Neuroimaging techniques can augment current methods of inspecting

the effect of prenatal alcohol exposure on cognitive functions

The goal of the current proposal is to study how moderate prenatal alcohol

affects learning in general and learning about alcohol at various postnatal ages.

A limited review of current knowledge on neurobiological and behavioral effects

of prenatal alcohol exposure will be followed by methods and specific hypotheses

of the current proposal. This paper will conclude with a discussion of what

implications this line of research has for prevention and treatment of individuals

with prenatal alcohol exposure.

Much research has been done on fetal alcohol syndrome (FAS) before but

especially since its recognition as such in the late sixties and early seventies (No

authors listed, 2000). FAS is diagnosed when children are born with growth

deficiencies, craniofacial abnormalities and central nervous system dysfunction

manifested by structural and functional (cognitive deficits) abnormalities. In

addition to children with FAS are children with similar symptoms but little or no

growth- or facial-abnormalities. These children have been labeled with what is

known as an alcohol-related neurodevelopmental disorder (ARND). Children

without the full criteria for FAS are said to have PEA or prenatal exposure to

alcohol, which results in FAE or fetal alcohol effects. Recently, in an attempt to

curb the apparently endless expansion of labels, the umbrella term fetal alcohol

spectrum disorders (FASD) has been applied to refer to the innumerable

combinations of physical and neuropsychological effects of prenatal alcohol

exposure (Riley & McGee, 2005). The variations commonly seen in the

phenotypes (both neuroanatomical and behavioral) of FASD that prompted so

many labels stem from differences in amount and pattern of maternal alcohol

intake and perhaps most importantly the gestational age at exposure. Other

mediating factors are the mother’s age, nutrition, use of other drugs, and the

child’s genetic composition.

In addition to the well-known facial phenotype of many individuals with

FASD are gross brain structure abnormalities. Of these is microencephaly, or

small brain, a consistent finding in FASD individuals that is usually inferred from

microcephaly (small head relative to body size). Initially using autopsied

individuals and later with magnetic resonance imaging (MRI) it was determined

that this microencephaly is not a global defect but rather results from specific

damage. Most consistently found is a reduction in size of the parietal lobes and

the junction at the parietal and temporal lobes. Reduced size of the frontal lobes

(specifically the left ventral frontal lobe) has also been noted. Finally, the right

temporal lobe is usually larger than the left temporal lobe in controls but this

asymmetry was reduced in FASD subjects (Riley & McGee, 2005). Analyses of

brain structure abnormalities have overwhelmingly found white matter to be

specifically affected (reduced) with a concomitant increase in gray matter.

In addition to these gross reductions in size, specific structural deficits

have also been seen in the corpus callosum, cerebellum, hippocampus and

basal ganglia. In severe cases, the corpus callosum is completely absent

(agenesis) but in most cases it is thinned. This thinning tends to occur in the

rostral- and caudal-most parts. Additionally, the corpus callosum is displaced in

three dimensional space compared to control children (Mattson, Schoenfeld, &

Riley, 2001). Another structure reduced in size subsequent to prenatal alcohol

exposure is the cerebellum, particularly in the anterior vermis. Death of the

Purkinje cells of the cerebellum may be responsible for this reduced volume as

these output cells are especially sensitive to the effects of alcohol. FAS children

also show a greater than normal (right is larger) asymmetry in the hippocampus

(Mattson, Schoenfeld, & Riley, 2001). The caudate nucleus is part of the basal

ganglia and also implicated in spatial memory (as well as higher cognitive

functions). This structure is reduced in volume after prenatal exposure to

ethanol.

General deficits in intellectual functioning as well as specific impairments

including learning and memory, language, attention, reaction time, executive

functions, motor skills, visuospatial functioning and social-emotional functioning

have been documented subsequent to prenatal alcohol exposure (No authors

listed, 2000; Riley & McGee, 2005). Most individuals with FASD are not mentally

retarded although lower IQ scores are commonly seen (Riley & McGee, 2005).

Verbal learning is one area that consistent deficits are seen though non-verbal

learning has also been studied. However, memory (especially implicit) in

individuals with FASD is relatively spared (No authors listed, 2000). Attentional

deficits make FASD subjects likely to get a diagnosis of ADHD. The attentional

difficulties seen in FASD, however, are more specific. The impulsivity is more

consistently found in children with an ADHD diagnosis. During tests of reaction

times, children exposed to alcohol had slower premotor and motor reaction times

than control children (Riley & McGee, 2005). In addition to, and perhaps

because of, these cognitive dysfunctions, people with FASD have a higher

incidence of delinquency, criminality, and mental health disorders (e.g.,

depression and suicide) (Kelly, Day, & Streissguth, 2000).

