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Prenatal malnutrition and development of the brain

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Neuroscience and Biobehavioral Reviews, Vol. 17, pp. 91-128, 1993 0149-7634/93 $6.00 + .00
Printed in the U.S.A. All rights reserved. 1993 Pergamon Press Ltd.

Prenatal Malnutrition and Development

of the Brain

PETER J. M O R G A N E , *~ R O B E R T A U S T I N - L A F R A N C E , ~ JOSEPH BRONZINO,f

JOHN TONKISS,$ SOFIA DIAZ-CINTRA,§ L. CINTRA,§
T O M K E M P E R $ A N D J A N I N A R. G A L L E R $

* Worcester Foundation for Experimental Biology, Shrewsbury, MA, ~fTrinity College, Hartford, CT,
$Boston University Medical School, Boston, MA, and §Instituto de Investigaciones Biomedicas,
National University o f Mexico, Mexico City, Mexico

R e c e i v e d 17 A u g u s t 1992

MORGAN'E, P. J., R. AUSTIN-LAFRANCE, J. BRONZINO, J. TONKISS, S. DfAZ-CINTRA, L. CINTRA, T.

KEMPER AND J. R. GALLER. Prenatal malnutrition and development of the brain. NEUROSCI BIOBEHAV REV
17(1) 91-128, 1993.-In this review, we have summarized various aspects as to how prenatal protein malnutrition affects
development of the brain and have attempted to integrate several broad principles, concepts, and trends in this field in
relation to our findings and other studies of malnutrition insults. Nutrition is probably the single greatest environmental
influence both on the fetus and neonate, and plays a necessary role in the maturation and functional development of the
central nervous system. Prenatal protein malnutrition adversely affects the developing brain in numerous ways, depending
largely on its timing in relation to various developmental events in the brain and, to a lesser extent, on the type and severity
of the deprivation. Many of the effects of prenatal malnutrition are permanent, though some degree of melioration may be
produced by exposure to stimulating and enriched environments. Malnutrition exerts its effects during development, not only
during the so-called brain growth spurt period, but also during early organizational processes such as neurogenesis, cell
migration, and differentiation. Malnutrition results in a variety of minimal brain dysfunction-type syndromes and ultimately
affects attentionai processes and interactions of the organism with the environment, in particular producing functional
isolation from the environment, often leading to various types of learning disabilities. In malnutrition insult, we are dealing
with a distributed, not focal, brain pathology and various developmental failures. Quantitative assessments show distorted
relations between neurons and giia, poor formation of neuronal circuits and alterations of normal regressive events, including
cell death and axonal and dendritic pruning, resulting in modified patterns of brain organization. Malnutrition insult results
in deviations in normal age-related sequences of brain maturation, particularly affecting coordinated development of various
cell types and, ultimately, affecting the formation of neuronal circuits and the commencing of activity of neurotransmitter
systems. It is obvious that such diffuse type "lesions" can be adequately assessed only by interdisciplinary studies across a
broad range of approaches, including morphological, biochemical, neurophysiological, and behavioral analyses.

Malnutrition and developing brain Prenatal protein malnutrition Gestational malnutrition

Nutrition and brain development Selective vulnerability of brain to malnutrition
Critical periods of brain development Malnutrition and developmental retardation Pre-brain growth spurt period
Brain growth spurt period Cellular and molecular aspects of malnutrition Effect of malnutrition insult on
neurogenesis Malnutrition and cell cycle kinetics Malnutrition effects on germinal cells in brain
Neuroplasticity following malnutrition insult Malnutrition and synaptic plasticity
Malnutrition and synaptic efficacy Malnutrition effects on hippocampal formation
Malnutrition and long-term hippocampal potentiation Malnutrition and dentate gyrus kindling
GABAergic interneuronal excitability in dentate gyrus Malnutrition and sleep-waking states
Malnutrition and hippocampal theta activity Malnutrition and theta shifting activity
Malnutrition and EEG ontogeny Malnutrition effects on dendritic spines Behavioral effects of malnutrition
Malnutrition and environmental stimulation Malnutrition effects on learning and cognition

Requests for reprints should be addressed to Dr. Peter Morgane, Worcester Foundation for Experimental Biology, 222 Maple Avenue,
Shrewsbury, MA 01545.

91
92
GENERAL INTRODUCTION AND
DELINEATION OF THE PROBLEM
THE physical, chemical, and physiological development of
the brain and consequent behavior in all higher species evolves
from the continuous interaction of genetic and numerous envi-
ronmental factors. Included among the latter are nutritional
and cultural variables, as well as disease and educational op-
portunities. This review is intended to provide an updated
integration of some broad principles, concepts, and trends
related to the effects of prenatal protein malnutrition on the
developing brain. In recent years there has been a tremendous
increase in awa~eness of the importance of nutrition to brain
development and behavior, and the resultant upsurge in re-
search has now provided some partial answers to major ques-
tions in this field. Thus, it is now clear that:
1. Nutrition plays a necessary role in the maturation and
functional development of the central nervous system, be-
ing concerned directly with providing energy and nutrients
needed for development of cellular structures and various
essential metabolic systems.
2. Malnutrition adversely affects many aspects of brain devel-
opment and function.
3. Malnutrition is but one of a critical group of environmental
factors affecting the development of the nervous system.
4. Some amelioration of the effects of malnutrition can oc-
cur, depending on a host of factors, including restoration
o f an adequate diet and environmental stimulation.
5. If the malnutrition insult occurs during various critical pe-
riods of central nervous system development, either in the
pre- or postnatal period, permanent effects on brain mor-
phology, physiology, and neurochemistry are produced
that impact higher nervous system functions, particularly
in the sphere of cognitive development (200).
It is important at the outset to point out that brain develop-
ment and cognitive maturation have a reciprocal influence on
each other. As indicated by Hudspeth and Pribram (141),
biological variables are not the only factors that produce ac-
celeration or deceleration of brain development, but cognitive
experience can also stimulate brain development and further
alter the time course of cognitive development. Of the various
types of malnutrition, intrauterine or gestational malnutrition
is considerably more common in developed countries than is
postnatal malnutrition. The etiology of intrauterine malnutri-
tion is complex and appears to be related to factors altering
placental passage of nutrients, including poor placental blood
supply to the fetus, maternal malnutrition, maternal disease,
and various genetic disturbances.
It has generally been held that maturation of the central
nervous system and development of optimum behavioral
capabilities depends on three critical factors:
1. heredity, i.e., inborn or genetic directives;
2. complexity and degree of environmental stimulation; and
3. adequate and balanced nutrition (285-287).
It is also clear that the genetic directives can only be carried
out if the nutritional status of the mother is adequate and if,
after birth, a stimulating and learning-directed environment is
available to the offspring. Malnutrition is one of the principal
nongenetic factors affecting the developing brain and may
adversely alter the organism's ability to interact and cope with
its environment (162). The effects of malnutrition on the de-
veloping central nervous system have been studied mainly be-
cause of the widespread incidence of infantile malnutrition
and the considerable evidence that many of its effects are
MORGANE ET AL.
permanent and are associated with impairments of higher
mental functions, including deficits in intelligence. It is clear
that abnormalities in prenatal human brain maturation result-
ing from malnutrition can manifest themselves as curtailed
mental development and other types of developmental disabil-
ities. However, more numerous, and ultimately more impor-
tant for society, are the borderline cases, i.e., those of so-
called suboptimal brain development, generally referring to
the nonfulfillment of the entire genetic potential of the indi-
vidual (315).
In our rat models we have studied the effects of both mod-
erate and severe chronic protein malnutrition insult with em-
phasis on the prenatal period. In these studies, rats were fed
one of three diets (Teklad, Madison, WI) containing 25%
(control), 8% (moderate malnutrition), or 6% (severe malnu-
trition) casein. Females, destined for breeding, were begun
on one of the three diets 5 weeks prior to mating and were
maintained on the respective diet through weaning of the lit-
ters. Breeder males were fed one of the diets for 1 week prior
to mating and were maintained on the respective diet during
the mating period. The composition of each of the diets has
been detailed in our previous studies (113,202,276). In our
early studies, litters were maintained on the diet of the mother
throughout life to examine aspects of combined pre- and post-
natal (i.e., concurrent) protein malnutrition (200,202). Con-
currently malnourished animals were designated 25/25, 8/8,
or 6/6 to indicate both the pre- and postnatal dietary regimen.
In our more recent studies, we have focused on the effects of
a purely prenatal protein malnutrition insult. This is accom-
plished by cross-fostering pups at birth from mothers fed the
6°10 casein diet to foster mothers fed the control (25%) diet.
These animals are designated 6/25, again to indicate both the
pre- and postnatal dietary condition. This paradigm institutes
dietary protein rehabilitation within 24-h of birth and provides
a means of delineating the effects of purely prenatal nutrition-
ally induced alterations. We have examined these 6/25 rats in
young adulthood, following a significant period of dietary
rehabilitation, using a variety of anatomical, physiological,
biochemical, and behavioral methodologies (200). In these
studies there was control of social context, i.e., litter size and
maternal care, by providing newborn protein-malnourished
rat pups with a foster mother displaying all maternal behav-
iors and by maintaining them in standard-sized litters of eight
pups. Reproductive success is not compromised in the prena-
tally malnourished animals, i.e., the diet did not affect gesta-
tional or litter parameters. We should emphasize the fact that
the mothers were protein deprived for 5 weeks prior to mating.
Hence, as noted below, the impact on fetal brain development
was more marked than that seen in purely prenatal malnutri-
tion and with considerably limited fetal brain "sparing" ef-
fects. In this regard, Zamenhof and Van Marthens (315) have
pointed out that malnutrition occurring only during preg-
nancy is considerably less harmful than malnutrition begun 1
month prior to mating and continued throughout pregnancy.
Thus, the level of maternal nutrient reserves at the beginning
of pregnancy is an important factor in determining the out-
come of malnourished pregnancy. Rosso (232) has also em-
phasized that the fetal consequences of inadequate maternal
intake of energy and protein during pregnancy are strongly
influenced by the nutritional status of the mother prior to
conception. His data indicate that the only types of maternal
malnutrition that affect brain growth are chronic severe mal-
nutrition and combined prepregnancy and gestational malnu-
trition.
It is clear from our own studies, as well as an examination
PROTEIN MALNUTRITION AND DEVELOPING BRAIN 93

of the literature, that these types of malnutrition insults do
not result in an obviously abnormal anatomical shape of the
brain, focal pathologies equivalent to clear-cut definable mac-
ro- or microscopic lesions (Fig. 1), or in gross mental retarda-
tion or psychopathology, but rather in permanent suboptimal
development, including, in humans, so-called minimal brain
dysfunction-type disturbances and learning disabilities (7). In
this regard, there is a long period between the end of organo-
genesis and the end of neurogenesis during which time inter-
ference with cell proliferation results in the loss of neurons
without resulting in gross malformations of brain structures.
Overall, this type of chronic, prenatal malnutrition appears to
retard the acquisition o f certain learning tasks (277) and to
reduce the animal's ability to adapt to change (276). We will
not discuss here those gross congenital malformations, which
largely reflect extensive cell death, nor drastic changes in mi-
totic rates, or mass failures or severe mistimings of cell migra-
tion and differentiation, which represent early ontogenetic
deficits and result in gross and severe abnormalities. In this
regard, the term "malformation" has most frequently been
used to imply a gross deformity, whereas, in reality, it may
also refer to distortions in the arrangements, numbers, and
ratios of cells and circuits that can only be defined by the use
of morphometric techniques. In general, definitive pathology
within specific brain regions is not a consistent feature of
mental retardation or learning disabilities. Thus, we are con-
sidering here a "distributed" or diffuse pathology. Diffuse in-
sults, such as malnutrition, appear to damage primarily prolif-
erative cell pools in the developing brain. They tend to affect
neuronal histogenesis, in large part by delaying cell prolifera-
tion, often secondary to modifications in protein metabolism
(31). Chronic malnutrition, of the types used in our animal
studies, usually results in the partial failure of basic neuronal
organizational processes, leading to some circumscribed orga-
nizational defects and, in humans, to functional abnormalities
in the general area of altered cognitive development. Thus,
derangements resulting from chronic malnutrition insults tend
to be subtle rather than overt pathologies. They largely involve
alterations in cell genesis, in cellular architectronic arrange-
ments, and in changes in connectivity and synaptic plasticity
that can only be assessed by quantitative anatomical studies
and functional analyses (10). As discussed below, these latter
anatomical and physiological parameters of modified brain
development are best examined in brain model systems, such
as the hippocampal formation, in which much of the internal
wiring and connectivity is understood, and which have, to
a considerable extent, already been quantitatively studied in
normal animals.
Relative to the above concepts of how insults affect the
developing brain, some aspects of malformations need to be
considered. In general, considerations of "malformations" in-
volve distinguishing between a primary malformation as a
morphological defect resulting from an intrinsically abnormal
developmental process, and a secondary malformation result-
ing from the breakdown of, or interference with, a normal
developmental process. These are two separate phenomena,
with conditions resulting from inherently abnormal develop-
ment (primary malformations) being most often genetically or
chromosomally determined, while interference with a normal
developmental process (secondary malformations) often has a
partial or total environmental basis. There are several general-
izations that can be made about malformations of the brain
as defined above, and an understanding of them may be of
use in determining the processes by which the malformation
occurs. First, malformations in general represent an arrest or
slowing of development of the brain and lead to a failure or
delay of the brain to progress to the next developmental stage.
Second, malformations are usually distributed along a contin-
uous spectrum of abnormality from gross distortions to small
imperfections or deviations in the programs of development,
which, as noted, can only be assessed by quantitative morpho-
logical methods. Third, the time in prenatal development
when an insult occurs appears to be a more decisive factor in
determining the ultimate brain defect than the type of insult.
Fourth, malformed brains may or may not be small in size or
reduced in weight. Fifth, there is usually no direct relation
between the type of insult and the resulting distortion of pat-
tern or histogenic abnormality of the brain.

INSULT PATHOPHYSIOLOGY BEHAVIORAL ALTERATIONS

Malnutrition ] Molecular and biochemical "lesions"

(type, severity, in developing CNS. May be partly reversible
total duration and [ if mild and of short duration involving only pathology. [
time instituted I a limited brain component during specific
in relation to developmental period. These are largely
developmental events microstructural and biochemical
n the bra n) subeellular events.

May ultimately result in histological lesions or

marked cellular imbalances (altered cell ratios).
Produced by cellular lesions in selectively
vulnerable brain areas or by effects on
particular lines of stem cells.

FIG. 1. Schema illustrating pathophysiology of neuronal insult. As noted in text, distributed

