Neonatal Alcohol-Induced Region-Dependent Changes in
Rat Brain Neurochemistry Measured by High-Resolution
Magnetic Resonance Spectroscopy
Shonagh K. O’Leary-Moore, Andrew P. McMechan, Matthew P. Galloway,
and John H. Hannigan
Background: Maternal drinking during pregnancy can lead to a range of deleterious outcomes in
the developing offspring that have been collectively termed fetal alcohol spectrum disorders
(FASDs). There is interest and recognized value in using non-invasive neuroimaging techniques
such as magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) to charac-
terize, respectively, structural and biochemical alterations in individuals with FASDs. To date, how-
ever, results with MRS have been inconsistent regarding the degree and⁄or nature of abnormalities.
Methods: High-resolution magic angle spinning (HR-MAS) proton (1H) MRS is an ex vivo
neuroimaging technique that can acquire spectra in small punches of intact tissue, providing clini-
cally relevant neurochemical information about discrete brain regions. In this study, HR-MAS
1H MRS was used to examine regional neurochemistry in frontal cortex, striatum, hippocampus,
and cerebellum of young rats previously exposed to ethanol as neonates. Key neurochemicals of
interest included N-acetyl-aspartate (NAA), glutamate, GABA, glutamine, creatine, choline and
Results: Daily neonatal alcohol exposure from postnatal day 4 (PN4) through PN9 signifi-
cantly reduced levels of NAA and taurine in the cerebellum and striatum, and induced sex-depen-
dent reductions in cerebellar glutamate when measured on PN16. In addition, myo-inositol was
significantly increased in cerebellum. The frontal cortex and hippocampus were virtually unaf-
fected by this neonatal alcohol exposure.
Conclusion: Results of this research may have implications for understanding the underlying
neurobiology associated with FASDs and aid in testing treatments in the future. Ongoing studies
are assessing the developmental persistence of and⁄or maturational recovery from these changes.
Key Words: Fetal Alcohol Syndrome, N-Acetyl-Aspartate, Glutamate, Animal Model (Rat).
during pregnancy, lead to a wide range of both behavioral
dysfunction and structural anomalies in the brain of humans
and animals (Riley and McGee, 2005) the most severe of
which is fetal alcohol syndrome (FAS) (Hoyme et al., 2005;
Jones and Smith, 1973). The estimated prevalence of FAS is
between 0.5 and 2.0 per 1000 live births (Abel and Sokol,
1991; Bertrand, et al., 2005; May and Gossage, 2001) and
may be even higher among at-risk populations (Abel and
ETAL ALCOHOL SPECTRUM disorders (FASDs),
which result from heavy maternal ethanol consumption
Hannigan, 1995; Ceccanti et al., 2007; Viljoen et al., 2005),
although the number of individuals affected by FASDs may
be as much as 8 times as high (Burd et al., 2003; Sampson
et al., 1997). Despite the known adverse effects of ethanol on
the developing fetus, a recent Centers for Disease Control
(CDC) study of drinking behavior reported that 10.1% of
pregnant women reported drinking during pregnancy, with
1.9% engaging in high-risk heavy frequent or binge drinking
Over the past decade, a number of neuroimaging studies
have characterized some distinct patterns of brain damage
associated with fetal alcohol exposure in humans, including
corpus callosum dysgenesis, ventriculomegaly, cortical and
cerebellar atrophy, and abnormalities in white matter
(reviewed by Riley et al., 2004; Roebuck et al., 1998; Spadoni
et al., 2007). Magnetic resonance spectroscopy (MRS) is a
non-invasive neuroimaging technique that allows the quantifi-
cation of various neurochemicals and is used both clinically as
well as in animal models to assess the integrity of neural tissue
following exposure to teratogens. In
ground water signals in the brain are suppressed to reveal
signals from brain neurotransmitters and other biochemicals
(Moore, 1998). The various MR-visible neurochemicals in
1H-MRS, high back-
From the Departments of Psychology (SKO, JHH), Obstetrics &
Gynecology (APM, JHH), Psychiatry & Behavioral Neuroscience
(MPG), and Anesthesiology (MPG); and C.S. Mott Center for
Human Growth & Development, Wayne State University School of
Medicine (JHH), Detroit, Michigan.
Received for publication February 28, 2008; accepted May 14, 2008.
Reprint requests: Dr. S. K. O’Leary-Moore, Fetal Toxicology
Division, Bowles Center for Alcohol Studies, University of North
Carolina at Chapel Hill, CB# 7178 Thurston Bowles Building Chapel
Hill, NC 27599-7178; Fax: (919) 966-5679; E-mail: somoore@
Copyright ? 2008 by the Research Society on Alcoholism.
Alcoholism: Clinical and Experimental Research
Vol. 32, No. 10
Alcohol Clin Exp Res, Vol 32, No 10, 2008: pp 1697–1707 1697
MRS at clinical field strengths up to 4 Tesla include N-acetyl
aspartate (NAA—which also includes some signal from
N-acetyl-aspartylglutamate), total choline (tCho—a combina-
tion of free choline, glycerophosphorylcholine and phospho-
rylcholine), and creatine (Cre—including phosphocreatine).
