen in the allelic expression of _Igf2_ in the female hippocampus, and in _Dio3_ allelic

expression in either sex (Supplementary Figures 3 and 4). However, total hippocampal expression of both _Dio3_ and _Ctcf_ were increased by the 5-Aza treatment, again confirming the

similarity to FAE effects (_Dio3_, F(1,24)=61.02, _P_<0.01; _Ctcf_, F(1,25)=4.52, _P_<0.05; Figures 5g and h). Metformin administered together with 5-Aza eliminated all of the 5-Aza

effects (Supplementary Figure 5). Body weights at weaning and in adulthood are shown in Supplementary Table 6. DISCUSSION The major finding of the present study reveals the therapeutic

potential of neonatal T4 and metformin treatments, administered after FAE, for reversing hippocampus-dependent cognitive deficits in adult offspring. The mechanism by which these treatments

are effective is via restoring the FAE-induced decrease in hippocampal _Dnmt1_ expression. Indeed, both FAE and direct inhibition of _Dnmt1_ caused a memory deficit, while reversing the

decreased _Dnmt1_ expression by neonatal T4 and metformin treatments normalized fear memory. These treatments also restored FAE-induced changes in hippocampal total expression of _Dio3_ and

_Igf2_, two imprinted genes known to affect hippocampal memory processes. However, only the male hippocampus showed allelic expression changes of _Dio3_ and _Igf2_ post FAE, of which only

_Igf2_ were normalized by both of these treatments. Dnmt1 is the major maintenance DNA methyltransferase that preserves DNA methylation patterns.48 Recently, Dnmt1 has been associated with

the etiology of autism,69 neurodegenerative diseases and other central nervous system disorders.65 Decreased transcript levels of _Dnmt1_ impairs learning and memory processes.70,71

Correspondingly, administering the Dnmt inhibitor 5-Aza impairs the formation of contextual fear memory.72 Our results are in agreement with these findings as both FAE and neonatal 5-Aza

administration decreased hippocampal expression of _Dnmt1_, in parallel with contextual fear memory. Conversely, the FAE-induced deficits in _Dnmt1_ expression and memory were both reversed

by neonatal T4 and metformin treatments. Similarly, choline deficiency and supplementation modulate _Dnmt1_ expression in parallel with learning and memory performance.73,74 The effect of

choline on _Dnmt1_ is relevant to this study, because preclinical studies suggest that choline administration is also beneficial for FAE-induced learning and memory deficits.5,6 _Dio3_ and

_Igf2_ are two imprinted genes with allele-specific methylation patterns conserved by Dnmt1.48 _Dnmt1_ expression is inversely correlated with the expression of _Dio3_ (ref.75) and linearly

with _Igf2._ 76 These patterns are confirmed in this study, where after FAE adult hippocampus showed increased _Dio3_ and decreased _Igf2_ expression concurrently with decreased _Dnmt1_

expression. Treatments with T4 and metformin reversed these effects and also normalized fear memory. Indeed, decreasing hippocampal levels of _Dio3_ and increasing that of _Igf2_ are known

to improve memory.21,28,77 As the FAE-induced deficit in _Dnmt1_ expression is also normalized by the treatments, and _Dnmt1_ is the major regulator of imprinting maintenance, the possible

involvement of other imprinted genes in reversing the memory deficit after FAE still exist. Sex specificity of the FAE effects and of the treatments was also observed in the allelic

expression of _Dio3_ and _Igf2_ in the hippocampus, as neither FAE nor the treatments altered allelic expression of these genes in adult females. This sex-specific effect of FAE on the

allelic expression of two imprinted genes with different imprinting regulators could be caused either by sex differences in the imprinting processes or by peripubertal estrogen-induced

compensatory mechanisms. Other developmental studies have shown sex-specific consequences of _in utero_ treatments in the expression of _Dio3_ (ref.78) and male-specific FAE effects have

also been observed for _Dio3_.12,28 Interestingly, structural sex differences have also been found in human FASD subjects, although the small sample size in that study precluded to identify

sex differences in memory performance.10 Nevertheless, smaller size of anterior hippocampus is found in FASD males, but not females, compared to controls. It appears that in general males