lesions resulting from malnutrition insult are subtle and not usually directly seen at light or
electron microscopic levels. They principally involve distortions of neuron ratios, cellular
atopias due to altered migration of neurons, alterations of regressive events such as cell death,
axonal pruning, and synaptic elimination, all of which are best examined by quantitative
morphological techniques (e.g., dendritic spine counts, degree of dendritic branching, cell ratio
analyses, cell counts, etc.). These types of pathologies can only be defined by morphometric
techniques and thus represent distortions in aspects of development rather than any type of
direct focal lesion.
94
In regard to point three, above, the idea that there are
times during development when the organism is particularly
vulnerable to insult is one that underlies the entire subject of
the effects of early insults on the developing brain (200). This
relates to the general concept of "critical periods" in develop-
ment, i.e., the type and magnitude of the effects of insults
depends on the stage of brain development at the time of the
insult (10). In particular, the term critical periods refers to the
peak period of activity of specific events such as neurogenesis,
gliogenesis, cell migration, cell differentiation, etc. The con-
cept of critical periods is based on findings that developmental
and organizational processes are altered most easily during
those periods when they are proceeding most rapidly. In this
regard, brain areas tend to develop by leaps and bounds rather
than as a slow, continuous process. As noted by Scott (241),
an organizational process cannot have a critical period if it
proceeds at a uniform rate; in other words, it can be modified
as easily at one time as at any other. On the other hand,
developmental processes cannot proceed at uniform rates and
thus all developmental processes exhibit critical periods, the
duration and significance of which depend upon the rates of
change in these processes. Scott (241) has also pointed out
that the course of these rate changes takes several forms, the
most common being that the process takes place rapidly for a
short period of time and then ceases completely. This latter is
true for most embryological and early developmental pro-
cesses. A second form is that the process initially proceeds
rapidly and then falls off to a low level, but never ceases
entirely, examples being social attachment processes in higher
animals or postnatal microneurogenesis of dentate granule
cells. Finally, the organizing process can proceed intermit-
tently at elevated rates, thus allowing for the possibility of
multiple critical periods, as is the case of learning in higher
mammalian forms. Since critical period concepts tend to dom-
inate the field of malnutrition and the developing central ner-
vous system, it is of use to keep these types of concepts in
mind. An insult occurring prior to the critical period should
produce no untoward effects, while an insult occurring during
the critical period should produce effects proportional to the
speed of brain organization. Once the organizational process
is complete, the occurrence of an insult would be expected to
have minimal impact. In general, critical periods relate to the
amount of plasticity that remains in the developing system at
the end of the period, i.e., after a truly critical period, only
limited plasticity remains. This is of particular relevance to
the developing central nervous system since "once only" events
refer to the fact that following completion of these organiza-
tional processes they do not occur again, usually due to the
absence of a host of factors essential for their occurrence.
Thus, when making interspecies comparisons of the effects of
environmental insult, the timing of fetal growth events must
always be made in relation to analogous gestational times in
order for animal models to be used to assess the human condi-
tion. Generally speaking, the same insult operating at differ-
ent times results in a different perturbation of brain morphol-
ogy. Regarding the fourth point above, that malformed brains
may or may not be small in size or reduced in weight, there
may be several types of malformations produced by malnutri-
tion, including altered histo-architectural arrangements and
topologies of neurons, as well as loss of neurons or failure of
neuronal migration and differentiation. In various instances
abnormal ratios of neurons may result, leading ultimately to
distortions of either macroneuronal or microneuronal circuits.
Altered neuron/gliai relations may also produce various ab-
MORGANE ET AL.
normalities, including the failure of neurons to properly mi-
grate and assemble into functional units. This is particularly
true if there is an effect on radial glial cells. For example,
radial glia provide a mechanism for sequential stacking of
functionally distinct groups of neurons of the same lineage
into radial columns. They are assembled in the telencephalon
prior to the earliest migratory wave of postmitotic neurons
(52). The fifth point is that there is usually no direct relation
between the type of insult and the resulting distortion of pat-
tern or histogenic abnormality of the brain. Thus, many types
of insults may produce quite similar effects on the brain, the
type of vulnerability to any insult, toxic or deprivational, ap-
pearing to be closely related to the timetable of developmental
events at the time of the insult. In the case of neurotoxic
injuries, however, there are many exceptions to this rule. As
pointed out by Reuhl and Lowndes (223), some specific toxic
insults can selectively affect particular brain regions, or even
specific cell types, with relative or complete sparing of other
regions or cell groups, regardless of the time in development
during which the insult occurs. However, this does not appear
to be the case with malnutrition insults.
Nutrition is probably the single greatest environmental in-
fluence both on the fetus and the neonate (221). An adequate
supply of essential nutrients is required for the maintenance
of growth, as well as for the normal development of all physi-
ological functions. In general, an adequate supply of nutrients
is essential to the fetus for three basic processes:
1. maintenance, i.e., the chemical work or work of transport
and concentration and the mechanical work required of
each cell for its steady-state existence;
2. growth, i.e., the net formation of new cytoplasm, mem-
branes, and organdies; and
3. differentiation, i.e., the changing of composition with
characterization of specific tissues (251).
It is clear that the process of growth requires the net input of
energy for cell division and cytoplasmic accretion, and that
these requirements are supplied by chemical energy. In the
case of the developing central nervous system, Williams and
Herrup (302) have pointed out that neurons are especially
greedy cells, since in resting humans 18°70 of circulating oxy-
gen is consumed by the brain even though it comprises only
2°7o of the total body weight. Therefore, neurons represent a
very high and fixed metabolic expense, and the cost of nour-
ishing neurons and associated glial cells may well be a factor
that constrains the total size of the neuron population. It is
likely that the basic cause of developmental fetal brain dis-
turbance is ultimately malnutrition in one form or another.
Clearly, the factors that determine brain growth and matura-
tion are multiple, but all appear to affect the common path-
way of delivery of oxygen and nutrients to the developing
fetus. The factors involved in intrauterine growth retardation
can be divided into three general categories, namely fetal fac-
tors, placental factors, and maternal factors (263). Fetal fac-
tors include genetic defects, hypoxemia, and often involve
agents such as drugs and alcohol that are directly toxic to the
fetus. Placental factors include reduced size and poor placen-
tal differentiation, both clearly relatable to fetal nutritional
deprivation. Maternal factors include malnutrition, vascular
insufficiency, and various toxemias that affect placental devel-
opment and function. Small placental size appears to be a
major determinant of intrauterine growth retardation involv-
ing a reduction of intrauterine blood flow to the placenta,
thus decreasing placental transport of oxygen and nutrients to
PROTEIN MALNUTRITION AND DEVELOPING BRAIN
the fetus. As pointed out by Bassett (24), placental tissue
weight, like fetal growth, is actively maintained by the rate
of nutrient delivery into the fetal circulation. There is also
considerable evidence for a critical time window during which
the conceptus is able to bring about changes in the uterine
wall that are necessary to support the development of a larger
placenta and a subsequent higher rate of fetal growth. In
terms of protein malnutrition, decreased placental efficiency
in delivering amino acids to the fetus appears to be a major
contributing factor to fetal growth retardation. Depression of
amino acid uptake and transplacental transport of amino
acids appear to be common factors in both malnutrition and
drug effects, such as those associated with nicotine, cocaine,
etc. (237). Thus, in fetal growth retardation there is a decrease
in all dimensions of fetal growth, indicating a common cause
involved in the development of all fetal organs. Therefore,
among the factors to consider in malnutrition are the nutri-
tional status of the mother, blood flow to the uterus, and
the size and transport capabilities of the placenta (57,59,247).
Intrinsic fetal factors also influence the fetal processing and
utilization of nutrients, although these factors are not well
understood. There is little question that, of all these causes,
maternal malnutrition, resulting in reduced placental growth
and subsequent placental insufficiency, is the principal factor
in perturbed development of the fetal brain (231,233). In sum-
mary, the ultimate effect of maternal dietary insufficiency,
often associated with underdevelopment of the placenta, is
malnutrition of the fetus.
The purpose of this review is to integrate some of our
knowledge regarding nutrition and brain developmental
events, with emphasis on the effects of prenatal protein mal-
nutrition. All nutrients, to a certain extent, influence matura-
tion of the nervous system, though protein appears to be the
component most crucial for development of neurological
functions. Many amino acids are precursors of enzymes, pep-
tide hormones, and neurotransmitters and, in many instances,
are themselves neurotransmitters. They are also the precursors
of the structural proteins that are essential for growth of body
tissues, including the brain. It is, therefore, obvious that the
broad involvement of amino acids in brain functions goes
considerably beyond simple support of protein synthetic func-
tion (221).
It is also important to consider several principal factors in
the definition and interpretation of the effects of malnutri-
tion. The first of these is the type of malnutrition, i.e.,
whether the deprivation represents an inadequacy of proteins,
calories, vitamins, or trace elements, among others. The sec-
ond aspect of malnutrition insult is the period in the organ-
ism's development, either during gestation or postnatally, dur-
ing which the malnutrition insult occurs. The third aspect to
be considered is the duration of the malnutrition, while the
fourth is the degree or severity of the insult (200). It is also
necessary to examine the influence of the malnutrition insult
at the molecular, cellular, systems, and cognitive levels in or-
der to adequately interpret the underlying basis and ultimate
significance of the effects. In this way the resulting behavioral,
learning, and cognitive disorders can be better considered in
terms of underlying mechanisms.
In the human situation, in particular, it is important to
stress that malnutrition has complex biological, psychological,
and sociological aspects. Because of these various factors, pre-
dispositions to malnutrition and behavioral practices associ-
ated with malnutrition tend to be passed from one generation
to the next (76,77). Consequently, these effects become per-
95
petuated and may even be cumulative over generations. To
overcome such generational effects requires combined eco-
nomic, educational, behavioral, and nutritional rehabilitation
extending considerably beyond the single generation.
MALNUTRITIONINSULTS:SOME PRINCIPLESAND DEFINITIONS
Overwhelming evidence indicates that fetal brain develop-
ment is jeopardized in offspring of pregnant women living at
or below the poverty level, not only in underdeveloped na-
tions, but also in highly developed countries such as the
United States (86,144,169,286). Given the high incidence of
malnutrition and undernutrition among pregnant women, it
is likely that a majority of infants and children in lower socio-
economic groups have sustained various degrees of prenatal
nutritional deprivation, usually followed by continuing mal-
nutrition in infancy and early childhood. Prenatal malnutri-
tion insults have not appeared to be as dramatic in their effects
as postnatal malnutrition insults due, in part, to the overem-
phasis by Dobbing (78,80) on the largely postnatal, so-called
"brain growth spurt" (see below). Even given a limited degree
of fetal brain sparing in utero (76,77), malnutrition insult
prior to this growth spurt period (Fig. 2) can disrupt certain
basic developmental and organizational features, resulting, in
varying degrees, to regional damage and, in particular, alter-
ations in plasticity (200). Hence, prenatally malnourished
newborns are likely to exhibit marginal development, are more
susceptible to continuing nutritional insults, and are less capa-
ble of adaptive functioning in the face of adversity or stress.
Overall, their adaptive capacities appear markedly dimin-
ished, with a concomitant reduction in flexibility and range of
behavior.
Relative to the above-mentioned general principles, it
should be emphasized that the brain is a subtle aggregate of
many organ subsystems. These are exquisitely integrated and
mutually interdependent, especially during early development,
when a wide variety of events must occur within sharply de-
limited time frames in order for operative links to develop
between neurons, ultimately resulting in the formation of
functional neural circuits. Thus, failures or limitations of neu-
ron production, and disturbances or mistimings of cell migra-
tions resulting in the inability to properly populate, organize,
and laminate columns of cells, as in cortical laminae, can lead,
to various degrees, in behavioral and learning deficiencies
(7,8). Alterations in these critical, largely prenatal, develop-
mental events are far more decisive in perturbing fundamental
brain organization and development than disturbances in
whole-brain growth spurts which, in large part, are postnatal
events representing late gliogenesis and myelinization of vari-
ous axonal systems. The basic prenatal organizational pro-
cesses represent crucial "once only," highly time-limited events
that, once past, show little or no "catch-up" potential (76,77).
We would again suggest that far too much attention has been
paid to gross spurts in brain weight gain, whereas more mi-
croevents, including many interrelated and interacting cellular
and molecular changes that occur prenatally, are of critical
significance, especially when considering the potential for re-
habilitation and eventual prognosis.
Some critical definitions of terms used in this field need
further attention. The word "malnutrition" encompasses all
forms of insufficient nutrition, particularly imbalanced food
intake, and includes a general reduction of caloric intake (of-
ten referred to as "undernutrition"), of proteins, carbohy-
drates, lipids, various trace elements, and vitamins. In most
96
Rat: Pre-Brain Growth Spurt Period
~ ~
~ -
Migration and
differentiation ~
Migration and
differentiation ~
Prenatal Period
FIG. 2. Schema showing various prenatal and postnatal developmental events in both the pre-braln growth spurt period and brain growth spurt
period. Note that postnatal events of gliogenesis and myelinization, as well as proliferation of rnicroneurons, overlap the velocity curve of whole
brain rates of weight changes.
instances these deficiencies occur in combination, whereas in
experimental studies of malnutrition, a specific type of malnu-
trition can be individually examined. In this regard, we em-
phasize that in using animal models we can investigate the
effects of specific nutritional inadequacies on brain develop-
ment. Extrapolations to humans may easily overemphasize the
actual contribution of an individual factor, since experimental
conditions tend to exaggerate the role of individual factors
that may not apply to other, more complex situations (142).
Most workers in this field originally turned to animal models
to investigate the effects of early undernutrition upon learning
ability and behavior because it was thought that by using ex-
perimental animals the confounding variables associated with
human malnutrition, i.e., combinations of social and environ-
mental factors, could be controlled. However, the procedures
currently used to produce postnatal nutritional deficiency in
rodents, such as separation of the young from the mother and
increased litter size, also induce major changes in the suckling
animals' social and environmental conditions, and these
changes may play an important part in the determination of
later behaviors (99). Early malnutrition and the variables com-
pounded with it also affect subsequent body size, motor abil-
ity, behavior toward food, ability to absorb and use food, and
the ability to adjust to food derivation, and responsiveness to
environmental stimuli. Thus, the influence of early undernu-
trition may involve effects upon these other variables that can
affect learning performance, rather than on learning capacity
per se (109). It has become apparent from many studies that
malnutrition may delay, i.e., retard, the development and
MORGANE ET AL.
Rat: Brain Growth Spurt Period
Late prenatal and early postnatal gliogenesis of oligndendroglla,
followed by m gration~ d fferantiation and formation of myelin.
Late prenatal and extended postnatal neurogenests of microneurons (granule cells of ]
de_._ntatellyrus, olfactory bulb and cerebellum), followed by migration and differentiation [
Velocity curve - Rate
whole brain weight gain
Birth Postnatal Period
maturation of the brain or permanently stunt development, in
varying degrees, of its component parts (200). Accordingly,
differential vulnerability of brain areas, based mainly on the
programmed timing of maturational parameters, is a key as-
pect to consider when developing neuronal models for inter-
disciplinary studies (see below). Importantly, there are differ-
ent timings of various developmental events with respect to
trimester of pregnancy, birth, and the early postnatal period
in the brain of different species that must always be taken
into account when extrapolating from animal models to the
human (Fig. 3). Clearly, in cross-species interpretations we
must examine comparative ages, specifically in terms of matu-
rational events in the brain. Birth occurs at different stages
of brain development across different species and, therefore,
interspecies comparisons must be based on stages of brain
development and not on chronological age.
In various studies of malnutrition and its effects on the
developing central nervous system, the term "retardation" has
also frequently proven to be misleading, as it suggests a patho-
genesis in which essentially normal, but slower, processes pre-
dominate, thereby indicating a development that is retarded
but otherwise normal. Used in this sense, "retardation" sug-
gests a full "catch-up" potential when the nutritional deficit is
corrected. Often, however, particular brain processes may be
stunted to the point of cessation or irretrievably deranged by
the time their "once-only" critical period of development has
passed and, in this sense, they are not simply retarded.
In the field of malnutrition insult to the brain the word
"growth" is also frequently used in an unclear or overly gen-
PROTEIN MALNUTRITION AND DEVELOPING BRAIN 97

BIRTH
Myelinization

RAT EarlyG l i o g e ~

Macroneurogenesis

I I t
0 7 14 21 7 14 21 28 35
DAYS

BIRTH Myellnization

EarlyGllogenesis
MAN
/~'~oneuroge n~~,,~..._~
Macroneurogenesis
___../
I I I I I I I I
0 3 6 9 3 6 9 12 15
MONTHS

FIG. 3. Schema comparing major developmental events in the brain of rats and humans. Developmental events are shown in
relation to birth, thus providing information on windows of vulnerability related to specific maturational events following
prenatal and postnatal insults. Arrows on myelinization, late gliogenesis, and microneurogenesis curves indicate their continua-
tion over extended postnatal periods.