The CNS structures most susceptible to in-utero alcohol exposure

(mentioned previously) have all been implied in the behaviors (e.g., attention,

learning and memory, movement, executive functions) affected by prenatal

alcohol exposure. For example, structural deficits in the corpus callosum are

related to functional deficits in verbal learning (Mattson, Schoenfeld, & Riley,

2001; Riley & McGee, 2005). Also, deficits in eye-blink conditioning are likely

rooted in cerebellar damage (Stanton & Goodlett, 1998). The link between

structural and behavioral abnormalities is indirect and weak without functional

neuroimaging techniques in a behaving individual. Furthermore, since many of

these techniques are invasive and difficult for a child to complete (i.e., they entail

not moving) not many studies have been conducted.

Nevertheless, positron emission topography (PET) was used with FAS

adults and adolescents at rest and found reduced activity in the caudate nucleus

and thalamus. As of 2001 there were no published studies using fMRI’s and

FASD subjects but a preliminary study suggested that the dorsolateral prefrontal

cortex was active in FASD subjects but not controls during a working memory

task (Mattson, Schoenfeld, & Riley, 2001). This was interpreted by the authors

as impaired processing. Due to their noninvasiveness and ability to deal with a

moving infant/child, electroencephalography (EEG) is the functional

neuroimaging technique most commonly used (with infants) in this area of study.

So far, EEG studies have been able to confirm (by reduced resting alpha wave

strength) the structural evidence that the left hemisphere is particularly

susceptible to the effects of alcohol an effect distinguishing FASD from Down’s

syndrome (No authors listed, 2000). An evoked response potential (ERP) called

P300 is delayed in the parietal cortex of children exposed to ethanol prenatally

(Mattson, Schoenfeld, & Riley, 2001). The authors of this study suggest that this

implies information processing deficits. Furthermore, alcohol early and late in

pregnancy has been associated with increased P1 and N1 latencies and

increased N2 latencies respectively, in response to visual stimuli (cited in

Slawecki, Thomas, Riley, and Ehlers, 2004).

Much of the work done with humans and animals includes massive doses

of ethanol prenatally. Indeed, much more is known about the effects of binge

drinking than moderate prenatal ethanol intake. As it is stated in one paper,

“although research has well established that heavy prenatal alcohol exposure

leads to neurobehavioral impairment, the effects of lower levels of alcohol

exposure are not as clear” (No authors listed, 2000, p 35). The goal of the

current proposal is to study how moderate prenatal alcohol affects

responsiveness to chemosensory aspects of alcohol, learning in general and

learning about alcohol (as a conditioned stimulus) at various postnatal ages.

Behavioral effects of early moderate alcohol exposure will be examined along

with possible neuroanatomical and neurochemical mechanisms in a rat model.

This animal model will generate specific hypotheses, which will be further

scrutinized using functional neuroimaging techniques (EEG) first on animals

followed by the use of human subjects.

There is a limited literature on functional neuroimaging on animals. One

article showed that ethanol exposure (6 g/kg/day for 5 days) prenatally increased

peak frequency of EEG readings in the frontal, and parietal lobes. Furthermore,

parietal N1 latencies to a tone were increased subsequent to this alcohol

exposure. The authors suggest that this may be an indicator of attention deficits

(Slawecki, Thomas, Riley, and Ehlers, 2004). This particular study did not

attempt to relate the EEG/ERP readings to a behavioral measure directly.

Hyperactivity was also measured but outside of the EEG techniques. Animal

models of prenatal alcohol exposure have largely converged on the same

findings as humans in terms of the structural brain abnormalities, behavioral

deficits and even facial dysmorphis. Using neurophysiological measures (such

as ERP) in conjunction with an alleged behavioral deficiency would, however, be

a way to solidify the suggestion that certain brain deficits are in fact related to

behavioral abnormalities. The use of exposure to ethanol’s chemosensory cues

(subsequent to prenatal exposure) concurrently with ERP techniques would be a

tremendous tool. Additionally, the present proposal plans to look at the effect of

prenatal alcohol exposure on postnatal learning in general and learning about

ethanol.

A quick look at the search “prenatal alcohol AND learning” reveals that

much work has been done on the effect of prenatal ethanol exposure on

postnatal learning. Spatial memory deficits are a very common area of research.

This is likely due to the consistency of these effects in both humans and rats.

Long-term potentiation is also affected by prenatal alcohol exposure. Motor

learning, such as eye-blink conditioning, also has deficits subsequent to alcohol

exposure in both humans and rats. Another consistent deficit specific to humans

was in verbal learning. It seems for verbal learning as well as in several other

paradigms that deficits tend to be in acquisition rather than memory (Riley &

McGee, 2005). Consistent with this was the finding that free recall is impaired

but not recognition; implicit memory was also spared (Mattson, Schoenfeld, and

Riley, 2001). Furthermore, reversal learning but not discrimination training is

affected by prenatal ethanol. Finally, and possibly relating to attentional deficits

in FASD individuals, habituation (as evidenced by cardiac orienting) is delayed

and slower after alcohol exposure in utero (Hunt & Phillips, 2004).