MRS spectra acquired with editing and modified pulse
sequences also detect GABA, and myo-inositol, and a com-
bined peak including glutamate and glutamine (GLX)
(reviewed by Stanley et al., 2000). All of these chemicals are
abundant in brain at concentrations sufficient for the detec-
tion limits of MRS, which is between 0.5 and 1.0 mM (cited
in Bonavita et al., 1999). MRS also has potential clinical
utility as a diagnostic tool for neurological or psychiatric
disorders (Moore, 1998; Moore and Galloway, 2002), and
possibly for fetal alcohol effects (Astley et al., 1995; Cortese
et al., 2006; Fagerlund et al., 2006).
Of particular interest are changes in levels of NAA which
has been implicated in a number of important functions dur-
ing development, including myelination and osmoregulation,
and is considered a biomarker of neuronal viability or density
since it is found only in neuronal mitochondria. A compre-
hensive review of NAA in brain was recently published by
Moffett et al. (2007). NAA levels are reduced in a range of
(Guimaraes et al., 1995), hypoxia-ischemia (e.g., Hoehn et al.,
2001; Malisza et al., 1999), experimental hydrocephalus
(Harris et al., 1997), neuronal tumors (Wilken et al., 2000),
autism (Kahne et al., 2002), and prenatal stress (Poland et al.,
1999). Other chemicals such as precursors and breakdown
products of membranes (choline, GPC, and PCh), those
reflecting energy metabolism like Cre, major inhibitory and
excitatory neurotransmitters GABA and GLU, and still
others important as osmolytes (myo-inositol and taurine) or
antioxidants (GSH and taurine), can also be measured using
MRS (see Moore and Galloway, 2002), and are of interest
after fetal alcohol exposure.
To date, only a few MRS studies have assessed the impact
of prenatal alcohol exposure on brain biochemistry in human
participants. Previous clinical studies in FASD have shown
evidence of increased NAA levels and NAA⁄Cre ratios in the
caudate nucleus (Cortese et al., 2006), findings that seem to
contradict what is known about the effects of prenatal alcohol
exposure in general and the function of NAA in brain.
Another recent MRS study reported further indication of
overall increases of NAA intensities in some regions but
decreases in NAA⁄Cre and NAA⁄Cho ratios in adolescents
and young adults with fetal alcohol exposure (Fagerlund
et al., 2006). These percent increases in NAA are consistent
with Cortese et al., 2006 and the ratio decreases seemed to be
due to increases in the signal of Cre and Cho after fetal alco-
hol exposure (Fagerlund et al., 2006). Finally, a preliminary
abstract reported no changes in NAA in hippocampus in per-
sons exposed prenatally to alcohol (Hamilton et al., 2003).
Animal models of fetal alcohol exposure and currently
available very high-field strength magnets for animal research
may help in interpreting the results of clinical MRS studies
because animal studies control or eliminate a number of medi-
ating factors that can affect a developing organism’s suscepti-
bility to alcohol-induced teratogenesis (Abel and Hannigan,
1995; Hannigan, 1996). MRS has already been used by Astley
et al. (1995) to investigate fetal alcohol-induced damage in
macaques after exposure to alcohol for various periods during
gestation. They reported that in the absence of gross
abnormalities in structural MRI, there was an exposure dura-
tion-dependent relationship between the amount of alcohol
administered during gestation and the Cho⁄Cre ratio. This
ratio also correlated with a composite index of ‘‘developmen-
tal impairment’’ (i.e., higher ratios related to greater impair-
ment) derived from an extensive battery of motor, social,
cognitive (e.g., attention, discrimination, learning & memory),
behavioral, and physical measures (i.e., growth & craniofacial
dysmorphia) (Astley et al., 1995).
In the current study, high-resolution magic angle spinning
1H magnetic resonance spectroscopy (HR-MAS
was used to examine brain biochemistry after neonatal alco-
hol exposure in rats. HR-MAS1H-MRS is an ex vivo version
of MRS that affords high spectral resolution comparable to
solution state spectroscopy in semi-solid biological tissue by
rapidly spinning samples around their axis with the axis main-
tained at the ‘‘magic’’ angle (h = 54.7?) relative to the static
magnetic field (B0). One advantage of this technique is that
small samples of unprocessed intact brain tissues are analyzed
directly, minimizing artifacts introduced by procedures such
as chemical extraction, membrane disruption, chemical deriv-
atization, or homogenization typical of other analytic tech-
niques (Cheng et al., 1997). The position of the sample
relative to B0reduces dipole-dipole couplings, chemical shift
anisotropy, and magnetic susceptibility changes, ultimately
resulting in better signal-to-noise ratios (Griffin et al., 2002).
These advantages increase specificity as well as confidence in
quantifying neurochemical information. Previous HR-MAS
studies show direct links between NAA levels and neuronal
loss in the neurodegenerative Pick’s Disease (Cheng et al.,
1997) and suggest that HR-MAS
experimental pathological investigation and can aid in inter-
pretation of clinical MRS data used to study neurological dis-
orders. Additionally, regional specificity in neurochemical
profiles in HR-MAS-derived spectra has been reported in our
laboratory (e.g., Bustillo et al., 2006; O’Leary-Moore et al.,
2007) and others (Tsang et al., 2005), and suggests that1H
HR-MAS is especially conducive to conducting regional anal-
yses of particular brain areas.
The current experiment used a well-validated rat model of
third-trimester-equivalent neonatal binge alcohol exposure
and assessed brain biochemistry in the young rat with
HR-MRS. Animals were assessed at postnatal day 16
(PN16), a week after cessation of alcohol exposure, in part to
avoid direct pharmacological effects of alcohol, but also to be
able to later characterize a developmental biochemical profile
after perinatal alcohol exposure.