eral manner. It is clear from most of the literature in this field
that the term brain "growth" usually refers to maturational
sequences involving an orderly succession of developmental
events taking place within different brain regions. This is very
different from "growth" that basically refers only to increases
in size a n d / o r weight, which only in toto reflect differential
and regional aspects of interdependent developmental pro-
cesses, such as the acquisition and differentiation of ceils. For
example, the velocity curves of brain "growth" developed by
Dobbing (77,79,83) primarily represent rates of total brain
weight change over time. Studies of total D N A content and
cholesterol levels underlying these curves (76,77,84) give little
hint of the regional processes underlying these changes in total
brain weight.
A few additional points need to be summarized relative to
malnutrition insults to the developing brain. It is well docu-
mented that malnutrition is only one of a group of factors,
including social and intellectual deprivation, emotional stress,
and poor mother-infant relationships, that operate synergisti-
cally to inhibit brain development (23,200,289). Chronically
undernourished animals are also usually understimulated and
show altered social and behavioral maturation. The malnour-
ished animal functionally isolates itself from learning experi-
ences by essentially withdrawing from interaction with the
environment (120,162,163,165,230). Postnatal malnutrition
and environmental deprivation tend to work synergistically to
functionally isolate the animal from the physical and social
environment. Thus, it becomes behaviorally withdrawn, apa-
thetic, and shows diminished social involvement with and ex-
ploration of the environment. Concurrent malnutrition, i.e.,
in effect at the time of experimental assessment, appears to
result in overall sensory deprivation, the effects usually not
being restricted to a particular modality (23). Generally, the
animal has difficulty coping with novel situations that require
adaptive responses and accommodations to the new environ-
mental demands. Obviously, the ability to perform various
learning tasks well is dependent on the animal's capacity to
attend to the specifics of the task at hand while inhibiting
irrelevant or interfering and distracting stimuli, thus allowing
the animal to form appropriate associations and execute mo-
tor responses. While it is now recognized that appropriate
dietary restitution and environmental enrichment may, in
some cases, partially alleviate some effects of prior dietary
insult, this is true only in cases where the insult was not severe
and did not result, due to the timing of the insult, in inexora-
ble effects on neurogenesis and gliogenesis, neuron and glial
migration, and differentiation.
The use of the hippocampal formation as a model system
to examine the effects of malnutrition (see below) is of special
interest, since the hippocarnpal formation plays a major role
in attentional processes (140,175) and regulates the animals'
interaction with the environment (145). In chronic malnutri-
98
tion insult, functional isolation from the environment ap-
pears, in part, to be associated with altered attentional pro-
cesses, resulting in a relative inability to fully receive or utilize
information from the environment. Again, these attentional
processes appear to relate to hippocampal activity (140,175)
that, as indicated below, shows derangements as a result of
prenatal malnutrition insult, Undernourished animals also ex-
hibit difficulty in making adaptive responses to difficult or
stressful situations. In highly nov61 situations, malnourished
humans fall to explore their environment, showing either inhi-
bition or aimless activity (23). Many of these effects are subtle,
such that malnourished animals may function reasonably nor-
maily until the deficit is unmasked by exogenous influences
such as stress, disease, or specific pharmacological agents. In
this regard, various anatomical deficiencies related to cerebral
conical thickness and glial-to-neuron ratios measured in com-
bined pre- and postnatal malnourished animals resemble, in
several ways, deficiencies resulting from sensory deprivation
or early developmental rearing in an impoverished environ-
ment. However, Bedi and Warren (30) and Bedi and Bhide
(29) have found no statistically significant interactions be-
tween nutrition and environment for these various morpho-
logical measures. They found that environmental enrichment
in later life cannot restore the anatomical deficits or remove
the distortions known to result and persist after a period of
undernutrition during early life.
VULNERABLE PERIOD HYPOTHESIS AND BRAIN GROWTH
SPURT CONCEPTS
One central theme that continues to pervade the entire field
of malnutrition and its effects on the developing brain is the
so-called vulnerable, i.e., critical, period hypothesis originally
put forth by Dobbing (76,77,80,82). This hypothesis states
that developmental processes in the central nervous system are
more vulnerable to dietary, as well as other insults, during the
time of their most rapid growth rates, i.e., during the period
Dobbing terms the "brain growth spun." As shown by brain
growth spurt analyses, where brain weight is plotted against
age, the brains of all species grow along a sigmoid trajectory
(78). The transient, near linear phase of rapid weight increase
illustrated by such curves is defined as the brain growth spurt,
which represents the peak velocity of whole brain growth (Fig.
2). There are multiple anatomical, physiological, and neuro-
chemical events underlying the brain growth spurt that are
extraordinarily active during this dynamic developmental pe-
riod. Every region of the brain, with its distinctive collection
of histological units, follows an intricately planned and pre-
cisely timed developmental sequence. Each brain region, in-
cluding its cellular units, fiber systems, and dendritic geome-
try, passes through similar sequences of development and, in
addition, has its own developmental timetable arranged in
relation to that of other associated regions to produce an inte-
grated whole. In this sense, each brain region has its own
growth spurt period. One of the major problems with the
concept of the brain growth spurt is that velocity curves of
total brain weight gain do not reflect many pre-brain growth
spurt critical periods (Fig. 2) such as early gliogenesis, macro-
neurogensis, early glial and neuronal migrations, nor do they
encompass the multiple interrelationships of the timetables
of growth within subregions of the developing brain. Thus,
velocity curves represent a very small, though highly dramatic,
period of true growth (weight change) of the brain, but do
not encompass other critical developmental processes that are
also highly susceptible to malnutrition insult.
MORGANE ET AL.
Among rodents, rats and mice are born before the brain
growth spurt has begun, while guinea pigs have nearly com-
pleted this stage at the time of birth. Among primates, the
brain growth spurt of the rhesus monkey is somewhat earlier
than in man, being largely prenatal, which accords well with
their greater behavioral maturity at birth than that found in
humans (82,83). From the point of view of their overall brain
growth spurt patterns, animals can be divided, somewhat arbi-
trarily, into prenatal, perinatal, and postnatal brain develop-
ers. These divisions have considerable utility in experimental
malnutrition as to when to apply the deprivation in relation-
ship to the chronology of the many underlying brain develop-
mental events that both precede Cure-brain growth spurt pe-
riod) and occur during the brain growth spurt period in
different species (Fig. 3).
Though the general utility of the brain growth spurt con-
cept is quite limited, the vulnerable period hypothesis is useful
in several regards, namely:
1. It links the consequences of brain insult to the timing of
the adversity in relation to the developmental, not chrono-
logical, age of the animal.
2. It specifies developmental age on the basis of brain devel-
opment, regardless of species differences in the timing of
birth.
3. It enables different species to be categorized into prenatal,
perinatal, and postnatal brain developers based on the po-
sitions of their peak velocities of brain growth relative to
birth.
4. It gives a visual impression in each species of the propor-
tion of the brain growth spurt that is prenatal or postnatal
based on the relative area beneath the velocity curves on
either side of birth (Fig. 2).
The brain growth spurt concept is, however, misleading in
several regards, since it gives the impression, in animal models
such as the widely used rodent models, that the insult must
occur largely during the early postnatal period in order to
most drastically affect the developing brain. In other words,
it largely ignores the entire prenatal period in rats and the
early prenatal period in primates. In both rats and humans,
the brain growth spurt curve does not include the early or
middle periods of neurogenesis and neuronal migration, nor
the period of early gliogenesis (Fig. 3). These periods cannot
be ignored since this pre-brain growth spurt period (Fig. 2),
which may be referred to as the cell division growth spurt, is a
transient window of neurogenesis for the total macroneuronal
population, which is ultimately responsible for the develop-
ment of the larger circuit formations and for the basic archi-
tectronic organization of the brain.
Our view, therefore, is that the brain is variously vulnera-
ble to insults during the entire period of pre- and early postna-
tal development. Thus, there are as many growth spurts dur-
ing these developmental periods as there are parameters to
measure. To focus on a single brain growth spurt, particularly
the one largely associated with the glial/myelinization phase
related to the most marked brain weight changes, can be quite
misleading. The biggest drawback of such a focused ap-
proach, seen most strikingly in rodent research of nutritional
insults, has been the implication of the postnatal period as the
single period of maximum vulnerability to insult. In all species
so far studied, the brain growth spurt begins at the time when
most of the adult complement of macroneurons (pyramidal
and stellate neurons) has been reached, so that insults occur-
ring during the brain growth spurt period would not be ex-
PROTEIN MALNUTRITION AND DEVELOPING BRAIN
pected to dramatically affect the macroneurons or early glio-
genesis, including genesis and development of radial glia. The
brain growth spurt encompasses the period of explosive multi-
plication of late forming glia (oligodendroglia) and the laying
down of myelin, as well as a major period of microneurogen-
esis (e.g., genesis of granule cells of the hippocampal dentate
gyrus, cerebellum, and olfactory bulb). Dobbing's concept of
the brain growth spurt, for instance, fails to consider the ef-
fects of prenatal malnutrition on dentate granule cell neuro-
genesis, while our group (74) and others (147,148) have shown
that this postnatal neurogenesis may be markedly affected by
prenatal malnutrition occurring well before the brain growth
spurt period in rats. This may result from effects on dentate
granule stem cells in the primary germinal cell matrices or on
secondary germinal matrices in the dentate gyrus. In this re-
gard, Zamenhof and van Marthens (315) have pointed out
that postnatal neuronal differentiation is likely to depend on
neuronal protein content at birth. Thus, such prenatal nutri-
tional insults appear to affect postnatal neurogenesis of micro-
neurons.
To reiterate our main assertion, there are many significant
critical periods of brain development outside the brain growth
spurt period defined by Dobbing. Thus, in malnutrition stud-
ies, emphasis has tended to be placed on the brain growth
spurt period to such a degree that other critical phases of brain
development, not measurable by such gross parameters as
changes in brain weight, have been largely ignored. These
considerations also bring into consideration the concept of
"brain sparing," which now requires reassessment. Dobbing
(76,77,82), in particular, put forward the view that the brain
is "spared" during the gestational phase of development, based
largely on the notion that the body shows a proportionately
greater weight loss than the brain as a result of malnutrition.
Moreover, if brain weight is calculated as a proportion of
body weight, brain weight is reduced less than that of any
other body organ system by malnutrition insult occurring dur-
ing various prenatal periods (306). However, decreased brain
weight resulting from strictly prenatal malnutrition in the rat
is associated with a significant deficit in neuron numbers
(312,314). In our studies, utilizing a 6070 casein diet, we found
that prenatal protein malnutrition resulted in significantly
lower brain and body weights than those obtained from ani-
mals raised on either an 8070 or 25070 casein diet (222). None-
theless, as expected, neuron losses and disarrays occurring
during this prenatal period were not reflected in gross brain
weight and size. Such gross weight losses are, however, seen if
the malnutrition insult is imposed during the more explosive
postnatal glial phase associated with oligodendrogliogenesis,
myelinization, and extensive cellular differentiation. Dobbing
(76-78,82) and Davison and Dobbing (68) have argued that
cell loss in the brain may be meaningless in terms of behavioral
function and mental development, and that huge numbers of
cells would have to be lost to significantly impair function.
There is a degree of reasonableness in these types of arguments
given the extensive amount of programmed ceil death that
occurs normally during development (211,302). Dobbing's
view of the relative unimportance of cell numbers may stem
from the fact that extensive cell death occurs during normal
development, but, as noted, this should be considered in a
totally different context from disruptions of cell genesis or the
selective cell death that occurs as a result of developmental
insults. One aspect pertaining to brain cells that reinforces the
importance of their numbers is the remarkable precision of
regulation of neuron number apparent during development of
99
neuronal populations. As pointed out by Williams (301), the
number of neurons in any brain region is determined by three
main factors:
1. the duration of the proliferative period as a whole, which
may vary across brain regions from days to months;
2. the duration of the cell cycle, which varies from hours to
days and can vary among cell types within the same region
and between ceils of different regions; and
3. the total number of stem or precursor cells from which a
neuronal population is derived.
It is of special interest that ratios of interconnected cells are
also regulated in a highly precise manner by naturally occur-
ring cell death. Overall, it appears that neuron numbers are
rigorously adjusted in an interactive manner by changes in
proliferative potential and the extent of cell death (302). Natu-
ral ceil death is a programmed process that adjusts and fine-
tunes the nervous system, whereas the loss of cells resulting
from insults represents a pathological process. Since neurons
are essential elements in circuit formation and organization, it
becomes a question of deciding how much cell loss and what
type of cell loss is sufficient to impair brain function and,
ultimately, behavior. Also, cell losses may be disproportion-
ate, thereby resulting in altered ratios of neuronal types and
also glial/neuron ratios that would thereby produce various
types of imbalances in the brain, such as between interacting
neuronal systems that are chemically (i.e., transmitter) spe-
cific. These changes may then affect relations between excit-
atory and inhibitory systems in the brain. For example,
changes in degrees of inhibitory activity in the dentate gyrus,
resulting from activity of interneuronal GABAergic elements,
would be important to consider in relation to some of the
physiological findings in our studies described below. We
would emphasize here that it is well accepted that the ratio of
different types of neurons within individual nuclei and ratios
of neurons in interconnected parts of the nervous system are
important determinants of neuronal performance (153,303,
313). Based on a variety of data from several types of prenatal
insults, Rodier (226-229) has emphasized that cell loss need
not be at all massive to alter critical functions in the brain.
In the rodent, malnutrition that precedes the brain growth
spurt entails prenatal deprivation (Figs. 2, 3). According to
the predictions of Dobbing's vulnerable period hypothesis, fe-
tal malnutrition in the rat or mouse should have less of an
effect on brain development than malnutrition occurring dur-
ing lactation (256,259). However, various studies have shown
that prenatal malnutrition, i.e., occurring prior to the brain
growth spurt, has a marked effect on both body and brain
weight and, in some cases, these effects are as severe as those
resulting from malnutrition during the growth spurt period
itself (34,46). Bush and Leathwood (46) found that prenatal
protein deprivation in mice produced birth weight deficits of
40-50%, while postnatal undernutrition led to weaning weight
deficits of 50-55%, so that in terms of bodily growth restric-
tion the degrees of reduction were approximately equivalent.
The long-term effects of prenatal deprivation on body
weights, regional brain weights, and regional DNA content
were actually greater than those produced by postnatal depri-
vation. These results are of interest since they indicate that
growth restriction before the brain growth spurt may have
nearly as much effect on adult brain and body weight as mal-
nutrition during the growth spurt itself. As pointed out by
Leathwood (158), rather than there being a clear-cut vulnera-
ble period coincident with the brain growth spurt, it is also
100
apparent that vulnerability lasts from conception to the end
of the growth spurt period [see also Herschkowitz and Rossi
(139)]. Using a 6070 casein diet only during the prenatal period,
we routinely observe weight deficits of 15-20070 compared to
controls (276,279). In contrast, Stephens et al. (267), using a
protein/calorie restriction during the suckling period, found
a body weight deficit of 66% compared to controls, Smart
(253,255), in reviewing the extensive literature on the effects
of malnutrition on behavior, has actually concluded that ges-
tation is the single-most vulnerable period for the effects of
malnutrition insult on later learning and memory perfor-
mance. In any event, rather than considering only transient
windows of special vulnerability, such as the Dobbing brain
growth spurt period, it is clear that the entire gestational pe-
riod and the early postnatal period represent a continuous
period of special vulnerability to insults.
ASPECTS OF NORMAL DEVELOPMENT OF THE CENTRAL
NERVOUS SYSTEM
Before we can begin to unravel the mechanisms of develop-
mental-restricting insults on the brain, it is useful to summa-
rize some aspects of normal brain development and their regu-
lating and controlling mechanisms. Thus, to interpret the
effects of malnutrition or other developmental insults on the
brain we must have a general understanding of normal struc-
ture, which includes organization in terms of cellular patterns
of arrangement as well as embryological development, both
at the cellular and molecular levels. Many of these mecha-
nisms are incompletely understood, particularly at the molecu-
lar and cellular levels, though various recent findings are be-
ginning to shed light on this important area. Studies on
molecular mechanisms of neural development, especially in
relation to growth factors and morphogens, are clearly the
wave of the future for understanding how changes in subcellu-
lar processes impact on brain function and behavior conse-
quent to insults during development.
The manner in which the central nervous system develops,
from the earliest embryological events through fetal, neonatal,
and young adult life towards the mature adult form, is known
to be reasonably similar in principle from one species to an-
other. There are marked species differences in gross propor-
tions, such as the comparatively large forebrain with increas-
ing cephalization in primates, which are accompanied by
increasing morphological complexity. However, in general
terms the broad developmental sequence of events does not
vary fundamentally among different mammalian species.
Many of the functional properties of the cellular units of
which nervous tissue is composed are also remarkably similar
in widely differing species. Rather, the only substantial differ-
ences with regard to brain development are represented by the
timing of birth in relation to the stage of brain maturation.
In general, histogenesis of the central nervous system in all
mammals can be divided into three principal stages:
1. organogenesis;
2. neuron and glial production; and
3. differentiation of immature neurons and gila (Fig. 4).
Given that there are transient periods of vulnerability re-
lated to various phases of the cell cycle (248), it is important
in any consideration of insults to the developing brain to note
that some toxic insults directly destroy progenitor cells while
other insults, such as malnutrition, alter the proliferation rates
of neural and glial stem cells. In each case, later develop-
mental processes that depend on the prior completion of
events that have been disturbed will themselves be secondarily
MORGANE ET AL.
affected. Malnutrition appears to more markedly affect devel-
opmental brain processes that are contemporaneous with it
and also various developmental processes that follow it. This
latter is important, since there is no overt lesion or destruction
of brain structures that are already in place, though this does
not imply that microlesions, including cellular disarrays and
biochemical lesions, do not compromise the functional organi-
zation of these structures. In fact, a host of minimal brain
dysfunction, learning, and behavioral disability-type disorders
appear to be related to distributed brain pathologies not
clearly discernible as specific lesions in particular brain areas
(7,8).
In general, brain development proceeds by the temporal
unfolding of an orderly succession of events that are regulated
by processes that are both intrinsic and extrinsic to the brain.
It should be kept in mind that brain development involves an
orderly succession of events, each growing out that which
precedes it. Delay in even a few isolated neurobiological events
causes a chain reaction capable of amplifying functional er-
rors, since misdirected, mistimed, or absent developmental
cues increasingly perturb the normally ordered progression
toward maturation of the brain and development of highly
complex expressions of intelligence. This development prog-
resses regionally in a rather precisely timed series of events,
each of which becomes a critical foundation for the next devel-
opmental period. Retardation or cessation of developmental
steps thus affect the succeeding steps in development, thereby
altering the ultimate organization of the brain. We will not
attempt here to review in detail the many overlapping events
occurring in morphogenesis of the central nervous system,
since these have been elaborated in detail in many previous
studies (26,32,114,130,182,183,218,235,250). However, in or-
der to discuss and interpret how insults to the developing brain
affect various aspects of maturation and ultimate brain organ-
ization, some general orientation to central nervous system
development will be outlined.
Various studies have indicated that the matrix cells, the
precursors of neurons, show well-known patterns of prolifera-
tion kinetics (204,248). Thus, there are various peaks of neu-
ronal replication (Figs. 3, 5) such that following organogenesis
the timing of cell division in the brain becomes more regional
with loss of the synchrony of cell division that is characteristic
of earlier development (263). The nuclei of matrix cells synthe-
size DNA in the inner portions of the matrix layer and the
matrix cells move to the surface of the ventricles to undergo
mitosis. Following division of the matrix cells, many of the
daughter cells remain in the matrix layer and undergo further
proliferation, whereas others migrate out to become neurons
(248). Relative to malnutrition insult, it is important to em-
phasize that matrix or precursor cells are DNA-synthesizing
cells and need more materials and energy for cellular prolifera-
tion than do other cells in the cerebrum during these critical
stages of development. Thus, they are likely to be more sus-
ceptible to malnutrition insult.
Another developmental aspect to consider is the impor-
tance of neuron ratios. There is considerable evidence that the
ratio of different types of neurons in specific brain areas and
nuclear formations and the ratio of neurons in interconnected
parts of the nervous system are major determinants of neu-
ronal performance (153,302,303). Zamenhof (313) has re-
cently shown that combined pre- and postnatally mildly mal-
nourished rats maintain the proportionality of cell numbers in
the cerebrum and cerebellum, indicating the strong mecha-
nisms operating to preserve this proportionality. It is also of
interest that neuron death appears to play a major role in the
PROTEIN MALNUTRITION AND DEVELOPING BRAIN I01