The effect of prenatal ethanol on later learning about ethanol is not as well

documented however, a related topic: the effect of prenatal ethanol on later

intake patterns has recently been reviewed (Spear & Molina, 20005). Some

studies do indeed look at the interaction of prenatal and neonatal learning about

alcohol but most use ethanol prenatally as a chemosensory cue rather than a

toxic substance (very low doses are used). This is because the focus of these

lines of work is to elucidate how prenatal learning about alcohol affects later

intake of the substance (a very important topic indeed). The present focus,

however, is to study the effect of prenatal alcohol exposure on postnatal learning

abilities. One study using pharmacologically relevant doses of ethanol prenatally

with this same aim found impairment on a passive avoidance task in the

adolescent offspring of exposed dams (Barron & Riley, 1990).

Researching possible mechanisms for alcohol-mediated structural and

functional impairments can help the future of prevention and treatment of FASD.

Cell death (necrotic or apoptotic) plays a major role in the underlying

mechanisms of damage to the alcohol-exposed fetus. Specifically, the

premature neural crest cell death may be linked to facial abnormalities of FAS.

One possible cause of the cell death could be free radicals formed by alcohol

metabolism. Free radicals break down the mitochondria of cells. In addition to

neuronal death, some cells do not properly develop or migrate (ectopic cells).

Growth factors are largely responsible for cell proliferation and are a possible

mechanism of alcohol’s effects. Astrocytes play a large role in cell migration.

Thus, if astrocyte formation is affected by alcohol, as research suggests, this

could be causing faulty cell migration. In addition to glia, alcohol also disrupts

(more specifically delays) the growth of serotonergic neurons. Serotonin and

glutamate are both involved in normal brain development and both systems are

negatively affected by alcohol. Additionally affected by alcohol are glucose

utilization, a cell adhesion molecule called L1, and gene expression.

Prevention is always a better option than treatment. Fortunately, this

disease is 100% preventable. Unfortunately, this fact has not changed incidence

of FASD drastically as the prevalence of drinking during pregnancy and even

FAS cases has increased in the last decade (No authors listed, 2000). This is

not however due to a lack of research in the area. The current proposal however

covers research areas that would have implications for treatment more so than

prevention. Much research in the area of treatment has already been done.

Experiments have found complex motor training initiated in adulthood can

alleviate some of the alcohol induced cerebellar damage (Klintsova, Goodlett, &

Greenough, 2000). Additionally, neonatal handling and enriched environments

also have beneficial effects. Pharmacologically, antioxidants, growth factor

proteins, and glutamate receptor antagonists (to name only a few) have been

shown to protect against or attenuate some of alcohols effects on the fetus

(Chen, Maier, Parnell, and West, 2003). Critical to any treatment or prevention

plan and central to the current proposal is the identification of brain structure

abnormalities with a corresponding mechanism of action.

References

Barron, S. and Riley, E. P. (1990). Passive avoidance performance following

neonatal alcohol exposure. Neurotoxicology and Teratology, 12, 135-138.

Chen, W. A., Maier, S. E., Parnell, S. E., and West, J. R. (2003). Alcohol andthe developing brain: neuroanatomical studies. Alcohol Research &Health, 27, 174-180.

Hunt, P. S., and Phillips, J. S. (2004). Postnatal binge ethanol exposure affectshabituation of the cardiac orienting response to an olfactory stimulus inpreweanling rats. Alcoholism: Clinical & Experimental Research, 28, 123-

130.

Kelly, S. J., Day, N., and Streissguth, A. P. (2000). Effects of prenatal alcoholexposure on social behavior in humans and other species.Neurotoxicology and Teratology, 22, 143-149.

Klintsova, A. Y., Goodlett, C. R., and Greenough, W. T. (2000). Therapeuticmotor training ameliorates cerebellar effects of postnatal binge alcohol.Neurotoxicology and Teratology, 22, 125-132.

Mattson, S. N., Schoenfeld, A. M. and Riley, E. P. (2001). Teratogenic effects ofalcohol on brain and behavior. Alcohol Research and Health, 25, 185-191.

No authors listed. (2000). Prenatal exposure to alcohol. Alcohol Researchand Health, 24, 32-41.

Riley, E. P., and McGee, C. L. (2005). Fetal alcohol spectrum disorders: anoverview with emphasis on changes in brain and behavior. 357-364.

Slawecki, C. J., Thomas, J. D., Riley, E. P., and Ehlers, C. L. Neurophysiologicconsequences of neonatal ethanol exposure in the rat. Alcohol, 34, 187-196

Spear, N. E., and Molina, J. C. (2005). Fetal or infantile exposure to ethanolpromotes ethanol ingestion in adolescence and adulthood: a theoreticalreview. Alcoholism: Clinical and Experimental Research, 29, 909-929.

Stanton, M. E., and Goodlett, C. R. (1998). Neonatal ethanol exposure impairseyeblink conditioning in weanling rats. Alcoholism: Clinical &Experimental Research, 22, 270.