Of the many neurochemicals that can be measured with
MRS we were particularly interested in those that are also
1H MRS has utility for
O’LEARY-MOORE ET AL.
visible at clinical field strengths and show sensitivity to neuro-
pathology. These included NAA, Cho, Cre and myo-inositol,
as well as glutamate and GABA because of their potentially
togenesis (see Cohen-Karem and Koren, 2003), the antioxi-
dants glutathione (GSH) and taurine were examined. We
hypothesized that levels of NAA and the antioxidants would
be decreased and that levels of Cho and myo-inositol would be
increased after neonatal alcohol exposure, hypotheses based in
part on previously observed effects of prenatal alcohol expo-
sure on MRS-visible neurochemicals in monkeys (Astleyet al.,
1995) and humans (Cortese et al., 2006; Fagerlund et al.,
2006). We focused on four brain regions of interest: the frontal
cortex, striatum, hippocampus and cerebellum (see Fig. 2).
Because alcohol exposure during the early postnatal brain
growth spurt in rats is known to significantly decrease the
bellum (e.g., Goodlett et al., 1998; Green et al., 2000, 2002),
prised of those cells (e.g., Green et al., 2000, 2002; Stanton and
Goodlett, 1998), we hypothesized that we would detect the
The basic design was a 3 · 2 factorial experiment with neonatal
ethanol Treatment (Non-intubated, Sham-intubated, or intubated
with 5 g⁄kg ethanol) and Sex (male and female) as factors. For
each subject, four brain regions were assessed: the frontal cortex
(Fr2 area), striatum, hippocampus, and cerebellum. The main
dependent variables were the MR-visible metabolites depicted in
Fig. 1 although we were primarily interested in NAA, Cho, Cre,
glutamate (Glu), GABA, taurine, myo-inositol, and glutathione
(GSH). Body weight gain during the intubation period from postna-
tal day 4 (PN4) to PN9 and body weight on PN16 were also
Subject Generation and Litter Management
Timed-pregnant female rats from Charles River Farms (Portage,
MI) arrived in the lab on gestational day 4 (GD4) and were assigned
randomly to have their litters be either intubated or non-intubated.
Pups from intubated litters were either sham-intubated (no fluid) or
ethanol-intubated (5 g⁄kg⁄d) during the neonatal period, while non-
intubated litters were undisturbed (except for daily weighing from
postnatal day 4 [PN4] - PN9) during the same neonatal period. Start-
ing on GD20, cages were inspected twice daily for births (usually on
GD22). On PN1, the day following birth, pups were weighed, exam-
ined for obvious gross physical abnormalities, and litters culled to 8,
retaining 4 males and 4 females when possible. This final litter size
optimizes growth of intubated neonates and maintains growth rates
comparable to non-intubated offspring (cf., Light et al., 1998). On
PN4, all pups were tattooed on their paws to aid identification during
the neonatal period.
Neonatal Ethanol Administration
Neonatal ethanol intubations began on PN4 and continued
through PN9. Pups were assigned randomly within each intubated
litter to be either sham-intubated or intubated with a total
of 5 g⁄kg⁄day ethanol during the intubation period. This dose of
alcohol was chosen because it produces high peak blood ethanol con-
centrations (BACs) of ?320 ± 6 mg⁄dl and it was found to induce
behavioral deficits in reversal learning tasks in young male rats
(PN28) in our lab (O’Leary-Moore et al., 2006). Other labs have also
reported persistent deficits in learning and behavior (e.g., Goodlett
and Johnson, 1997; Goodlett and Lundhal, 1996; Goodlett and
Peterson, 1995) and cell loss after comparable dosing (e.g., Goodlett
and Horn, 2001; Goodlett and Lundhal, 1996; Goodlett et al., 1998).
Approximately half of each intubated litter received alcohol and half
were sham-intubated. Alcohol-intubated subjects were intubated
twice daily with 2.5 g⁄kg ethanol, 2 hours apart, with a milk solution
typically used for artificial rearing, supplied by Dr. Goodlett at Indi-
ana University-Purdue University Indianapolis (IUPUI) (Goodlett
and Johnson, 1997).
On PN4, intubated litters with dams were brought to the labo-
ratory from the vivarium and pups were removed from their
mother as a litter and placed under a warming lamp to maintain
body temperature. Each subject was weighed and the appropriate
volume for the intubation was calculated [volume of intuba-
tion = weight (g) * 0.0278 (ml⁄g)]. Alcohol was administered at a
concentration of 11.33% v⁄v. The same volume of milk and alco-
hol solution was used for each intubation on a given day.
Because this is a period of rapid growth, animals were weighed
and intubation volumes were re-calculated each day. Next, the
milk–alcohol solution was drawn into a syringe connected to a
measured piece of PE-10 tubing. This tube was lubricated with
corn oil and inserted down the esophagus and into the stomach
of the rat pup. Milk was infused directly into the stomach over
10 seconds and pups were then returned to their dam as a litter
after all pups from a given litter were intubated. The total elapsed
time to intubate a litter was ?8 minutes. Correct tube placement
in the stomach was verified by observing filling of ‘‘milk bands.’’
Sham-intubated pups received all of the same handling as alco-
hol-exposed pups but no milk was infused during the intubation.