MACRONEURONAL
NEUROGENESIS

EARLY
GLIOGENESIS

M1CRONEURONAL NEUROGENESIS
BRAIN
DEVELOPMENTAL LATE GLIOGENESIS
EVENTS
DENDRITIC GROWTH and DIFFERENTIATION

SYNAPTOGENESIS

z MYELINIZATION
z~
Maternal malnutrition can }
r~ z
result in failure to conceive or
failed implantation with
z,,~ resorption
BIRTH WEANING

EMBRYONIC FETAL PERIOD SUCKLING PERIOD POST-WEANING PERIOD ADULTHOOD

PERIOD (Period of macroneuronal (Period of microneuronal TO MATURITY IPeriod of late
(Period of histogenesis, early histogenesis, late ( Period of contin ned microneurogenesis, and late
TIME of organogenesis) gliogenesis, gliogenesis, micraneuronal microneuronal histogenesis, myelinization)
MALNUTRITION cell migration and early and oligodendroglial cellular differentiation,
INSULT differentiation) migration and synaptogenesis and
differentiation, myelinization)
myelinization and
synaptogenesis)

POTENTIAL EFFECTS Abnormalities Altered genesis of Altered genesis of Altered genesis of microneurous E.s~nlially no
of M A L N U T R I T I O N of form macroneurons and radial early microneurons, cellular differentiation, permanent
INSULT on GROSS and shape gila. Disturbances of cellular differentiation, synaptogeoesis, and effects
BRAIN and CELLULAR of brain neuronal migration synaptogenesis, late myelinization
STRUCTURE gliogenesis,
and myelinlzatlon

FIG. 4. Schema of brain developmental events in relation to time of malnutrition insult indicating potential effects of malnutrition on gross
brain and cellular organization.

precise final adjustments of ratios of interconnected neurons
(302). As noted above, the remarkable precision of regulation
of cell number apparent during the development of neuronal
populations strengthens the view that neuron number matters
a great deal in establishing normal brain function.
As opposed to most other tissues in the body, neurons in
the central nervous system cannot divide after the definitive
neuron is formed. On the other hand, most glial cells do retain
this capacity to divide throughout life. Neurons, however, are
postmitotic cells that have completed their final synthesis of
DNA and are incapable of reentering the cycle of cell division
(302). Thus, the number of neurons in different parts of the
central nervous system is, with the exception of the late divid-
ing microneurons (e.g., granule cells of the cerebellum, olfac-
tory bulb, and hippocampal dentate gyrus), apparently deter-
mined at early stages of development. Because mature neurons
have little or no capability to change type or to migrate to
other locations, they apparently cannot be regulated about
some optimum at maturity. The production and ultimate de-
ployment of neurons, accordingly, has to be done precisely
the first time as there appears to be no second opportunity
for these events to reoccur. The number of neurons that are
generated can be regulated in two ways, either by altering the
number of stem cells or by altering the number of descendants
that each stem cell produces (Figs. 6, 7). Factors that might
alter the number of stem cells operate early in neurogenesis,
whereas insults that change the number of descendants can
operate later, when the lineages are expanding, with the earlier
insults showing more drastic and marked effects. Changes in
stem cell number and descendent number may be controlled
either by mechanisms that are intrinsic to the population or by
mechanisms that are extrinsic. Intrinsic mechanisms, basically
genetic directives, are not easily altered by changing the devel-
opmental environment. Extrinsic mechanisms include the in-
fluence of the nutritive environment, hormones, and interac-
tions with other cells on the kinetics, commitment and survival
probabilities of stem cells and their descendants, as well as
the recruitment of still uncommitted cells to differentiate into
one of several potential subtypes. Importantly, glial cell num-
bers appear to be proportionally adjusted to match the neuron
populations (302).
As pointed out by Williams and Herrup (302), development
of the nervous system can be seen as occurring in three major,
overlapping phases. The first phase is the development of the
genetic nervous system, which relates to issues such as how
many neurons the stem cells are destined to produce, what
targets these cells need to survive, which ceils are programmed
to die, etc. In the second phase, the embryonic nervous system
emerges as the genetic instructions begin to interact within a
cellular milieu. The interaction of its parts then defines the
shape and size of the nervous system. Neuron numbers are
then adjusted interactively by changes in proliferative poten-
102 MORGANE ET AL.

DAYS FOLLOWING CONCEPTION BIRTH DAYS FOLLOWING BIRTIt

BRAIN AREA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 IS 16 17 18 19 20 21 I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
I I I I I l I I I t I I I I I I I I I i i ! I I I i I I i I I I I t i i i a i I I I

Hippocampal Formation

Hippocampus Proper
(PyramidalCells and
Intemeurons)
Dentate Gyrus
(Granule Cells)

Cerebral Neocortex

Lamina 1
Lamina II
Lamina Ill
Lamina IV
Lamina V
Lamina VI

Cerebellum
Granule Cells
Basket Cells

Approximate Time of Origin of Neurons in Several Brain Areas in the Rat

FIG. 5. Schema showing times of origin of neurons in hippocampal formation, cerebral neocortex, and cerebellum of the rat in relation to
postconception, birth, and postnatal periods. The open-ended tracings for dentate gyrus granule cells and cerebellar granule cells indicate
continued neurogenesis during the postnatal period.

tial and degree of programmed cell death. The third phase
of development begins when the brain becomes functionally
organized and the animal begins to deal with its surrounding
environment. During this period neuronal organization is re-
fined in terms of shape, number, and distribution, with axons
being rearranged or pruned, dendrites growing, branching or
retracting synapses being adjusted and fine-tuned, and recep-
tor densities being established in association with neurotrans-
mitter production. Relative to the development of neurotrans-
mitter systems, there is considerable evidence that, prior to
synapse formation, monoamines may be released as a type of
hormone serving as morphogenetic or developmental signals
helping to regulate neuronal proliferation, migration, differ-
entiation of dendrites and axons, synaptogenesis, and even
cell death (142). Huether (142) also notes that increased for-
mation of monoamines in the malnourished brain may repre-
sent an overall effect of monoamines during brain develop-
ment in promotion of differentiation processes. Mattson and
Hauser (181) stress that one of the main roles of neurotrans-
mitters is to regulate the formation of neural circuits. They
also appear to play a major role in synaptogenesis and adap-
tive modification of neural connections during development.
Mattson and Hauser also point out that glutamate released
from entorhinal axons plays an active role in synaptogenesis.
Alteration of neurotransmitter activity by malnutrition thus
becomes a part, not only of function in mature neuronal cir-
cuits, but also plays a morphogenic role during development.
Connections develop primarily after birth and attain their
specificity by pruning of dendritic and axonal branches,
which, having served a purpose in brain development, are then
disposed of in the programmed chronology of development.
Interestingly, as discussed below, malnutrition insult has been
shown in our studies to disrupt this normal pruning process
(71-73). Lewis (166) has hypothesized that the effects of mal-
nutrition may be to reduce the extent of normal elimination
of otherwise redundant synapses, thereby allowing restoration
of the normal synapse to neuron ratio. It thus appears likely
that so-called regressive events in brain development, such as
cell death, axonal and dendritic pruning, and synapse elimina-
tion, may all be altered by malnutrition. In our studies we
have shown that one effect of malnutrition is to prevent the
normal pruning of dendrites and dendritic spines that occurs
during postnatal development, and the studies of Lewis (166)
indicate that the same process may occur in the case of synap-
tic elimination. These processes can then be considered plastic
events aimed at ameliorating the effects of malnutrition.
Many of these types of interactions, related to environmental
interplay and degree of stimulation or enrichment, may con-
tinue throughout the life of the animal. In our studies of
prenatal protein malnutrition effects on the developing brain,
PROTEIN MALNUTRITION AND DEVELOPING BRAIN 103

NormalDiet

NormalProgenitorCell
Populationand Cell
CycleTime
Multipotential
~mbryeuicStem
Ceg

Prenatal
Malnutrition

I ReducedProgenitorCell PopulationDuring
Prolongationof Cell CycleT

Mitotic PeriodsUntil Final Cell Division

(Hypoplasia)With P'rolongalionof
Cell CycleTime
i ~

FIG. 6. Flow chart schema indicating effects of malnutrition on neurogenesis. Note delayed cell division and prolon-
gation of cell cycle time resulting in hypoplasia of neuronal cell lines and depression of neuronal (or glial) cell numbers.

we are concerned largely with the developing embryonic/fetal
nervous system. Also, the environmental interaction (third)
phase is also of special interest in relation to degrees of reha-
bilitation possible given the continued postnatal plasticity of
the central nervous system when exposed to positive environ-
mental stimulation.
A brief summary of major developmental events in the
brain is useful for interpreting the sections that follow. The
development of individual cells (neurons and gliai cells) can
generally be divided into three main phases: proliferation, mi-
gration, and differentiation, during which they acquire their
neuronal or glial phenotype and develop interneuronal and
neuronal-glial relations (Fig. 4). With respect to the issue of
cellular migration, young neurons must move according to a
rigid temporal schedule along specific pathways in order to
arrive at their final locations in synchrony with their afferent
synaptic partners (219). The importance of the time factors
cannot be emphasized strongly enough, because if migration
were slowed by insults to the brain, young neurons might
arrive at their destinations too late to recognize a specific
target fiber. As to assemblies of neuronal circuits, these de-
pend on the proliferation and differentiation of nerve cells
being closely coordinated in time and space. Normal circuit
development also depends on the formation and differentia-
tion of both nerve and gliai cells occurring in an interrelated
fashion. It is clear that a major task of the developing brain is
to produce the correct pattern of connections during a highly
limited period of time. The development of transmitter func-
tions, such as transmitter synthesizing enzymes, transport and
release mechanisms, and specific receptors, principally occurs
postmigrationally. As noted, however, some neurotransmitter
functions may develop during the predifferentiation phases
and play morphogenic roles. Nerve cell-specific proteins, such
as the proteins involved in synaptic transmission, are devel-
oped mainly during the postmigrational phase. However,
other proteins, such as the neural cell adhesion molecules,
which have been extensively studied in the past decade (88-
91), involved in the guiding of neurons and establishment of
specific cell-cell contacts, are expressed very early in the devel-
opmental process. Cell migrations and eventual postmigratory
differentiation have been shown to he mediated by complex
and coordinated interactions between special cell adhesion
molecules on cell membranes and on the extracellular matrix.
Reuhl and Lowndes (223) have shown that there is a consider-
able relevance of cell adhesion to both neurotoxicology and
to neuronal insults. As shown by Winick and Rosso (307) and
DeBassio and Kemper (69), migration of cells in the brain
is markedly delayed by different types of malnutrition. The
possibility exists that some of these effects may be mediated
by alterations in cell adhesion molecules. Finally, the time
sequence in which different neuronal and glial cell types and
brain regions are developed is of interest, since neurons and/
or regions that are more differentiated secrete factors that
guide and control the development of yet undeveloped groups
of neurons and brain regions. Factors secreted from these
regions may be in the form of proteins, peptides, and/or neu-
rotransmitters, the most well-studied factor in this respect be-
ing nerve growth factor.
104 MORGANE ET AL.

Macroneuean
Progenitor Macroneurons
Cell (Pyramidal and Stenate Cells)

NeuroblastsUndergoing ~ 0 ~ O
Mitos~'~-'-'---'l~ 0 ~ O
Neuronal Bipotential
Lineage Neuronal
Progenitor Lineage O ~ O
Cell Cell Microneuron Microneurons
Progenitor Cell (Granular Cells)

RadialGila RadialG l i ~
ProgenitorCell Cell

IA Glial
J Progenitor
Cell Type1Aslr~c.vte
O =-(3
Mitosis Lineage ~ O e
Progenitor f Type2 Aslrocyle "
Cell O ( ProgenitorCell
Type2 Aslroc?leand
Oligodendrocyle
\~ 0 ~ 0
ProgenitorCell Oligodendrocyle Oligodendrocyte
ProgenitorCell

FIG. 7. Schema of neural and glial cell lineages occurring during development of the brain. Glial cell lineages are based on the
studies of Cameron and Rakic (52).