demonstrate greater _in utero_ vulnerability.79 FASD also shows higher prevalence in boys,55 confirming to this general finding. Male-specific vulnerability may relate to the sequence of

sex-specific timing of re-methylation and imprint establishment and the period of alcohol exposure. As a result, sex differences in the effect of FAE on allele-specific expression of

imprinted genes could occur. The sex-specific effects that emerge after puberty could be the consequence of estrogen-induced compensatory regulation of _Dio3_ and _Igf2_ expression,80,81 or

the sex specificity of their epigenetic regulation during this period.82,83 The divergence of the total expression from the sex-specific allelic expression further suggests hormonal

regulation of either the allele-specific or the total expression of _Dio3_ and _Igf2_. Interestingly, opposite to the current findings, female specificity in the total expression of _H19_, a

long noncoding RNA reciprocally imprinted with _Igf2_, has been found without sex differences in allelic expression.84 As estrogen responsive elements are present in both the _Dio3_ and

_Igf2_ imprinted regions (Supplementary Figure 6), estrogen-induced increases in maternal allelic expressions of these genes could compensate for the FAE effects in the female hippocampus.

Limitations of the current study include the timing of the treatments, although we have changed it from maternal21 to neonatal T4 administration to reverse the detrimental effects post FAE.

Ideally, the time frame of treatment should be expanded all the way to puberty, as it is argued that neurodevelopmental changes still occur during puberty.83 Furthermore, different alcohol

exposure regimens and species could be investigated for determining the general efficacy of these treatments. These future explorations will provide further confirmation for the

translational value of T4 and metformin in the treatment of FASD. Another interesting finding, although not a limitation, in this study is the pair-feeding effects on the hippocampal

expression of _Igf2_. Recently described consequences of restricted feeding indicate major changes in adult hippocampal total and allele-specific expression of _Igf2_ similarly to that found

in the present study for pair-feeding.47 The significance that metformin reversed the allele-specific expression changes caused by FAE, but not by pair-feeding, suggests that FAE effects

are reversible (at least in this time frame), while those of PF are not. This latter suggestion is in agreement with the conclusions of the Dutch famine study,85 where altered methylation of

the _IGF2_ imprinting control region and _IGF2_ expression persist way into adulthood. In the current study, both neonatal T4 and metformin administration mitigated FAE-induced hippocampal

fear memory deficits, even when administered after the termination of _in utero_ ethanol exposure. The convergent effect of neonatal T4 and metformin treatments in reversing this memory

deficit in parallel with increasing hippocampal _Dnmt1_ expression suggests methylation maintenance as a common mechanism of these treatments. As neonatal treatments restored hippocampal

_Dio3_ and _Igf2_ expressions in addition to those of _Dnmt1_, it is possible that both of these treatments affect memory via elevating _Dnmt1_ and consequently _Dio3_ and _Igf2_ expressions

in the _in utero_ ethanol-exposed adult offspring. We believe these results are the first to show that post-FAE T4 or metformin are potential therapeutic agents for FASD when administered

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famine in humans. _Proc Natl Acad Sci USA_ 2008; 105: 17046–17049. CAS  PubMed  Google Scholar  Download references ACKNOWLEDGMENTS We thank Sarah Chung, Wendy Luo, Kathryn M Harper, Jeanie

K Meckes and Laura J Sittig for their contributions to this manuscript. This work was supported by NIH AA017978 to EER. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Psychiatry

and Behavioral Sciences, The Asher Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA E Tunc-Ozcan, S L Wert, P H Lim & E E Redei * Department of Cellular

and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA A Ferreira Authors * E Tunc-Ozcan View author publications You can also search for this author

inPubMed Google Scholar * S L Wert View author publications You can also search for this author inPubMed Google Scholar * P H Lim View author publications You can also search for this author

inPubMed Google Scholar * A Ferreira View author publications You can also search for this author inPubMed Google Scholar * E E Redei View author publications You can also search for this

author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to E E Redei. ETHICS DECLARATIONS CONFLICT OF INTEREST The authors declare no conflict of interest. ELECTRONIC

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Tunc-Ozcan, E., Wert, S.L., Lim, P.H. _et al._ Hippocampus-dependent memory and allele-specific gene expression in adult offspring of

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