An additional milk-only intubation was given to alcohol-exposed
pups 2 hours after the second intubation daily (plus 2 additional
milk-only intubations on PN4) to help replace calories lost during
intoxication (Goodlett and Johnson, 1997).
Brain Dissection and Tissue Collection
Regional brain tissues were collected at PN16, 1 week after ter-
mination of alcohol administration. Animals were killed, whole
brains rapidly removed and placed into a chilled brain matrix (on
ice) and sliced into 2 mm-thick coronal slices. The left and right
hippocampus (CA1 region), striatum (dorsolateral region), and
frontal cortex (Fr2 region), as well as the anterior cerebellar
vermis, were microdissected using a punch technique (2.1 mm
dia.-punches) from the slices containing the various regions of
interest. Relative to bregma, slices were taken for frontal cortex
(+2.70), anterior striatum (+1.20), hippocampus ()4.20), and cer-
ebellum ()10.30) using Paxinos and Watson (1998) atlas coordi-
nates (Fig. 2). Punches were then immediately frozen on dry ice
and stored at )80?C until the time of HR-MAS analysis. No
additional tissue processing occurred. This procedure consistently
produced tissue samples weighing ?4 to 5 mg.
HR-MAS1H MRS Methods
Tissue samples were removed from the freezer, weighed quickly,
and placed into a Bruker zirconium rotor (2.9-mm dia., 10-ll capac-
ity) containing 4 ll PO4buffer (pH = 7.4), formate, and [3- (trim-
ethylsilyl)propionic-2,2,3,3-d4 acid]; sodium salt (TSP) as the
0.0 ppm reference. The rotor was then immediately placed into the
ned at a temperature of 4?C and the rotor was spun at a MAS rate of
NEONATAL ALCOHOL AND1H HR-MAS MRS
4.2 kHz @ 54?7¢. A CPMG (Cheng et al., 1997) rotor-synchronized
pulse sequence (TR = 3500 ms, bandwidth 8 kHz, 16 k complex
points, 128 or 256 averages) was used. All neurochemical data
were standardized to their respective non-intubated controls within
each brain region before any group analyses were commenced.
For all samples, TSP served as the external chemical shift reference,
resonating at 0.00 ppm. D2O was used to lock on the center
Spectral Analysis and Quantification of Neurochemicals
Spectra were analyzed using LCModel software that uses a lin-
ear combination of model spectra from a custom basis set gener-
ated with 27 individual phantom standard solutions (Provencher,
2001). Cramer-Rao bounds were also computed to estimate the
precision with which LCModel was able to fit the data (Cavassila
et al., 2001). Cramer Rao bounds were typically below 10% and if
they exceeded 25% or if signal-to-noise ratios (SNR) were below
15 for a given spectra, data from those neurochemicals and⁄or
samples were not included in further analyses. Neurochemical data
were standardized (nmol⁄mg tissue) and expressed as a proportion
of non-intubated controls. Total choline (tCho) was calculated as
(Cho + GPC + PCh). In addition, we examined the ratio of
NAA to Cre because prior clinical research reported changes in
NAA⁄Cre after prenatal alcohol (Cortese et al., 2006; Fagerlund
et al., 2006) and clinical MRS often uses such ratios to detect neu-
ropathology and sometimes psychiatric disease (cf., Danielsen and
Ross, 1999; Moore and Galloway, 2002). Most typically Cre is
used as the denominator of these ratios since it is assumed to be
relatively stable and impervious to pharmacological modification
(but see below). The following ratios of neurochemicals were also
calculated: NAA⁄Cre, NAA⁄tCho, and tCho⁄Cre, since they are
often used to interpret clinical scans where absolute quantification
of neurochemical concentration is not available (Danielsen and
Body weight gain during the early neonatal period was assessed
using a 3 (Treatment group) · 2 (Sex) · 6 (Day) repeated measure
analysis of variance (ANOVA). To assess body weight at the time
of tissue collection on PN16, a 3 (Treatment group) · 2 (Sex)
ANOVA was used. Student–Newman Keuls (SNK) post-hoc tests
were used to follow-up any significant main effects. For MRS
outcomes, while significant effects of brain region were evident
for nearly all neurochemicals examined (p’s < 0.05), brain region
differences were not the focus of this study. Thus, neurochemical
data were analyzed within each brain region using separate 3
(Treatment group) · 2 (Sex) ANOVAs for each neurochemical.
Data are expressed as a proportion of non-intubated controls. An
alpha value of 0.05 was considered significant and maintained for
Effect of Neonatal Ethanol Exposure on Body Weight
As expected, there was a significant main effect of Day
[F(5,960) = 8022.88,p < 0.05]as well assignificant
Fig. 1. HR-MAS1H MRS spectrum from striatum of an adult male rat at 11.7 T (ALA, alanine; ASP, aspartate; BET, betaine; CHO, choline; CRE, crea-
tine; GABA, gamma-aminobutyric acid; GLU, glutamate; GLN, glutamine; GSH, glutathione; GPC, glycerophosphocholine; GLY, glycine; LAC, lactate; mI,
myo-inositol; NAA, N-acetyl aspartate; NAAG, N-acetyl aspartyl glutamate; PCh, phosphorylcholine; PEA, phosphorylethanolamine; SUC, succinate; TAU,
O’LEARY-MOORE ET AL.