MALNUTRITION AND ITS RELATION TO DEVELOPMENTAL
EVENTS IN THE BRAIN
Nutritional inadequacies affect the brain's organizational
capacities and eventual functional capabilities in several ways.
First, the fundamental organizational play may be disrupted
in varying degrees such that subtle alterations in topology and
altered cell ratios lead to failures to properly organize the
brain into a functionally integrated whole. Altered morpho-
logical organization, changes in biochemical parameters, and
physiological dysfunctions occur, ultimately leading to disrup-
tions in several aspects of behavior. Secondly, since highly
programmed overlapping and sequential developmental pro-
cesses occur in all brain areas, distortions in their orderly
development resulting from malnutrition may be exacerbated
by alterations in the animal's interaction with the environment
secondary to the malnutrition. Thus, with these types of mal-
nutrition, withdrawal and nonattentiveness to environmental
stimuli may themselves impact on brain developmental pro-
cesses. Third, learning performance may be influenced by
other behavioral changes, including attentional changes and
altered emotionality, during ontogeny. Therefore, disruption
of learning, from whatever cause, early in development may
affect the animal's ultimate behavioral repertoire. There is
little question that malnutrition may produce behavioral
changes that are incompatible with the incorporation of envi-
ronmental information necessary for optimum cognitive
growth and the development of social maturity. As outlined
by Montagu (196), social malnutrition, including functional
isolation and an inadequate sensory diet, result in what he
terms sociogenic brain damage, which tends to compound
brain alterations resulting from physical malnutrition.
Normal fetal growth, including that of the central nervous
system, depends in large part on an adequate supply of essen-
tial nutrients such as amino acids, glucose, trace elements,
and vitamins, as well as an adequate supply of oxygen. The
major conduit for these nutrients is the placenta, which in the
human becomes a functional system at approximately 12
weeks of gestation (94). Any conditions that impair placental
function adversely impact the nutritional status of the fetus
(24,143,263). It is clear that factors which determine fetal
growth and development are multiple and appear to affect the
common pathway of delivery of nutrients and oxygen to the
fetus. There is evidence from both animal and human studies
that during the prenatal period there are at least two types of
intrauterine malnutrition. The first results from a poor mater-
nal diet before a n d / o r during pregnancy, while the second
type of intrauterine malnutrition results from placental insuf-
ficiency resulting in diminished transfer of nutrients to the
fetus. Rosso and Kava (233) have shown that both uterine and
placental blood perfusion are markedly reduced in rats that
were food restricted during pregnancy when compared with
well-fed normal animals. Human placenta data (143,305) sug-
gests that maternal malnutrition affects placental growth by
decreasing the DNA content, i.e., cell numbers. Morgan and
Naismith (197), Rosso (232), and Winick et al. (308) have
shown that malnutrition affects fetal growth by interfering
with cell division and/or protein synthesis. In principle, the
supply of amino acids is expected to have an important influ-
ence on protein metabolism and protein composition in the
brain, since precursor availability is often the rate-limiting
factor in metabolic reactions. Changes in amino acid levels
may be expected to have greater influences on protein meta-
PROTEIN MALNUTRITION AND DEVELOPING BRAIN
bolism of the developing brain in which metabolic controls
are not fully developed than in the fully developed mature
brain (156).
Interestingly, various types of nonnutritional, i.e., environ-
mental, insults to the brain appear to produce most of their
effects ultimately on nutritional status. For example, toxic
agents may not only be directly toxic to the fetus but also to
the placenta (237). Placento-toxicity occurring regardless of
nutritional status can thereby lead to selective fetal malnutri-
tion. If we assume normal fetal utilization of nutrients, ade-
quate fetal nutrition then depends on an adequate and bal-
anced maternal intake and a normally functioning placenta.
In human studies, Fisher (94) has found that ethanol, for
example, interferes with all three phases of fetal nutrition,
i.e., maternal supply, placental transfer (particularly amino
acid transfer), and fetal utilization. G a r n e t al. (115), Stein
and Susser (266), and Warshaw (294) have reported that ciga-
rette smoking is one of the most powerful determinants of
intrauterine growth retardation in the developed world, result-
ing in brain weight deficiencies of 150-200 g in adults. They
pointed out that the effects of cigarettes are related to de-
creased nutrient intake probably secondary to alterations in
placental blood flow.
Several key factors appear to play major roles in determin-
ing how malnutrition may adversely impact on brain develop-
ment. First and foremost is the developmental state of the
maturing brain when the insult occurs. The importance of
timing is emphasized by the fact that many types of insults
given at similar developmental time periods produce many of
the same type of brain alterations and functional abnormali-
ties, since they impact on a particular developmental event. A
second factor that underlies considerable work in this field,
particularly regarding rehabilitation of function, is the reor-
ganization of the anatomical substrates of function, i.e., on
the capacity of neuronal circuitry to rearrange itself following
neuronal insult. This restorative plasticity or capacity to adapt
by reorganizing the brain, both anatomically and functionally,
may, in part, underlie the capacity to show various degrees of
adaptive response to neuronal insult. It is clear that neuronal
insults, including malnutrition, alter the plastic capacities of
the brain, both in terms of pathway and circuit reorganization
and, especially, by changes in synaptic efficacy. Considera-
tions of neuronal plasticity are critical in interpreting the ini-
tial response to the original insult and, especially, in assessing
later potential for amelioration of functional deficits. There is
little question that altered plasticity contributes to the out-
come of nutritional imbalances on the developing central ner-
vous system. The role of neuroplasticity is not well understood
because it acts not prior to, nor together with, but only after
the nutritionally caused defect of the developing brain. At
present we have only limited ideas as to what extent plasticity
involving structural and functional rearrangements contrib-
utes to rearranging the connectivity of the brain and alters
synaptic efficacy so as to ameliorate the insult-induced de-
rangements. This is an area of considerable concern in the
case of the hippocampal model system, there neuroplasticity
in terms of fiber and circuit rearrangements and changes in
synaptic efficacy have been so well studied. This also relates
to the issue of the mechanisms through which neuronal prop-
erties and connections are modified during learning, given
that environmental stimulation may offset some of the de-
rangements resulting from prenatal malnutrition. Poor envi-
ronmental stimulation, i.e., narrowed environmental experi-
ences, also appears to slow learning-dependent synaptogenesis
105
(286). There seems to be little question that experience-
dependent synaptogenesis operates as one plausible memory
mechanism (132,138,291). This is important given the effects
of malnutrition on the hippocampal formation and the fact
that many enduring effects of prenatal malnutrition involving
learning and memory require plastic changes at the synaptic
level. As discussed below, one of the physiological findings
we have noted is a diminution in neuroanatomical and physio-
logical plasticity in the dentate gyrus after malnutrition insult
(17,41,42,61,74).
Developmental abnormalities in cell numbers, cell ratios,
failures in organization of neuronal assemblies, including ca-
pacity to form and maintain neuronal circuits, may be lost
forever following malnutrition insult. However, later changes
in synaptic efficacy, including those related to increased den-
dritic arborization, increases in dendritic spines, and altered
synaptic function may result in partial amelioration of the
effects of earlier insults to the brain. A third factor as to how
malnutrition affects the developing brain deals with interac-
tions of the brain with the environment, and this is of major
importance. For example, enforcing activity of the central
nervous system during critical periods potentiates adaptive
processes so the organism remains adaptable and flexible. In
this regard, learning is recognized as progressing in stages,
each of which becomes the foundation for the next develop-
mental period. By not responding to early stimulation, the
malnourished animal becomes less able to benefit from later
experiences. As indicated in a host of studies, a stimulating or
enriched environment, i.e., one that is socially positive and
learning-driven, may, in part, compensate for some of the
original deficits resulting from nutritional insult by altering
synaptic efficacy in systems that are not irreparably damaged
by the original insult (99,196). Van Gelder (286) has pointed
out that, as postnatal maturation progresses and the genetic
directives for neuronal development diminish, rehabilitation
becomes more difficult and, with biological development
slowing down, the environmental stimulation needed to fine-
tune the intellectual capacity of the brain assumes a dominant
importance. Malnutrition almost always occurs in a social sit-
uation where such an enriched environment is lacking and,
under such conditions, it appears that little in the way of
rehabilitation can occur. Nevertheless, the plastic capacities
of the brain appear to be one hope for partially modifying
the consequences of early malnutrition insult. The earlier the
rehabilitative attempts the better, since if they occur during
the period of late gliogenesis and microneurogenesis the best
chance at functional rehabilitation is achieved. The main rea-
son for this is that considerable microneurogenesis and some
gliogenesis continues well into the period of postnatal develop-
ment (Fig. 3). Altman (7,8) has emphasized that the microneu-
rons or granule cells appear to be the neurons of adaptation
based on evidence that interference with numbers of these
neurons during development reduces the animal's adaptive ca-
pacities in the face of novel situations and stress. He has also
noted that patterns of interaction between the developing or-
ganism and its environment are particularly affected with this
system of microneurons is incompletely developed or dam-
aged. Further, Altman (7) proposes that the frank brain pa-
thologies can be considered as damage of "hard wired" sys-
tems and relate to well-defined neurological symptoms, but
that minimal brain dysfunction-type syndromes result from
the quantitative reduction in the total population of late-
generated microneurons without any visible brain pathology.
Before discussing the several specific means by which real-
106
nutrition insult affects the developing brain, some aspects of
maturational distortions in the brain need to be considered.
As Lewis (166) has emphasized, direct knowledge of the neu-
ropathology of the undernourished human brain and also ani-
mal brains is, to a considerable extent, rudimentary, especially
at the cellular and molecular levels of analysis. Nevertheless,
certain developmental considerations help in understanding
how malnutrition insult perturbs developmental sequences in
the brain. The carefully orchestrated temporal patterns of
primitive neuronal migration into different brain areas, the
patterns of neuronal alignment, and position in laminated,
nucleated, and reticulately organized brain structures are criti-
cal elements in normal brain development. Distortions in the
coordination of these maturational processes in different
brain components appear to be a significant factor in the ef-
fects of insults on the developing brain (200).
Some general aspects of the effects of prenatal malnutri-
tion in the rat can be summarized as follows:
1. There are reduced numbers of cells in the placenta.
2. There is reduced brain cell number at birth.
3. There are reduced numbers of brain cells at weaning.
4. There is an increased response to postnatal malnutrition in
animals that were also prenatally deprived.
In general, malnutrition appears to impose a limitation on the
complexity of neuronal circuits and, thereby, presumably, on
the ultimate functional capabilities of the brain (166). The
topology of the dendritic network following rehabilitation has
been shown to be markedly different in malnourished animals
compared to that of controls (184-186). Lewis (166) notes that
the effect of previous malnutrition may be to reduce the nor-
real elimination of otherwise redundant synapses. This latter
is in line with our studies in combined pre- and postnatal
malnutrition in the nucleus raphe dorsalis (71), locus coeruleus
(72), and visual cortex (73), in which we showed a failure
of normal pruning of dendrites in malnourished animals. In
general, these findings point to the likelihood that synapse to
neuron ratios are markedly altered by malnutrition insults. In
our studies we have noted that the terminal two-thirds of the
apical dendritic tree of dentate granule cells respond idiosyn-
cratically to prenatal protein malnutrition (61,74). In our 6/
25 diet model (6070 casein diet during pregnancy with cross-
fostering of the pups at birth to mothers fed a 25070 casein
diet) study (74), we found greater effects of malnutrition on
dendritic spine number and apical dendritic branching than in
our combined pre- and postnatal malnutrition study using the
8/8 diet model (8070 casein diet during pregnancy with cross-
fostering to a mother on the same diet who gave birth on that
day) (74). In our paradigms, there is no random fostering of
all pups born on the same day from the same diet groups with
litter size standardized at eight pups. This brings into question
some aspects of restorative plastic reactivity of the outer two-
thirds of the dendritic segments of dentate granule cells given
that long periods of postnatal restoration using a 25070 casein
diet failed to ameliorate the effects of the prenatal insult. It is
of interest that the lower-order dendritic segments of dentate
granule cells (proximal third) are more resistant to protein
deprivation than higher-order segments (distal two-thirds). It
will be important in future studies to determine, as Scheibel
(239) has noted, whether combining nutritional and environ-
mental rehabilitation may, to some extent, restore the plastic
reactivity of the outer dendritic segments.
In this section, we will now summarize several main find-
ings of studies concerned with how malnutrition affects the
developing brain and develop some principles that can be de-
MORGANE ET AL.
rived from such studies. In studies of prenatal malnutrition,
Zamenhof et al. (314) found reduced cell division in all fetal
organs studied, including the brain, and similar changes were
seen in the placenta itself. It should again be stressed that the
magnitude of the effect on cell division appears to be directly
related to the rate of cell division occurring at the time the
insult is applied. Further, given the considerable misinforma-
tion in the literature regarding fetal "sparing" following mater-
hal malnutrition, Zamenhof et al. (314) also stressed that the
maternal-placental barrier in the rat is not effective in protect-
ing the fetus from the effects of maternal protein restriction.
Relative to the issue of brain cell number, several studies
are of particular interest. Animals subjected to prenatal mal-
nutrition alone have been shown to have a 15070 reduction in
total brain cell numbers at birth (304). Interestingly, animals
subjected to purely postnatal malnutrition show a similar 15-
20070 reduction in cell numbers at weaning (306). On the other
hand, animals deprived both prenatally and postnatally show
a 60070 reduction in total brain cell number by weaning (305).
Thus, the latter produces a significantly greater effect than
the sum of effects produced during either the pre- or postnatal
period of cell proliferation. It then appears that the duration
of the insult is of particular importance and that prenatally
deprived animals not only lose or fail to generate a percentage
of brain cells (approximately 15%), but that other developing
cells are apparently made more vulnerable by the prenatal
insult, so that malnutrition continued postnataily has a greater
effect than strictly postnatal malnutrition by itself. Addition-
ally, as emphasized by van Gelder (286), chronic nutritional
insufficiency in the mother before pregnancy, associated with
underweight, results in an infant at greater risk than when
malnutrition is confined only to the gestationai period, since
the mothers show decreased nutritional reserves and plasma
volumes and an inadequacy of uterine and placental per-
fusion. It is clear from these types of studies that the type
of vulnerability to an insult is closely related to the total dura-
tion of the insult, which, therefore, overlaps many timetables
of developmental events occurring during the period of the
insult.
In particular, malnutrition appears to have especially
marked effects on cell acquisition in the developing brain.
Effects so far demonstrated include:
1. distortion or lengthening of the cell generation cycle;
2. reduction o f germinal cell numbers postnatally, particu-
larly, in the secondary germinal matrices;
3. an increase in numbers of degenerate postmitotic cells in
germinal zones;
4. slightly prolonged persistence of the cerebellar external
granular layer and subependymal cell plate;
5. permanent deficits in brain cell numbers; and
6. lasting changes in cellular composition of the brain, largely
affecting ratios of different cell types, both neuronal and
glial.
Some types of structural alterations may show modest degrees
of recovery with dietary rehabilitation and environmental
stimulation, but, in general, the topological disorders tend to
remain permanent with varying degrees of behavioral alter-
ations. In humans, these have been found to be mostly in the
sphere of attention deficits and learning disabilities.
Studies of effects of dietary deprivations to date indicate
that stem cell division processes and migrating and early dif-
ferentiating neurons are especially vulnerable. There is also
limited data regarding the issue of whether particular cell
types, neurons or glia, are selectively harmed. Altman (7,8)
PROTEIN MALNUTRITION AND DEVELOPING BRAIN 107

has provided considerable evidence that microneuronal hypo-
plasia in animals serves as a model of human minimal brain
dysfunction. In his view, minimal brain dysfunction results
from the quantitative reduction in the total population of the
late generated microneurons without any clear pathological
"lesion" in the brain. The minimal brain dysfunction syn-
drome in rats associated with hippocampal microneuronal hy-
poplasia includes hyperactivity and learning disabilities (27).
Altman (7,8) points out that this may involve memory deficits,
attentional disorders, or abnormal response tendencies (e.g.,
disruption of spontaneous alternation). The syndrome of hy-
peractivity, possibly involving decreased response inhibition,
and learning disabilities suggests certain attentional problems
may underlie poor discrimination learning performance in ani-
mals with hippocampal microneuronal hypoplasia. Regarding
this hypoplasia, Lewis et al. (167), Bedi (28), and our group
(74,75) have shown that prenatal malnutrition leads to a re-
duction of granule cell number in the hippocampal dentate
gyrus. Prenatal malnutrition has been shown to significantly
prolong the length of the cell cycle of brain matrix cells (248)
(Fig. 8) and to alter the durations of specific components of
the cell cycle without markedly changing its length (168,213).
Importantly, these types of studies of effects on the cell cycle
are most likely to provide our best cellular and molecular clues
as to the fundamental derangements produced by malnutri-
tion, as well as other insults, to the developing brain. In this
same context, it is of interest that Trevarthan et al. (281) point
out that as much as 40°70 of the potential DNA content of
the brain and other vital organs may never be made in the
developing human fetus if the mother's protein intake is below
a critical level during pregnancy.
Each major process of brain development, including neu-
rogenesis and gliogenesis (Fig. 7), migration, differentiation,
synaptogenesis, myelinization, and cell death involves several
sequential steps, all of which may be to some degree vulnera-
ble to malnutrition insult (Fig. 4). As one example to illustrate
such possibilities, we may note the several developmental steps
leading to myelin formation, which is so critical to the even-
tual maturation and function of pathways in the nervous sys-
tem. As pointed out by Miller (193), although a metabolic
insult to one or more of these steps may potentially result in
permanent myelin deficits, designating a precise age of special
vulnerability is still problematic, Also, myelinization does not
occur simultaneously in various brain areas, so that the tem-
poral heterogeneity of the whole process of myelinization var-
ies across all brain structures and regions. Perhaps even more
interesting is that functional systems of the brain, rather than
brain areas or particular nuclear formations, as well as lami-
nar or reticular organized structures, involve several intercon-
nected anatomical sites that myelinate at different rates. One
or more developmental steps that culminate in myelin deposi-
tion may be affected by malnutrition. These involve glial lin-
eage cells, specifically oligodendroglial progenitor cells, the
proliferation phase of these cells, the migration and differenti-
ation phase of these ceils, the expression of myelin compo-
nents, myelin membrane production and axon ensheathment,
and, finally, the slowing and cessation of myelination related
to the number of myelin lamellae and axon diameters. All of