Treatment · Day [F(10,960) = 71.94, p < 0.05] and Treat-
ment · Sex · Day interactions [F(10,960) = 2.91, p < 0.05]
on weight gain during the neonatal period. Further, there
were main effects of neonatal Treatment [F(2,192) = 85.95,
p < 0.05] and Sex [F(1,192) = 15.38, p < 0.05], and a signif-
icant neonatal Treatment · Sex interaction [F(2,192) =
5.04, p < 0.05]. Post hoc analyses revealed that neonatal
alcohol-exposed rats weighed significantly less than both
sham- and non-intubated controls, and sham-intubated rats
weighed less than non-intubated controls during the intuba-
tion period. Males weighed significantly more than females
(p’s < 0.05).
At PN16, SNK post hoc tests following the significant main
effect of neonatal Treatment [F(2,64) = 18.09, p < 0.05],
showed that alcohol-exposed rats weighed significantly less
than sham- and non-intubated control groups, and that the
sham-intubated group weighed significantly less than the non-
intubated controls. In addition, a significant neonatal Treat-
ment · Sex interactionwas
p < 0.05]. Sham- and alcohol-exposed males weighed less
than non-intubated control males, whereas alcohol-exposed
females weighed less than both the sham- and non-intubated
control females (p’s < 0.05).
found[F(2,64) = 3.93,
Alcohol exposure during the first postnatal week signifi-
cantly altered a number of neurochemicals at PN16 in both
male and female rats with the majority of effects being seen in
both the cerebellum and striatum. The regional data are
presented for each neurochemical.
N-Acetyl-Aspartate (NAA). The 3 (Treatment) · 2 (Sex)
ANOVAs revealed that neonatal alcohol exposure signifi-
[F(2,62) = 8.178, p < 0.05] (Fig. 3A). No significant main
effects or interactions with Sex were found. Post-hoc tests
revealed that alcohol-exposed animals had significantly lower
levels of NAA than both non-intubated and sham-intubated
controls. Additionally, NAA levels were significantly reduced
in the striatum [F(2,62) = 4.207, p = 0.019] compared to
both sham-intubated and non-intubated controls. There were
no significant changes in NAA in the hippocampus or frontal
cortex (p’s > 0.05 see Fig. 3). Although significant decreases
were evident in absolute concentrations of NAA in alcohol-
exposed animals compared to both control groups in the cere-
bellum and striatum, significant decreases in NAA⁄Cre were
only seen in cerebellum when data were collapsed on Sex
compared to NI control (p < 0.05; Table 1). The NAA⁄tCho
ratio was higher in alcohol-exposed subjects in the hippocam-
pus compared to NI Controls [F(2,36) = 3.356, p < 0.05]
NAAin the cerebellum
Creatine (Cre). In the cerebellum there was a significant
Treatment group · Sex interaction for Cre [F(2,62) = 3.640;
p < 0.05] but no main effects were apparent. Analyzing
males and females separately, sham-intubated male rats had
higher Cre levels than non-intubated controls (p’s < 0.05),
but alcohol-intubated rats did not differ from either group. In
the striatum, a main effect of neonatal Treatment [F(2,62) =
3.753, p < 0.05] revealed that the alcohol-intubated groups
had lower levels of Cre than Sham-intubated controls, but
neither group differed from non-intubated controls. No
Fig. 2. Brain tissue samples (punches) from regions of interest (Paxinos and Watson, 1998).
NEONATAL ALCOHOL AND1H HR-MAS MRS
differences in Cre were apparent in the frontal cortex or hip-
pocampus; data are shown collapsed on Sex (Fig. 3A).
Choline-Containing Compounds (Cho⁄GPC⁄PCh⁄tCho).
There were no significant differences in levels of free choline
(Cho) after neonatal alcohol exposure in any region
(p’s > 0.05). However, a main effect of Sex in the striatum
indicated that males had higher Cho levels than females
[F(1,62) = 4.013, p < 0.05]. Further analyses revealed that
GPC levels were significantly decreased in the hippocampus
of neonatal alcohol-exposed animals compared to non-
intubated controls [F(2,37) = 3.572, p < 0.05], and levels of
Fig. 3. Mean NAA (A), Cre (B), GABA (C), and taurine (D) as a percent of NI controls. Data are shown collapsed on sex; gray bars are sham controls
and black are EtOH treated. The dark line represents the NI control values (mean = 1.0). *Significantly different than NI and sham controls; ^Significantly dif-
ferent than sham controls.
Table 1. Mean Metabolite Ratios After Neonatal Alcohol Exposure in Each Brain Region
Ratio GroupFrontal CtxStriatum HippocampusCerebellum
0.58 (0.02 )#
Means are shown collapsed on Sex. *Significantly different than NI and sham controls, p < 0.05. #Significantly different than NI controls,
p < 0.05.
O’LEARY-MOORE ET AL.
PCh in frontal cortex were significantly lower in alcohol-
exposed animals than in sham-intubated but not non-intubat-
ed controls [F(2,49) = 3.638, p < 0.05]. We also analyzed
total choline (tCho) because the choline peak that is MR-
visible at typical clinical field strengths (i.e., £3.0 Tesla) con-
tains contributions from the three compounds that comprise
tCho—free choline (Cho), GPC and PCH. Total choline was
unchanged by neonatal alcohol exposure in all regions,
although a main effect of neonatal alcohol treatment
approached significance (p = 0.06) and post-hoc tests were
significant in the hippocampus. We also analyzed the ratio of
tCho to Cre. In hippocampus, tCho⁄Cre was significantly
compared toboth non-intubated
controls [F(2,34) = 4.601, p < 0.05; Table 1]. In striatum,
tCho⁄Cre was increased ?18% in neonatal alcohol-exposed
animals compared tonon-intubated
[F(2,21) = 3.647, p < 0.05; Table 1].
Significant decreases in levels of Glu were evident in cerebel-
lum after neonatal alcohol exposure compared to both non-
intubated and sham-intubated controls [F(2,62) = 6.135,
p < 0.05]. There was also a significant neonatal Treat-
(Glu), Glutamine(Gln),and GABA.
ment · Sex interaction in the cerebellum [F(2,62) = 3.956,
p = 0.024], and follow-up analyses revealed that sham-
intubated males had higher levels of Glu than both non-
intubated and alcohol-intubated males [F(2,34) = 5.199,
p < 0.05] whereas neonatal alcohol-exposed females had
significantly reduced levels of Glu than either non-intubated
or sham-intubated females [F(2,28) = 5.183, p < 0.05]
(Fig. 4A). There were no significant main effects of alcohol on
glutamine (Gln) levels in any brain region assessed. In addi-
tion, there were no statistically significant effects of neonatal
alcohol exposure on MR-visible levels of GABA (all
p’s > 0.05) although GABA levels tended to be decreased
(?6%) in the striatum (Fig. 3C).
Other Compounds. A number of other neurochemicals
were also affected by neonatal alcohol exposure in the cerebel-
lum, including myo-inositol and taurine. GSH levels were
unchanged by neonatal alcohol exposure in all regions
(p’s > 0.05). Levels of myo-inositol were increased by neona-
tal alcohol in the cerebellum [F(2,62) = 3.691, p < 0.05] and
a Treatment group · Sex interaction approached significance
(p = 0.06) (Fig. 4B). Finally, levels of taurine were signifi-
cantly reduced by neonatal alcohol exposure compared to
both control groups in the cerebellum [F(2,62) = 6.207,
p < 0.05] and striatum [F(2,62) = 4.667, p < 0.05; Fig. 3D].
The focus of the current experiment was to examine the
effects of binge-like neonatal ethanol exposure in the rat,
modeling third-trimester exposure during the brain growth
spurt in humans, on regional neurochemistry using an ex vivo
proton (1H) MRS technique. The high-resolution ‘‘magic’’
angle spinning MRS method (HR-MAS1H MRS) utilizing
an 11.7-Tesla magnet, allowed resolution and measurement
of all compounds that are visible in typical clinical MRS scans
at <3.0-Tesla such as NAA, Cre, and Cho. A number of
other neurochemicals such as antioxidants (e.g., taurine &
glutathione) and neurotransmitters (e.g., glutamate & GABA)
that can only be resolved using high-resolution MRS tech-
niques or specialized pulse sequences, were also able to be
assessed. This is the first study to use this method to examine
CNS neurochemistry after neonatal ethanol exposure. In line
with our hypotheses, neonatal alcohol exposure from PN4 to
PN9 significantly altered neurochemistry in a brain region-
dependent manner. The manner in which these neurochemi-
cals were changed and possible implications of such changes
are discussed below.
Neonatal alcohol exposure in rats significantly reduced
levels of NAA in the cerebellum and striatum, but not in
hippocampus or frontal neocortex. The finding of reduced
NAA in the cerebellum was in line with the hypotheses that
levels of NAA would be reduced, consistent with the marked
Fig. 4. Mean cerebellar Glu (A) and myo-inisitol (B) as a percent on NI
controls in male and female offspring. *Main effect of treatment differs signifi-
cantly from NI and sham controls p < 0.05; #Treatment · Sex interaction,
p < 0.05; @Treatment · Sex interaction p = 0.06.
NEONATAL ALCOHOL AND1H HR-MAS MRS
reduction in the number of cells in this region after neonatal
alcohol exposure (e.g., Goodlett et al., 1998; Green et al.,
2002). The present NAA results are consistent, in general,
with previous clinical MRI studies in adolescents showing
particular sensitivity to prenatal alcohol exposure of both
cerebellum (Riley et al., 2004; Roebuck et al., 1998; Sowell
et al., 1996) and striatum (Archibald et al., 2001; Cortese
et al., 2006; Mattson et al., 1996). However, there are a few
inconsistencies among the small number of previous MRS
studies of the effects of prenatal alcohol exposure on human
brain NAA levels. For example, MRS had revealed either
increases (Cortese et al., 2006) or no changes (Fagerlund
et al., 2006) in striatal NAA levels, NAA percentages, and⁄or
NAA⁄Cre ratios. Fagerlund et al. (2006) reported decreases
in the NAA⁄Cre and NAA⁄Cho ratios in individuals with
FASDs in the cerebellar dentate nucleus, but not in the cere-
bellar vermis or hippocampus. To date, Cortese et al. (2006)
have only reported data from caudate nucleus, whereas
Fagerlund et al. (2006) also reported increases in NAA
(as percent of controls) in thalamus and frontal and parietal
neocortex, and decreases in NAA⁄Cre in corpus callosum,
anterior cingulate, parietal cortex and frontal white areas.
Despite decreased NAA in cerebellum and striatum, signifi-
cant decreases in the NAA⁄Cre ratio were seen only in the
cerebellum when collapsing on Sex, indicating that the use of
ratios alone might not be the best way to interpret sex-specific
neurochemical effects of developmental alcohol exposure.