G I Phase
(Presynthetic Phase
approx. 7.5 hours

S Phase

Normal Diet GestationalMalnutrition

(approximately18 hour cellcycle) (approximately20.5 hour cellcycle)
FIG. 8. Schema indicating effectsof prenatal/postnatal malnutrition on cellcycle time derived largely from data of
Shimada et al. (248). This figure shows malnutrition effects as delaying cell division with prolongation of total cell
cycle time. Studies by Shimada et al. (248) showed that prenatal malnutrition significantlyextended the generation
time of matrix cellsin mice with malnutrition, resultingin a 14% prolongation of the generation time of these neuron
precursor ceils,thus resultingin a decreased production of neurons. Studies in rats by Lewis et al. 068) with malnutri-
tion extending from the 6th day of pregnancy through lactationresultedin marked prolongation of the D N A synthetic
phase of the cellcycle with concomitant shortening of the G~ phase. Cell cycle times were unchanged up to the end of
the firstweek of lifeand then significantlyprolonged from day 12 to day 22. Disappearance of the externalgranular
layer was found to be delayed and cellnumbers in both germinal layers (lateralventricularsubependymal layer and
cerebellargranular layer)were significantlyreduced.
108
these types of critical developmental events are under the con-
trol of several regulatory factors and are variously susceptible
to insults to the central nervous system. A key question in this
and other aspects of developmental events in the brain is the
possibility for rehabilitation. For example, can the entire se-
quential process of myelinization be reinstituted beyond the
particular developmental period when it normally occurs in
different neuronal pathways? As Miller (193) has pointed out,
we cannot at the present assume that all steps of arrested
development are reversible. In the case of myelinization, the
various regulating factors in the myelinization process would
have to be added as a supplement to any dietary rehabilitation
and, even then, we cannot be sure that a process involving
steps of development could be reestablished outside the special
window of opportunity period for each process. Similar com-
plex developmental steps, all potentially susceptible to malnu-
trition insult, also occur in neurogenesis, gliogenesis, neuronal
and gliai migration, as well as during aspects of neuronal and
glial differentiation. Detailed consideration of these is beyond
the scope of this review, but must be considered as part of the
overall picture.
BRAIN MODEL SYSTEMS FOR STUDIES OF INSULTS TO THE
CENTRAL NERVOUS SYSTEM
Concept o f Selective Vulnerability
The use of particular brain models to study the effects of
insults is extremely important if progress is to be made in
understanding how insults compromise the organization and
function of the central nervous system. Brain models can, for
instance, be used to provide information on how generalized
or nonspecific insults such as malnutrition affect localized
populations of neurons. In malnutrition and many other types
of neural insults, there is little doubt that the pathological
effects are "diffuse" in nature and are not represented by spe-
cifically focal centers of more intense pathologies. In the selec-
tion of brain areas for study, therefore, we need information
on the differential severity of the effects of the insult. The
areas most affected would then constitute the best candidates
for a model system. The issue of selective vulnerability arises
even within a particular brain model system, e.g., the hippo-
campal formation. For example, there is selective vulnerability
of discrete neuronal subpopulations within the hippocampal
formation during ontogeny that reflects largely the pre- or
postnatal ontogeny of the cell group (Fig. 5). Further, the
molecular bases for selective vulnerability, based on events
occurring in short time frames, and for its variations during
ontogeny are prime areas for future studies. This would per-
tain particularly to neurotrophic factors and their perturba-
tion by neuronal insults. These factors, which may be highly
susceptible to neuronal insult, drive ceils to particular fates
and are also survival and differentiation factors (190). Neu-
ronal death and inhibition of mitotic activity are not the only
expressions of vulnerability in the developing brain. Thus, the
establishment of synaptic connections, the competition for
fields of innervation by axons from different neuronal popula-
tions, and programmed elimination of excess synaptic termi-
nals are all normal growth processes that can be affected by
insults to the developing brain (295).
In large part the vulnerability of the brain results from its
complex, programmed chronology of development. Interfer-
ence with any part of this developmental sequence at the time
of its most active periods of development may produce irre-
versible effects on brain organization. For example, Bal~zs et
al. (21) have shown an abnormally high rate of cell degenera-
MORGANE ET AL.
tion occurring in the germinal layers of the brain following
combined pre- and postnatal malnutrition. Such disturbances
in cell generation from the very fountainhead of neurogenesis
clearly leaves the brain permanently deficient in cell numbers
and, ultimately, in fundamental organization and circuit for-
mation. There is little question that alterations in the pro-
grammed chronology of development relates to distortions in
the ultimate integrative organization of the central nervous
system, which, in turn, lead to abnormalities in brain function
and behavior.
Specific Brain Model Systems: Emphasis on the Hippocampal
Formation
General concepts and anatomical changes induced by mal-
nutrition. A general overview of the main features of the hip-
pocampal formation will now be summarized, as well as as-
pects of its functional organization, as a prelude to discussing
our studies on how malnutrition impacts this structure. The
hippocampal formation is a prominent archicortical structure
that comprises a major neuronal complex in the brain of all
mammals. It forms a division of the limbic system that com-
prises a large number of neural components thought to be
involved in the regulation of autonomic and somatic behaviors
and has widespread connections with many regions of the
forebrain and brainstem. Being the subject of intensive inves-
tigations in recent years at the anatomical, physiological, bio-
chemical, and behavioral levels, it has become a common
model system in which to study the consequences of a wide
variety of insults to the brain (145,200). Its role in motivation
and emotional behaviors, and especially in learning and mem-
ory processing, has implicated the hippocampal formation as
a key structure whose abnormal functioning reveals elements
in animal models akin to various forms of human minimal
brain dysfunction and mental retardation (7,8).
With respect to the question of selective brain vulnerabil-
ity, it should be noted that certain brain areas, in particular
the hippocampal formation, have been extensively studied by
our group (17,19,20,39-42,198-200) and others (28,147,148,
154,167,209) following malnutrition insult. The most produc-
tive brain regions for analysis have been those neuronal sys-
tems that are relatively well understood with regard to their
anatomical organization, including basic internal circuitry and
synaptic relationships, as well as having a favorable cytoarchi-
tecture and local circuit organization for interpreting electro-
physiological recordings (103,268). Various forms of neu-
roplasticity lend themselves especially well to analysis in the
hippocampal formation, and that is another principal reason
we use this brain area as our model system. We emphasize
that synapses in malnourished animals appear less capable of
supporting plasticity and that our studies summarized below
have the major goal of examining various forms of hippocam-
pal neuroplasticity that appear to underlie attentional pro-
cesses and relations with the external environment (145). The
hippocampal formation shows a special form of electrical
activity, i.e., theta activity, which may play a role in gating
information flow into and the processing of information in
the hippocampal formation. Several studies have indicated
that theta activity reflects a functional state of the brain that
facilitates learning and memory formation (192,195,214). Its
study in malnourished animals is, therefore, of considerable
interest. Moreover, long-term potentiation, a candidate mem-
ory storage mechanism and an example of brain plasticity,
is especially well expressed and studied in the hippocampal
formation (1,14,15,43,174,208,236).
PROTEIN MALNUTRITION AND DEVELOPING BRAIN
Because of its special anatomical organization, including
lamination of its inputs and stereotyped geometry of synaptic
relationships, the hippocampal formation offers particular ad-
vantages for physiological analysis. Thus, stratification of in-
puts to the hippocampal formation allows extracellular re-
cording techniques to be employed, particularly field potential
studies, which provide meaningful information about synaptic
function (35). Of special interest is that different principal
components of the hippocampal formation show markedly
different developmental histories with particular cell groups
showing either predominantly prenatal or postnatal neurogen-
esis. (Fig. 5). This difference in cell ontogenesis allows for
assessment of insults on particular cell groups showing maxi-
mum rates of neuro-ontogenesis at different times during the
pre- and postnatal periods. However, even here, species differ-
ences are important to keep in mind. For example, it has
recently been found that approximately 80°70 of dentate gran-
ule cells in the rhesus monkey are generated before birth, as
compared to only about 15070 in the rat (70). In contrast to
the considerable and perhaps lifelong postnatal neurogenesis
of dentate granule cells in the rat (65,265), there appears to be
less evidence for production of neurons in the dentate gyrus
of the monkey after puberty (87). Thus, although there is
some postnatal neurogenesis in the dentate gyrus in the mon-
key, it appears to be less pronounced and to occur earlier
than in the rat. An important aspect of this is that prenatal
malnutrition in primates, including humans, may more di-
rectly affect granule cell neurogenesis than in the rat, where
dentate granule cell neurogenesis occurs most heavily in the
postnatal period. With regard to the use of the hippocampal
brain model system to study insults to the brain, it should also
be emphasized that there are many cell types comprising the
hippocampal formation, even though the two principal types
are the pyramidal neurons of the hippocampus proper and
the dentate granule cells of the dentate gyrus. Interspersed
throughout the hippocampal formation are also many varie-
ties of GABAergic interneurons, classified into at least five
types according to the studies of Ribak and Seress (224). The
developmental characteristics of these GABA interneurons
have been described by Seress and Ribak (244,245), Seress et
al. (246), and Liibbers et al. (171). Considering only pyramidal
cells and dentate granule cells is clearly an inadequate and
highly limited description of the anatomical separation of
functions in the hippocampal formation (103).
The hippocampal formation is a key component of brain
circuitry linking neocortical formations and lower brainstem
areas. It lies in a strategic position to process information
derived from the polymodal association areas, including the
frontal and temporal neocortical formations, and to redistrib-
ute it to cortical and subcortical regions involved in the modu-
lation of many complex behavioral processes. Importantly,
the hippocampal formation shows a remarkable degree of de-
velopmental, adaptive and restorative plasticity, and, accord-
ingly, has been widely used as a model system to understand
the biology of neural insult, its functional consequences, and
the extent and underlying mechanisms of neural and behav-
ioral plasticity (16,220,293).
The primary internal circuitry of the hippocampal forma-
tion, the trisynaptic circuit, is based on a series of sequential
synapses that relay information through its different compo-
nents. Interneurons in the hippocampal formation, in particu-
lar several types of GABAergic basket cells (224), serve to
modulate local synaptic transmission within the structure (Fig.
9). One of the most interesting and important properties of
hippocampal circuitry is that synaptic efficacy or strength is
109
enhanced through repeated stimulation (1,12-15,25,35,43,
208,236). Within the hippocampal formation there is a deli-
cate interplay of excitatory and inhibitory processes that ap-
pear to integrate and process the information of its many
synaptic inputs. The involvement of these intrinsic, as well
as extrinsic, modulatory systems that regulate the activity of
intrinsic hippocampal circuitry in high-order cognitive pro-
cesses and their special vulnerability to a broad range of in-
sults makes them important focal points for studies of behav-
ioral and neurological insults.
Relative to the study of malnutrition and its effects on
mental development, particularly learning and memory, it is
of special interest to examine the ontogeny of electrical events
in the brain that are known to be related to information input
and information processing, e.g., ontogeny of long-term po-
tentiation and theta activity, among others, in the hippocam-
pal formation and related structures. For example, Myhrer et
al. (205) have recently reported that levels of glutaminergic
activity in the lateral entorhinal cortex are altered by differen-
tial environmental stimulation and these alterations correlate
significantly with learning and retention of a visual discrimi-
nation task. With regard to the hippocampal model system,
an important example of electrogenesis is theta activity, which
appears to play a key role in modulating hippocampal activ-
ity and is clearly related to arousal and attentional states
029,140,155,175,217,239). This becomes of special interest
since information inflow into the hippocampal formation is
strongly vigilance state dependent (18,309-311).
With respect to the vulnerability of the hippocampal for-
mation to malnutrition insult, a variety of anatomical and
behavioral alterations have been reported following malnutri-
tion insult, particularly following postnatal malnutrition in
the rat (53,54,93,154,167,168,209,249). Learning and spatial
memory deficits correlated with anatomical changes in the
hippocampal formation have been reported following com-
bined pre- and postnatal malnutrition (147,148). Of particular
interest is the study of Lewis et al. (167) showing that prenatal
malnutrition reduces the thickness of the granular cell layer
of the dentate gyrus in rats and moderately prolongs the total
cell cycle time by substantially lengthening the DNA synthetic
phase and markedly curtailing the G~ phase of the cell cycle
(Fig. 8). The cell acquisition rate in the dentate gyrus was
shown to be markedly diminished, resulting in a severe deficit
in dentate granule cell numbers by the second postnatal week.
Bedi (28) has also shown that following food restriction from
day 16 of gestation, through lactation and into the early post-
weaning period, there was a significant reduction in numbers
of dentate granule cells when the rats were examined at 70 to
220 days of age. Our group (75) has recently found a signi-
ficantly reduced thickness and area of the molecular layer,
granule cell layer, and polymorph layer of the hippocampal
dentate gyrus in prenatally protein-malnourished rats. Impor-
tantly, such changes may exert their effects by altering the
normal afferent and efferent relationships of the dentate gy-
rus, which may underlie some of the functional changes seen
in malnutrition. As reviewed below, our group has found nu-
merous physiological changes in the dentate gyrus of the hip-
pocampal formation following prenatal malnutrition. These
will now be summarized, as will the behavioral changes seen
after prenatal protein malnutrition.
Effects of protein malnutrition on electrophysiological pa-
rameters of brain function. One major focus of our studies
dealing with the effects of prenatal protein malnutrition on
the developing brain has been the assessment of changes in
synaptic plasticity in brain regions, such as the hippocampal
110 MORGANE ET AL.

MEDIAL SEPTUM
(Cholinergic Fibers)

MEDIAL SEPTUM
Feed - Forward
Inhibition
+ @
(GABA Projection
Fibers)

BASKET DENTATE
CELL GRANULE
GABA interneuron CELL

MEDIAN RAPHE

Inhibition

LOCUS
COERULEUS
(Noradrenergic
Fibers)

+ MOSSY FIBERS
CA3

FIG. 9. Schema of basic circuits in dentate gyrus showing the main input pathway Oaerforant path) from entorhinai
cortex to molecular layer of dentate gyrus where it forms synapses en passage on apical dendrites of dentate granule
cells and dendrites of basket cells (GABAergic interneurons). Feed-forward and feed-back inhibitory circuits are
indicated. The activity of GABAergic interneurons is modulated by several major extrinsic inputs from the medial
septum, median raphe, and locus coeruleus, which have been conclusively shown to synapse on cell bodies of the
GABAergic interneurons ( 101,102,105,133,134,240).