Although we present ratio data here, one of the strengths of
this method is the ability to quantify individual neuro-
chemicals and not rely on ratio data alone (e.g., NAA⁄Cre;
NAA⁄Cho, and Cho⁄Cre). Because Cre has been considered
to be relatively stable, it has often been used as a denominator
in ratios in MRS research (Danielsen and Ross, 1999; and see
Jansen et al., 2006). However, the fact that Cre varies in devel-
opment and, in this case, across alcohol treatments, means
that absolute quantification is preferable (e.g., Tkac et al.,
2003; Yao et al., 1999). Other studies have also indicated that
the use of Cre as an equalizing denominator can increase vari-
ability (Li et al., 2003).
Based on previous work in humans showing some
increases in Cho levels but not in Cho⁄Cre ratios in FASDs
(Fagerlund et al., 2006), and in monkeys showing elevated
Cho⁄Cre ratios after prenatal alcohol exposure (Astley et al.,
1995), we hypothesized that Cho levels would be increased
after neonatal alcohol exposure in rats as well. In a typical
clinical MRS scan at £3.0 Tesla, the total Cho resonance
peak (tCho) is comprised of relatively low levels of free
Cho plus substantial contributions from phosphorylcholine
(PCh, a membrane phospholipid precursor) and glycer-
ophosphorylcholine (GPC, a breakdown product). In the
current study, Cho, GPC and PCh were readily resolved and
each of these compounds measured. The sum of the choline
peaks (tCho) and a tCho⁄Cre ratio were also computed to
compare with existing literature. Neonatal alcohol exposure
reduced levels of GPC in the hippocampus and of PCh in
the frontal cortex compared to sham-intubated controls. In
hippocampus, levels of tCho were marginally reduced
and the tCho⁄Cre ratio was significantly lowered in
alcohol-exposed subjects. In striatum, the tCho⁄Cre ratio
was significantly elevated in neonatal alcohol-exposed rats
compared to non-intubated controls, a finding that is consis-
tent with one previous MRS study in macaques (Astley
et al., 1995) but at odds with Fagerlund et al. (2006) who
did not report increased Cho in FASD individuals in the
striatum but did in other regions (e.g., anterior cingulate,
parietal cortex, thalamus, corpus callosum, and cerebral
Fagerlund and colleagues suggested that elevations in
choline reflected an enhanced turnover of Cho-containing
membrane constituents. This may be taken to indicate that
altered membrane stability in glia cells persists postnatally
after prenatal alcohol exposure (Fagerlund et al., 2006).
In the current study, that PCh and tCho⁄Cre were decreased
in the hippocampus while the tCho⁄Cre ratio was increased in
striatum is consistent with dysfunctional membrane turnover
in the young perinatal alcohol-exposed brain.
Glutamate (Glu), Glutamine (Gln), and GABA
This is the first study to assess glutamate (Glu), glutamine
(Gln), and GABA using MRS following perinatal alcohol
exposure. Sex-dependent decreases in Glu levels were seen
in female subjects after neonatal alcohol exposure in the
cerebellum. Male sham-intubated subjects actually had higher
levels of Glu compared to other groups. Since there are matu-
rational increases in levels of Glu (Raman et al., 2005; Tkac
et al., 2003; Yao et al., 1999), the reduced Glu levels in neona-
tal alcohol-exposed females in the current study could reflect
a developmental delay. Alternatively, reductions in Glu might
reflect cell loss in the cerebellum. In fact, it has been reported
that prenatal and neonatal alcohol exposure significantly
reduces the number of granule cells in the cerebellum (e.g.,
Maier and West, 2001; Maier et al., 1999). The precise reason
for sham-intubated subjects to have higher levels of Glu
remains unknown, but it could be related to the stress of
the sham-intubation procedure itself. Levels of Gln were
unchanged in any brain region after neonatal alcohol
Previous studies using a variety of other techniques have
investigated the role of glutamatergic systems in animal mod-
els of FASDs. Rawat and colleagues reported that Glu levels
in the whole fetal rat brain were elevated after prenatal alco-
hol exposure compared to controls and remained elevated at
PN5 and PN10 in pups whose mothers continued to receive
alcohol during lactation (Rawat, 1977). Conversely, others
reported that whole-brain Glu concentrations were signifi-
(Griezerstein and Aldrich, 1983). Regional differences in
effects on Glu are evident in the brains of fetal alcohol-treated
O’LEARY-MOORE ET AL.
animals as well. For example, prenatal alcohol exposure
reduced3H glutamate binding in whole brain and hippocam-
pus but not in neocortical regions (Farr et al., 1988; Kelly
et al., 1986). The varying findings are difficult to reconcile
because of differences in experimental techniques as well as
the unique cellular nature of MR-visible Glu, representing
both neuronal and metabolic pools. Nonetheless, our results
along with these other reports are consistent with the hypo-
thesis that glutamatergic function is disrupted following
perinatal alcohol exposure.
No significant alterations in GABA levels were seen
following neonatal alcohol exposure. The absence of such an
ethanol effect on MR-visible GABA levels differs from previ-
ous reports showing increased levels of whole brain GABA
after prenatal (Mena et al., 1982) or fetal binge alcohol expo-
sure (Maier et al., 1996) but are consistent with regional anal-
yses showing near-normal levels of GABA in the amygdala
and striatum (Moloney and Leonard, 1984) after pre- and⁄or
postnatal alcohol exposure, and others reporting no changes
in whole brain GABA after prenatal alcohol exposure in fetal
rats (Espina et al., 1989).