formation, that have been implicated in aspects of infor-
mation storage. Thus, we have applied electrophysiological
techniques in an effort to gain an understanding of synaptic
activity-driven modifications in the dentate gyrus of the hip-
pocampal formation and their alteration by malnutrition in-
sult. The two main types of synaptic modifications we have
examined are long-term potentiation and kindling. Various
features of long-term potentiation indicate it to be a leading
candidate for the synaptic substrate of the suspected adaptive
functions of the hippocampal neural networks. Further, due
to the basic organization of the intrinsic hippocampal cir-
cuitry, such that the GABAergic interneurons synapse on prin-
cipal cells (in this case the granule cells of the dentate gyrus),
and are themselves modulated by several major extrinsic sys-
tems (Fig. 9) from the medial septum (101,133,134,240), me-
dian raphe (102,137), and locus coeruleus (105), it is important
to examine the effects of malnutrition insult on levels of inhi-
bition on the granule cells by using paired-pulse methods.
Long-term potentiation is a long-lasting enhancement of
synaptic efficacy or "strength" induced by high-frequency
stimulation, as well as by kindling. It has received consider-
able attention as a model for memory formation since it shares
several main features with the memory process, including
rapid onset, extended duration, and further strengthening
with repetition of stimulation. Alterations of these forms of
synaptic plasticity by prenatal nutritional insult may provide
information on how this insult affects the developing brain
leading to long-term problems in attention, learning, and
memory. Because the paths of information flow in the hippo-
campal formation are altered according to the vigilance state
of the animal (309-311), it is of importance to examine degrees
of facilitation and inhibition in the hippocampal formation
using paired-pulse techniques. Relative to gate control theories
of information inflow, it is of interest to examine possibilities
that gating phenomenon, which partially underlie memory
processes during particular behavioral states, may be altered
by prenatal malnutrition insult.
Having reviewed various aspects of how malnutrition may
affect neuronal proliferation, migration, differentiation, and
cell death, we will now examine the implications of some
quantitative electrophysiological studies that examine the ram-
ifications of such altered or aberrant development on subse-
quent neural circuit function. Of particular interest, with re-
gard to the effects of malnutrition on the hippocampal
formation, are our studies indicating reduced complexity of
dendritic arborization with alterations in spine number and,
thereby, synaptic connectivity (61), and changes in neuro-
transmitter levels (202). Recently, Chen et al. (60) reported a
twofold increase in basal serotonin efflux from hippocampal
slices in prenatally malnourished rats compared to controls.
PROTEIN MALNUTRITION AND DEVELOPING BRAIN
Increased serotonin turnover in the hippocampal formation
in malnourished rats has also been found by Smart et al. (261)
and Fuenmayor and Garcia (106). The results of such studies
suggest that diet-induced alterations to structural and neuro-
chemical development should then be examined by quantita-
tive electrophysiological techniques designed to measure spe-
cific aspects of neural circuit function in these animals. We
should note here the relative paucity of electrophysiological
studies of the hippocampal formation, especially related to
prenatal malnutrition, that exist in the literature, and the fact
that the cross-laboratory interpretation of studies that do exist
is hampered to some degree by the variety of dietary para-
digms, timings of the insult, and the age and species of the
animals under study. We have applied a number of techniques
to examine diet-induced alterations to neural circuit activity
within the hippocampal formation. The following discussion
focuses on examinations utilizing several methods to assess
functions within the hippocampal formation and the possible
implications of these for behavior of the malnourished animal.
Our animal preparations are chronically implanted with
stimulating and recording electrodes at various ages from 10
postnatal days up to adulthood (Figs. 10, 11). Early studies
investigating the effects of a combined pre- and postnatal un-
dernutrition on the developing EEG (125,128) reported a 2-
3-day delay in the appearance of the high-amplitude EEG
patterns associated with sleep behaviors from its normal ap-
pearance at 10 days of age to 12 days of age (126,127). In
addition, a permanent change in the peak frequency of EEG
recorded from the sensorimotor cortex was found in malnour-
ished animals, with the peak shifting to the higher end of the
frequency window (2-4 Hz) in these animals, while this peak
remained at the low end of the frequency (0.4 Hz) in normally
nourished animals. Our power spectral studies of the develop-
ing EEG (38,198) established that the most striking effect of
combined prenatal/postnatal dietary protein deficiency was
found in the maturation of the rhythmic slow activity pattern
(theta activity) generated from hippocampal field CA1 and
the dentate gyrus during REM sleep. A typical theta rhythm
with a mean frequency of approximately 5 Hz is apparent in
the hippocampal EEG of well-nourished animals by postnatal
day 16. This peak frequency increases from approximately 5
to 7 Hz between postnatal days 16 to 30 (55,56). Spectral
analysis of the EEG obtained during REM sleep has shown
that, as normal rats mature to 45 days of age, there is a devel-
opmental shift in the peak frequency of this theta activity
from 4.4 to 6.7 Hz (38).
Our analysis of peak theta frequency measures in combined
pre- and postnatally malnourished rats indicates that the de-
velopmental shift normally associated with this measure is
markedly retarded in these animals. The degree of this retar-
dation was found to depend upon the severity of the dietary
protein restriction (Fig. 12). Thus, the peak theta frequency
obtained from animals of our 8/8 diet was found to he signifi-
cantly slower at 14 and 18 days of age, while measures ob-
tained from animals of both 6/6 and 6/25 diet groups were
found to be significantly slower at 14, 18, 22, and 30 days
(198) than those obtained from animals fed the control (25/
25) diet. These findings indicate that a protein malnutrition
insult, instituted during gestation and continued after birth
(8/8 and 6/6 diet groups), results in delaying the functional
maturation of neural systems involved in the generation and/
or modulation of hippocampal theta activity, and that the
duration of this delay is dependent upon the severity of the
dietary insult. Furthermore, since the results obtained from
rats of the 6/25 diet group were similar to those obtained
111
from rats of the 6/6 diet group, it appears that the prenatal
component of the dietary insult is the crucial period involved
in the retardation of theta frequency maturation.
Of particular interest with respect to these results is the
fact that normal maturation of peak theta frequency parallels
several critical anatomical and biochemical developments
within the hippocampal formation. For example, it is well
established that the afferent fibers that originate in the medial
septum must be functional for the generation of hippocampal
theta activity during REM sleep (116,131,157,212). Although
innervation of the hippocampus by the medial septum has
been shown to be present at birth in the rat (194), this innerva-
tion is physiologically immature and does not approach func-
tional maturity until at least postnatal day 25 (64,66,191,194).
Thus, the developmental retardation of particular EEG pat-
terns, especially theta rhythm, resulting from various forms
of malnutrition may be reflective of nutritionally related de-
lays in the functional maturation of brainstem reticular sys-
tems responsible for driving hippocampal EEG patterns, as
well as the septo-hippocampal theta-pacing system. The possi-
ble implications of delayed theta development are discussed
later in this section following examination of two further stud-
ies of nutritionally related alterations to hippocampal activity.
In an effort to quantitatively assess the effects of malnutri-
tion on the efficacy of neuronal transmission within the hippo-
campal formation, some studies have examined the ability of
malnourished animals to establish and maintain long-term po-
tentiation (LTP) (17,41,149). LTP is defined as the use-depen-
dent strengthening of synaptic transmission induced by short
periods of appropriate activity (44). Enhancement of synaptic
efficacy can be quantified by measuring the increase in the
population EPSP (an indication of the level of synaptic activa-
tion) and population spike (an indication of the degree of
population discharge) components of the extracellular field
potential (Fig. 13). Since the first description of LTP in the
hippocampal formation (36), it has been recognized as a
model of neuronal plasticity and is a leading candidate mecha-
nism for the adaptive synaptic reorganizations underlying in-
formation storage in the brain (49,85,152,271). Thus, determi-
nation of the capacity of malnourished animals to establish
and support the synaptic modifications associated with en-
hancement of synaptic efficacy and neural transmission pro-
vides significant information related to long-term learning and
memory dysfunctions reported in such animals.
The first study of the effects of malnutrition on long-term
potentiation was by Jordan and Clark (149) and employed a
50% restriction of the dietary intake to the mother during
pregnancy and lactation. They reported that, as adults, their
malnourished animals were capable of establishing potentia-
tion of the population spike component but that this potentia-
tion decayed to prepotentiation levels within 6 h, while control
animals maintained potentiation significantly beyond this pe-
riod. No information regarding the EPSP component was re-
ported in this study. In an initial acute study by our group,
Austin et al. (17) reported that anesthetized adult animals of
the 6/25 diet group responded to tetanization of the perforant
with significant enhancement of both the population EPSP
and population spike components of the dentate field re-
sponse. Enhancement of the EPSP component in 6/25 ani-
mals was found to be significantly lower than that of controls
at all time periods investigated. In addition, this measure de-
cayed to baseline levels significantly sooner in 6/25 animals
compared to controls, indicating a decreased ability to main-
tain EPSP potentiation in these prenatally malnourished ani-
mals. In recent studies, we have examined the developing ca-
112
PROTEIN MALNUTRITION AND DEVELOPING BRAIN
pability of 6/25 animals to support LTP of the perforant
path/dentate granule cell synapse in behaving animals (41,42).
In these studies, tetanizing trains were applied to the perforant
pathway and evoked field potentials were recorded from the
dorsal blade of the ipsilateral dentate gyrus at the level of the
granule cell soma. Results in 90-day-old animals indicate that
EPSP slope measures are enhanced to a significantly greater
degree in the malnourished group, while enhancement of the
population spike is delayed for several hours and remains sig-
nificantly below levels attained by controls over the period
tested. At 30 days of age, 6/25 animals show significant en-
hancement of the population EPSP, but this is accompanied
by a reduction in the population spike measure that was main-
tained over 24 h. The delayed enhancement (seen in 90-day-old
animals) and actual reduction (in 30-day-old animals) of the
population spike measure obtained from 6/25 animals in the
face of significantly enhanced population EPSP measures sug-
gests that systems modulating dentate granule cell excitability
may be differentially effected by the malnutrition insult. In
other words, for this system to show enhanced synaptic activa-
tion, as indicated by potentiation of the EPSP component of
the field potential, without subsequent enhancement of gran-
ule cell discharge, would require that the granule cells be hy-
perpolarized. This hyperpolarization could be accomplished
if tetanization of the perforant path/granule cell synapse in
6/25 animals resulted in potentiation of a population of
GABAergic interneurons that make synaptic connections with
the granule cell soma (242,243). Such potentiation of feed-
forward inhibition within the dentate gyrus has been reported
to accompany induction of LTP in well-nourished rats (152).
In this regard, it is important to note that threshold levels of
these GABAergic interneurons have been shown to be well
below the threshold for granule cell discharge (51) and, thus,
capable of affecting granule cell excitability in the absence of
granule cell activation.
To investigate the possibility that alterations in local circuit
modulation of granule cell excitability might underlie the
changes seen in LTP in malnourished animals, we have uti-
lized a paired-pulse stimulation paradigm. Application of
paired-pulse stimulations to perforant path over a range of
interpulse intervals (IPI) results in recording two extracellular
field potentials from the granule cell population. The first
response represents the basic field potential, while the second
indicates the net effect of modulatory inputs on granule cell
excitability. It has been shown in our laboratories (20,42), as
well as others (3,150,151,180,272), that by varying the IPI
over the range of 10-1500 ms, a paired-pulse index (PPI),
reflecting the effect of interneuronal modulation on granule
cell excitability, can be determined. The PPI is defined as the
relationship between the population spike amplitude obtained
as a result of the first pulse (PI) of the pair and that resulting
from the second pulse (P2) of the pair, i.e., PPI = P2 - P~
or, to present the PPI in terms of the percent change between
the two population spikes, PPI = (P2 - PI/P~) x 100. Elec-
trophysiological and pharmacological studies have established
three specific phases to this modulation. 1) An early inhibitory
phase occurring at IPIs between 10-40 ms. This inhibition is
GABA mediated, resulting from both direct activation of the
FIG. 10. Photograph of 16-day-old behaving rat taken during chronic brain recording and stimulation procedures. Stimulating electrode is in
perforant path and recording electrode is in dentate granule cell layer. These experiments involve long-term stimulation and recording of dentate
granule cell evoked field potentials to examine the ontogeny of long-term potentiation, paired-pulse inhibition and facilitation, and kindling as
the rat develops from day 15 through early adulthood. Many of these studies are carried out across the various vigilance states of active and
quiet waking, slow wave sleep, and REM sleep in order to assess vigilance state gating of information inflow into the dentate gyrus (see text).
113
interneurons by perforant path contacts (feed-forward inhibi-
tion) and activation of the interneurons by recurrent collateral
projections (feed-back inhibition) of the granule cells (Fig. 9).
2) A period of enhanced granule cell excitability at IPIs be-
tween 50 and 200 ms. The mechanisms responsible for this
effect are not well understood, though it has been demon-
strated to occur in slice preparations (210), indicating that it
depends solely on local circuitry. 3) A period of late-onset
inhibition occurring at IPIs between 300 and 1000 ms. This
late inhibitory phase appears to be mediated by a calcium-
dependent potassium conductance (207,272) and does not ap-
pear to be GABA dependent as it is insensitive to GABA
antagonists (282,283). Since this effect can be produced by
orthodromic, but not antidromic, stimulation, it has been sug-
gested that it may involve feed-forward neurons directly acti-
vated by the perforant pathway (206).
We have recently completed a paired-pulse study in adult
6/25 animals across several behavioral states (20). The data
indicate that both the magnitude and duration of interneuro-
nally mediated inhibition of granule cell excitability are in-
creased in these animals. Possible alterations underlying the
increased inhibition in malnourished rats include:
1. increased excitability of GABAergic interneurons;
2. reduction of GABA reuptake capacity; and
3. alteration to extrahippocampal inputs modulating activity
of the GABAergic interneurons.
Relative to this latter, it is possible that nutritionally related
alterations to systems modulating granule cell excitability play
a significant role in the failure of malnourished animals to
maintain LTP of the perforant path/dentate granule cell syn-
apse, and we are presently examining these possibilities.
The final model we have used to evaluate neuroplastic re-
sponse and hippocampal function following malnutrition
came about from early results of our group indicating that
malnourished animals were more susceptible to epileptiform
seizure (39,98). As a result of these earlier studies, we have
examined the susceptibility to, and development of, hippo-
campal kindling in malnourished animals. The kindling phe-
nomenon was first described by Goddard et al. (118) and re-
fers to the progressive intensification of electrographic and
behavioral responses associated with seizure activity. Kindling
is induced by the regular application of initially subconvulsive
stimulus trains to susceptible brain structures, particularly
those of the limbic forebrain. The effect has been shown to be
essentially permanent and to involve transsynaptic alterations
that result in a net increase in transmission along pathways
activated by the stimulus (269,270). Because of the enduring
nature of the changes induced by kindling, it has also been
used as a model of hippocampal plasticity (117), which may
be related to memory storage, i.e., for formation of an en-
gram or memory trace (I 19,203).
In our initial study (39) we reported that 6/25 animals
exhibit seizure thresholds significantly below those of con-
trois, indicating a greater susceptibility to epileptiform activ-
ity. In contrast, 6/25 animals were found to require more than
twice the number of kindling stimulations before developing
the generalized motor convulsions indicative of the fully kin-
 =

~ - ,,~
t..y .,

~~.: ~ ;~ ,I

114
PROTEIN MALNUTRITION AND DEVELOPING BRAIN
died state. While it may seem paradoxical that a delay in the
rate of kindling is associated with enhanced levels of excit-
atory transmission, it has been reported that depletion of nor-
epinephrine results in lowering seizure threshold and enhanc-
ing kindling rate (9,187-189). In this regard, Soto-Mayano et
al. (264) have recently reported significant elevation of norepi-
nephrine release in prenatally malnourished rats. Thus, it is
possible that the retardation in expression of the more severe
motor manifestations of kindling may be a function of ele-
vated norepinephrine levels we have found in malnourished
animals (202). Another possibility for the retardation of kin-
dling rate in malnourished rats may result from the kindling-
induced enhancement of either or both feed-forward or feed-
back inhibitory mechanisms, i.e., potentiation of GABAergic
interneuron activity (Fig. 9). Potentiation of inhibitory activ-
ity within feed-forward and feed-back systems modulating
dentate granule cell discharge is well documented from LTP
studies (50,51,92) has also been reported to accompany kin-
dling (42,178) and has been suggested as a possible mechanism
for the apparent decrease in dentate granule cell excitability
that occurs in kindling in normal animals (176,178). A strong
enhancement of inhibitory modulatory activity at the periph-
ery of the seizure focus, termed surround inhibition, would
tend to halt or retard the propagation of epileptic bursting
activity to secondary structures, such as the motor cortices
7-
5.
.sg
[-
~e
I I I I
14 18 22 30
Postnatal Age (Days)
FIG. 12. Graphs showing development of peak theta frequency dur-
ing REM sleep at days 14, 18, 22, and 30 postnatally. Peak theta
frequency measures are shown in control (25/25; solid squares), mod-
erately malnourished (8/8; shaded box), and severely malnourished
(6/6; open squares) rats. *, Indicates significant differences (p <
0.01) relative to the 25o7o (control) diet group. N = 6 at each time
point for 6, 8, and 25% casein diet animals.
FIG. I 1. Photograph of adult behaving rat undergoing long-term stimulation and recording procedures in the first limb of the hippocampal
trisynaptic circuit (perforant path to dentate granule cell pathway). Studies of long-term potentiation, paired-pulse inhibition and facilitation,
and kindling are carried out in these preparations. Note field potential record on oscilloscope screen indicating population EPSP and population
spike (see Fig. 13 indicating components of field potential).
115
and brainstem, whose participation is a necessary prerequisite
for the appearance of the more severe behavioral manifesta-
tions of generalized seizure. The possible mechanisms underly-
ing this delay in kindling development were next examined by
assessment of long-term potentiation effects and changes in
the paired-pulse index induced by kindling. In this way, we
examined malnutrition effects on neuroplastic responses by
evaluating several aspects of neural function simultaneously
as a means of determining the interrelationships between these
measures during neuroplastic reorganization.
A number of studies have reported the occurrence of po-
tentiated neuronal transmission during the course of kindling
in normal animals and have suggested that a long-term poten-
tion-like mechanism may underlie the neuroplastic alterations
in synaptic transmission seen during kindling (122,123,
176,177,269,270). Results from our analysis of dentate gran-
ule cell field potentials during perforant path kindling indi-
cated that measures of both EPSP slope and population spike
amplitude show significant potentiation in animals of both
diet groups. However, the level of this potentiation was found
to be significantly greater in animals of the 6/25 diet group
(40). Measures of both population EPSP and population spike
amplitude obtained from 6/25 animals were enhanced contin-
uously throughout the kindling period. The results of the pop-
ulation spike amplitude/EPSP slope measures obtained from
animals of the 6/25 diet group suggest that the potentiated
transmission measures reflect a hyperexcitability of the gran-
ule cell population resulting in a heightened tendency toward
the synchronized bursting activity characteristic of epileptic
discharge.
Of particular interest, with respect to these findings, is the
fact that the potentiation of both population EPSP and popu-
lation spike amplitude measures obtained from 6/25 animals
decayed to prekindling levels within 2 weeks after cessation of
kindling stimulations. Such decay was not found in animals
of the 25/25 diet group and suggests that the dietary insult
has a profound effect on mechanisms responsible for the
maintenance of the neuroplastic changes associated with the
permanence of the kindling effect in control animals. This is
important in light of previous findings of our kindling work
(39) and again suggests that the prenatal dietary insult has a
more marked effect on mechanisms responsible for the main-
tenance of enhanced synaptic transmission seen in both LTP
and kindling than on those mechanisms involved in the initial
establishment of such enhancement.
Since the initial reports of Maru et al. (179) that kindling
in normal animals results in enhanced paired-pulse depression
in the dentate gyrus, a number of investigators have utilized
paired-pulse stimulation as a technique to examine changes in
local inhibitory activity at various hippocampal sites and un-
der a number of pharmacological and pathological conditions
(2,282,283,290). For these reasons we included examination
of the paired-pulse index during the kindling paradigm. Re-
sults of these studies dearly indicate that perforant path kin-
dling results in enhanced inhibition of granule cell response to
the second pulse of a pulse pair in a continuous fashion during
the kindling process (42). The degree of this enhanced inhibi-
tion was found to be significantly greater in animals that were
prenatally protein malnourished. Additionally, these effects
116 MORGANE ET AL.

population
spike
offset

population _1
spike U
onset .Is

EPSP
onset
d population spike
amplitude

EPSP slo }e C
population
1
spike
minima
FIG. 13. Graphic representation of the evoked waveform recorded from the den-
tate granule cell population. The population EPSP slope is defined as the slope of
the line segment AI to B1. The population spike amplitude is defined as the
amplitude of the line segment C to C1. Onset and offset times are defined as the
respective turnover points A, B, C, and D.