Taurine is of considerable interest because in addition to
having antioxidant properties, it plays roles in osmoregula-
tion and neuromodulation and acts as a GABA-A agonist
(Aerts and Van Assche, 2002). Moreover, taurine deficiency
can lead to growth retardation and impaired CNS function
(Aerts and Van Assche, 2002). In the current study, cere-
bellar and striatal taurine levels were reduced significantly
by neonatal alcohol exposure. Because oxidative stress is
one proposed mechanism of alcohol-induced teratogenesis
(Abel and Hannigan, 1995; Michaelis, 1990) and previous
studies have shown that treatment with antioxidants may
ameliorate some of the adverse effects of perinatal alcohol
exposure (reviewed by Cohen-Kerem and Koren, 2003),
further studies exploring whether taurine supplementation
would ameliorate fetal alcohol effects could provide fruitful.
Since taurine levels in humans are currently beyond detec-
tion in typical clinical MRS, the utility of HR-MAS
MRS as an experimental technique for assessing disease
progression or therapeutic intervention is highlighted. Lev-
els of glutathione (GSH), another MR-visible antioxidant,
were unchanged in any brain region by neonatal alcohol
exposure though previous reports have indicated increased
GSH in the cerebellum after neonatal alcohol exposure
(Smith et al., 2005).
Neonatal alcohol exposure during the brain growth spurt
significantly altered regional brain neurochemistry. In general,
the majority of effects were localized to cerebellum and stria-
tum including deficits in Glu, NAA, and Cre, consistent with
other teratogenic effects of ethanol in these brain regions.
Although results of the current study differ from previous
human or non-human primate studies assessing neurochemis-
try after perinatal alcohol exposure, a direct comparison is
difficult given different ages of either exposure or testing, as
well as of ethanol exposure patterns. However, given the pau-
city of MRS studies of perinatal ethanol-induced neurochemi-
cal changes at any age, it is premature to expect a consensus.
Future experiments should strive for comparable studies of
specific neurochemicals accounting for homologous brain
regions, developmental stage, strict diagnostic criteria and,
where possible, similar patterns of ethanol exposure, MR
acquisition parameters, and means of determining neuro-
chemical concentrations (i.e., absolute levels vs. ratios or
percentages). Developmental stages of assessment are particu-
larly important because NAA levels vary systematically
during development and across brain regions (e.g., Horska
et al., 2002; Huppi and Inder, 2001; Tkac et al., 2003). Since
NAA levels increase over normal early development, the cur-
rent results with NAA—and perhaps with other metabolites
as well—may reflect either lowered (or elevated) levels of
NAA and⁄or developmental delay. Further analyses of neo-
natal alcohol-exposed rats at different ages with HR-MAS1H
MRS are in progress and will help to clarify the interpretation
of these results.
Strengths of the current study are that it was conducted in
a laboratory setting with precise control over the timing and
pattern of alcohol exposure, and uses a high-resolution MR
method with a much more powerful magnet than is typically
available for human studies. Although the ex vivo brain
specimens used in this HR-MAS
dissected and frozen as quickly as possible, and they were
not homogenized, extracted or processed in any way, rapid
neurochemical changes can occur post mortem which could
affect neurochemical data. Also, the small punch method for
collecting brain specimens is very precise and well localized
so that the risk of ‘‘partial volume’’ effects is low, that is, the
possibility that an MRS signal from a tissue punch or voxel
includes signals from more than one type of tissue (i.e., gray
matter, white matter or CSF). However, such effects for
smaller regions, such as the hippocampus, in young animals
cannot be ruled out. Related to this point, metabolite con-
centrations are affected by the amount of tissue versus CSF
and this might vary across treatment conditions. These
are considerations that should be taken into account when
interpreting data when tissue samples are considered to be
In addition to elaborating the developmental profile of neu-
rochemical changes after perinatal ethanol exposure, MRS
studies provide a paradigm to test potential treatments of
FASDs such as dietary supplementation with antioxidants
such as taurine or vitamin E (after Cohen-Karem and Koren,
2003; Heaton et al., 2000), or other nutrients (e.g., choline;
Thomas et al., 2004, 2007), or environmental enrichment and
exercise (Hannigan et al., 2007). In summary, the current
study shows that HR-MAS1H MRS, is a powerful, clinically
relevant tool that shows great promise for helping to advance
the understanding of FASDs, for identifying a potential CNS
biomarker of fetal alcohol effects, and for evaluating treat-
ment effectiveness for the FASDs.
1H MRS method were
NEONATAL ALCOHOL AND1H HR-MAS MRS
This research was part of a Ph.D. dissertation completed at
Wayne State University by Dr. O’Leary-Moore and was
funded in part by an NRSA Predoctoral Fellowship from
NIAAA (F31 AA015224) awarded to Dr. O’Leary-Moore,
R01-DA-016736 (MPG), and by the Brain Research And
Imaging Neurosciences Research Division (BRAINS) in the
department of Psychiatry & Behavioral Neurosciences at
Wayne State University School of Medicine. We thank
Dr. B.M. Cortese for comments on an earlier version of this
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NEONATAL ALCOHOL AND1H HR-MAS MRS