appear to be irreversible, as evidenced by the fact that they
were obtained in mature animals following an extended period
of dietary protein rehabilitation commencing at birth.
The changes in synaptic and paired-pulse measures pre-
sented here suggest that the prenatal protein insult results in
altering the balance between excitatory and inhibitory activity
within the dentate gyrus. These alterations result in enhanced
susceptibility to seizure initiation, as evidenced by the lower
kindling threshold, while inhibiting the development of motor
manifestations and acquisition of the fully kindled state. The
more rapid rate of decay of this enhanced inhibition, observed
in animals of the 6/25 diet group following the kindling pro-
cess, suggests that these animals have a reduced capacity to
maintain the neuroplastic alterations induced by kindling. As
noted, our previous work has indicated that prenatal protein
malnutrition results in an inability to maintain long-term po-
tentiation of excitatory transmission at the level of the perfor-
ant path/granule cell synapse (17).
Taken together, the results of our electrophysiological
studies point to significant changes both in neural circuit func-
tion (paired-pulse index) and neuroplastic response (LTP and
kindling) within the hippocampal formation of malnourished
rats. In the final analysis, we need to ask what implications
these alterations have for the animal's ability to sense and
interact with its environment? Electrophysioiogy alone cannot
provide complete answers to this question. But, the results
summarized above do provide some insights, which form the
basis for developing testable hypotheses. Relative to this, hip-
pocampal theta activity is of particular interest in studies of
malnutrition due to its relationship to learning and memory
functions (155,239,288). Theta activity is apparent in the hip-
pocampal EEG during two specific behaviors, REM sleep and
active waking with voluntary movements. Hippocampal theta
activity recorded during REM sleep has been shown to be
dependent upon cholinergic innervation arising from pace-
maker cells of the medial septum (104,240). The peak fre-
quency of this cholinergic component of theta activity remains
constant during REM sleep and has been termed "tonic" theta.
In contrast, the theta activity recorded during voluntary move-
ments has been shown to be insensitive to atropine, i.e., is
noncholinergic, and the peak frequency of this theta rhythm
(phasic theta) shifts in relation to the specific activity
(129,159). Gray et ai. (129) have expressed the view that di-
rected locomotion toward a reward is associated with a 9-10
Hz frequency; consumption of the acquired reward lowers
this frequency to 6-7 Hz, and an intermediate frequency of
7.7 Hz was obtained when the animal was in the goal area
during nonreward situations. This intermediate frequency was
also seen during initial exploration of the test alley. From this,
there are sufficient reasons to hypothesize that the specific
frequency of theta activity is significant for the processing of
incoming information. This is an important consideration in
light of our findings that malnutrition affects the shifting of
theta frequency (see below).
One of the more interesting alterations we have seen in
malnourished animals relates to measures associated with
REM (theta) sleep in early postnatal development (38). These
include a significant reduction in the percent time that mal-
PROTEIN MALNUTRITION AND DEVELOPING BRAIN
nourished animals spend in REM sleep prior to weaning, a
significant reduction in the duration of individual REM sleep
episodes, and a concomitant increase in the interval between
REM sleep episodes. These findings occur at a time in devel-
opment when the animals' eyes have just opened and volun-
tary exploratory behaviors are beginning to be exhibited. If
the occurrence of REM theta, and more importantly the fre-
quency of this theta rhythm, which, as indicated above, shows
a developmental retardation in malnourished animals, are sig-
nificant to the processing of information regarding the envi-
ronment, then early interactions between the animal and its
environment may be affected as a result of the altered theta
activity. As to the relation between behavioral state and theta
frequency, we have findings indicating that such theta gating
functions are markedly altered by prenatal protein malnutri-
tion. These data indicate that, as adults, control animals (25/
25) exhibit a typical shift in peak theta frequency between
active waking with voluntary movements and REM sleep. In
contrast, animals of the 6/25 diet group fail to show a signifi-
cant shift in this peak theta frequency measure between these
two behavioral states (199,201). These results suggest that the
prenatal dietary insult alters the ability of malnourished ani-
mals to modify the frequency of hippocampal theta rhythm
to match the requirements of particular behaviors. The fact
that the dominant frequency of atropine-sensitive theta gener-
ated during REM is not different between dietary groups may
indicate that the septo-hippocampal cholinergic system re-
mains relatively unaffected in the face of the prenatal insult.
However, the large apparent difference between diet groups
with respect to the theta frequency generated during active
waking behaviors suggests that there may be alterations to
the noncholinergic theta generating system. In this regard,
Buzs~iki and Eidelberg (51) have postulated that the rhythmic
firing of GABAergic interneurons affects, in an oscillatory
manner, the excitability of the principal neurons due to the
wide ranging connections of the interneurons. This mecha-
nism could provide the basis for the local inhibition of princi-
pal neurons contributing to theta activity and, at the same
time, explain why nonrhythmic projection cells also show
phase-locking to the theta cycle. In any event, derangements
of theta shifting activity between the behavioral states of REM
and active waking could alter the neuronal gating functions
that modulate information flow into and through the hippo-
campal formation. Also of interest in this regard is the work
by Vanderwolf and Baker (284) indicating that when serotonin
synthesis is blocked in well-nourished animals by injections of
PCPA, the character of atropine-insensitive theta that accom-
panies active waking is altered. The particular alteration that
accompanies P C P A treatment is a shift in the peak frequency
observed in these animals during active waking. Thus, another
interpretation of our results is that prenatal dietary insult has
impacted the serotonergic innervation of the hippocampal for-
mation arising from the median raph¢ in a manner that pre-
vents the theta frequency shift seen in control animals. In
this regard, Blatt and Rosene (unpublished observations) have
shown a decreased serotonergic innervation of the hippocam-
pal formation in prenatally protein-malnourished rats (6/25).
On the other hand, Chen et al. (60) report an enhanced seroto-
nin release in the hippocampal formation in 6/25 animals.
We have reported the existence of significantly higher levels
of presumably GABA-mediated inhibition during nontheta
behaviors (inactive waking and slow-wave sleep behaviors) in
6/25 animals in adulthood (20). These results suggest that
the functional activity of the prenatally arising GABAergic
interneurons may be adversely impacted, either directly or
117
indirectly, by the malnutrition insult. Since the activity of the
GABAergic interneuron population of the dentate gyrus is
under significant modulation by 1) feed-back activation from
the dentate granule cells themselves, 2) extrahippocarnpal in-
puts arising from the medial septum (33,101,133,134,240), lo-
cus coeruleus (105), and median raphe (102,137), each of
which provide the basis for powerful disinhibition of granule
cell activity, and 3) by direct activation from the perforant
pathway, nutritionally induced changes in any of these inter-
actions would result in significant alterations to continued
signal transmission. It is possible that the activity of one or
more of these systems may be affected by the prenatal nutri-
tional insult, resulting in changes either to signal entry, via
gating functions, or to transmission through the hippocampal
formation. Thus, alterations in any of these modulatory sys-
tems by malnutrition insult may change the gating function
that determines information input and processing.
In summary, the results of our electrophysiological studies
to date point to an overall increase in the activity of inhibitory
systems modulating dentate granule cell activity in prenatally
malnourished animals. Among the possible explanations for
these findings are that prenatal malnutrition results in in-
creased excitability of GABAergic interneurons, alterations in
GABA reuptake mechanisms, or alterations in the activity of
extra-hippocampal modulatory inputs synapsing on GABAer-
gic interneurons. Each of these possibilities would be consis-
tent with the findings of our LTP, paired-pulse, and kindling
studies. Overall, our results indicate that malnourished ani-
mals exhibit a relative inability to maintain the long-term neu-
roplastic changes associated with LTP in well-nourished ani-
mals, and that these alterations are not reversed by dietary
rehabilitation instituted at birth. The apparent inability of
dietary rehabilitation to reverse these effects strongly indicates
that nutritionally mediated alterations that occur early in de-
velopment have far-reaching postnatal effects on the organiza-
tion and subsequent functioning of brain structures, such as
the dentate system of the hippocampal formation. Further
research efforts in this vein presently include ontogenetic stud-
ies to establish the age at which LTP can first be elicited and
maintained by malnourished animals, developmental studies
of kindling and paired-pulse responses, and interdisciplinary
studies to examine the anatomy, neurochemistry, and behav-
ior associated with these models of activity-driven neuroplas-
ticity in both the adult and developing animal.
Taken together, the results of anatomical and electrophysi-
ological studies suggest that prenatal protein malnutrition re-
suits in"
1. a relative denervation at the level of the perforant path/
dentate granule cell connection, especially in the distal two-
thirds of the granule cell dendritic plexus (61,74);
2. significant alterations in local circuit modulation of gran-
ule cell excitability (41,42); and
3. alterations in vigilance state-dependent gating of informa-
tion inflow into the hippocampal formation (20).
These factors would tend to limit hippocampal accessibility to
incoming information, and to alter processing of such infor-
mation within the hippocampal formation. The fact that mal-
nourished animals fail to shift theta frequency during atten-
tional situations lends further strength to the notion that
prenatal protein malnutrition irreversibly alters neuronal in-
teractions such that the animal cannot fully receive or appro-
priately process information arriving at the hippocampal for-
mation from polymodal sensory organization structures.
Thus, environmental information, which the well-nourished
118
animal uses to frame its responses and interactions with the
surrounding environment, may never be processed, or may be
inadequately or inappropriately processed by the malnour-
ished animal. The possible ramifications of such altered pro-
cessing are considered across a range of overt behaviors in the
following section.
Neurobehavioral approaches to the study of diffuse insults
to the brain during development. As reviewed above, there
has been considerable interest in the possible consequences of
environmental insults (e.g., malnutrition, toxicants, or drug
exposure) experienced during early development. The effects
of such insults on the brain are generally "diffuse," meaning
that the insulting agent or event does not preferentially target
any one specific area or particular aspect of growth and devel-
opment, but rather impacts a variety of developmental pro-
cesses. Study of the effects of diffuse insults, especially as
they relate to neurobehavioral development, is particularly
complex and requires the adoption of carefully conceived ap-
proaches. In rodent models, the strategies that have com-
monly been employed can be summarized as follows:
I. effects on physical maturation (e.g., incisor eruption,
emergence of fur, eye-opening, ear-opening, testes descent,
vaginal patency, etc.);
2. effects on reflex development, including acquisition of be-
havioral milestones such as startle response, pain response,
righting reflex, cliff avoidance, visual placing, etc.;
3. effects on a battery of behavioral tests ("smorgasbord" ap-
proach) in order to identify types or categories of behaviors
consistently affected by the insult;
4. use of a brain model system selected on the basis of results
from a battery of tests, to serve as a focus of, or a prelude
to, a multidisciplinary investigative approach; and
5. use of a multidisciplinary approach in order to identify
underlying causes of behavioral change.
Behavioral findings can drive the other disciplines, or findings
from other disciplines can serve to direct the focus of behav-
ioral tests.
We will begin these discussions with a general historical
overview of the use of these approaches in the field of malnu-
trition research. Early investigations examined the physical
growth and development of rat pups following malnutrition
imposed by different methods, applied at different times with
respect to development (e.g., prenatal, postnatal, or both),
and with various durations and severity. The results of these
investigations have been covered in previous reviews (81,
200,202,254) and will not be considered in detail here. It is
sufficient to note that malnutrition generally results in im-
paired growth and physical maturation, and induced delays in
reflex ontogeny. Several of these effects appear to be more
pronounced in rat pups malnourished during the suckling pe-
riod than in pups malnourished only in utero (256). Thus,
prenatal malnutrition has been shown to cause a delay in the
emergence of early developmental landmarks, e.g., unfolding
of the pinnae, whereas postnatal malnutrition retards later
developing ones, e.g., ear-opening (296). In terms of reflex
ontogeny, postnatal malnutrition has been found to signifi-
cantly delay the appearance of the righting response (where
the animal rapidly returns to all-fours after being placed on
its back), auditory startle (definite startle reaction in response
to a loud sharp noise), visual placing (reaching toward an
object when suspended by the tall and lowered close to it),
and free-fall righting (subject turns in mid-air to land on all-
fours when released from a "back-downward" position). In
contrast, it has been shown that malnutrition restricted to the
MORGANE ET AL.
gestational period does not result in significant delays in any
of these measures (256). Although prenatal malnutrition has
less obvious effects on reflex ontogeny than postnatal malnu-
trition, it is clear that this situation does not hold true for
more complex behaviors, such as learning. In an extensive
review of over 130 experiments dealing with the effects of
prenatal, early postnatal, or postweaning malnutrition on
later learning performance in rats, Smart (253,255) concluded
that gestation was the single-most vulnerable period for the
effects of malnutrition. His conclusion was reached on the
basis that performance deficits were observed in 56°70 of the
learning experiments conducted on animals with a history of
malnutrition limited to the prenatal period, while such deficits
were observed in only 32070 of the experiments conducted on
animals malnourished during the suckling period and 0070 of
tests conducted on animals malnourished in the postweaning
period.
Smart's review (255) is a useful place to begin a consider-
ation of the "smorgasbord" approach to behavioral assess-
ment (i.e., the use of a range of behavioral tests) because even
when one category of behavior is under consideration, one
may reach very different conclusions based on the results of
visual discrimination tests, spatial learning tests, active avoid-
ance tests, etc. Thus, the disadvantage of the smorgasbord
approach is that it can lead to a lack of reliability and consis-
tency among the findings from different laboratories using
different behavioral tests. Eventually, this leads to a call for
standardization of test procedures and techniques as a means
o f reaching consensus on the precise consequences o f a diffuse
neuronal insult. For example, in the later 1970s, a Collabora-
tive Behavioral Teratology Study was conceived in an attempt
to standardize screening procedures for postnatal dysfunction
following prenatal toxicant or drug exposure (45). Such an
endeavor was undertaken due to the extreme lack of consis-
tency, reliability, and sensitivity of the test methods employed
at that time for the screening of potential behavioral terato-
gens. The advantages of the smorgasbord approach become
apparent when considering early malnutrition research. While
beset with methodological problems and inconsistencies, such
as differences in mother-infant interaction between experi-
mental groups, and extra-nutritional factors associated with
the procedures used to induce the malnutrition, etc. (112,
161,216), the smorgasbord approach has been extremely use-
ful in identifying behaviors that are rather consistently im-
pacted by malnutrition during development. Consistent be-
havioral findings following developmental malnutrition
include:
1. an increased responsiveness in food or water-getting situa-
tions, when subsequently deprived (136,257,259). This may
result from an increased responsiveness to reward in gen-
eral (278);
2. an increased responsiveness (or reactivity) to painful or
aversive stimuli (100,164,172,260);
3. disruption in home-orienting behavior (95-97,107,108,
111);
4. a tendency to show greater response perseveration (95,
262,274);
5. hyperactivity in long-term measures of activity, such as in
residential mazes (22,135); and
6. increased aggression (165,273,298) and generally increased
social interaction, such as contact between individuals
(275,297-300).
Demonstrating behavioral deficits following a diffuse in-
sult (280), while extremely important, may still only provide
PROTEIN MALNUTRITION AND DEVELOPING BRAIN
somewhat limited information. Primarily, it demonstrates
that the insulting agent or event has a significant impact on
the development of a particular aspect of behavior without,
necessarily, providing detailed information on the mecha-
nism(s) underlying the deficit. While this may seem obvious,
it is surprising how few studies have attempted to go further
than simply demonstrating behavioral deficits. One recently
adopted approach to the problem has been to concentrate
upon well-established behavioral findings and to use drug
probes to help elucidate possible mechanisms. For instance,
since malnutrition increases responsiveness, or reactivity, to
aversive situations [e.g., Frankova and Barnes (100), Levitsky
and Barnes (164)] and anxiolytic drugs appear to affect neural
systems regulating aversive behavior (124), the hypothesis that
postnatal malnutrition alters later responsiveness to anxiolytic
drugs has been tested. The evidence overwhelmingly indicates
that a low protein diet during the suckling and early postwean-
ing periods generally decreases the later response of rats to
anxiolytic drugs (4-6,37). Thus, the notion that the early insult
induces long-lasting changes in those neuronal systems under-
lying emotional behavior is greatly reinforced. There is dearly
considerable work to be done to unravel the precise neurochem-
ical, neurophysiological, and/or neuroanatomical changes gen-
erating such results, but this interdisciplinary approach ap-
pears to be most promising in this regard. In a recent study
that also investigated the possibility that early malnutrition
alters later responsiveness to drugs, Cohen et al. (62) found
that a brief period of postnatal malnutrition imposed by peri-
odic separation from the dam or by rearing in abnormally
large litters of pups reduced later behavioral responses to mor-
phine- and naloxone-precipitated withdrawal. The precise ex-
pression of these effects, however, was dependent upon the
method used to impose the malnutrition and the sex of the
subject.
In addition to the smorgasbord approach, another key ap-
proach to the study of the effects of pre- and early postnatal
malnutrition on behavior has been to adopt particular brain
model systems. This approach limits the range of available
behavioral tests by focusing attention upon specific types or
categories of behavior assumed to be subserved by a particular
brain area. Although there may be many brain regions that
are impacted by malnutrition, the hippocampal formation and
the cerebellum have frequently been singled out as model sys-
tems for the study of various brain insults. Significant ana-
tomical deficits in the cerebellum generated by both pre- and
postnatal malnutrition (58,67,292) suggested that motor coor-
dination, a behavior associated with the cerebellum, is mark-
edly impaired. Although some studies have reported data in
support of the predicted motor incoordination (11,146,173)
other investigators found no evidence of motor dysfunction
following malnutrition insult (110,135,252,258). Thus, at best,
the results are inconsistent and fail to provide clear-cut evi-
dence for functional consequences of the cerebellar deficits.
Furthermore, claims of selective sensitivity of the cerebellum
to malnutrition have recently been challenged (215). Taken
together, these findings suggest that the cerebellar model sys-
tem may not be the system of choice for such studies.
As noted above, the hippocampal formation shows signifi-
cant neuroanatomical changes in response to malnutrition
(61,74,148) and exhibits marked functional alterations upon
assessment of quantitative physiological measures (17,20,41,
42,200,201), and shows effects on behaviors commonly
thought to be hippocampally mediated. In their studies of
postnatal malnutrition, Castro and Rudy (53) found that dis-
tal cue utilization in the Morris water maze, conditional spa-
119
tial discrimination, short-term memory (54), and Pavlovian
trace conditioning (234) underwent ontogenetic delays. These
findings are all consistent with retarded development of the
hippocampal formation. In our studies on prenatal protein
malnutrition, which initially focused on the behavior of the
mature rat, two findings emerged as consistent with the view
that some hippocampaUy mediated behaviors are compro-
mised. These are, impaired acquisition of a differential rein-
forcement of low rates (DRL-18 s) operant schedule (277) and
increased resistance to extinction during reversal of a pre-
viously learned alternation response on a T-maze (276). How-
ever, two other behaviors that would have been expected to
be impaired as a result of hippocampal dysfunction have been
notable for their lack of significant alteration following prena-
tal malnutrition. These are, working memory (276) and distal
cue learning in the Morris water maze (unpublished observa-
tions). We have previously considered the possibility that,
since our experimental rats experience the malnutrition insult
during the fetal period of development only, there is consider-
able time for compensatory neuronal reorganization and ad-
aptation to occur during the early postnatal and postweaning
periods (279). The view that insults occurring during gestation
may be capable of considerable functional recovery is one
which is widely held (225) and supported by experimental find-
ings. For example, prefrontal lesions given to juvenile mon-
keys produce deficits in delayed spatial responses and alterna-
tions, while similar lesions given to monkeys on gestational
day 106 fall to produce these effects (121). It is possible that
some hippocampally mediated behaviors may be capable of
near normal functional development following prenatal mal-
nutrition, e.g., distal cue learning (unpublished observations),
while others cannot, e.g., performance of DRL schedules
(277). If this is a valid hypothesis, the question which arises is
how is it that some hippocampally mediated behaviors appear
unaffected in light of significant and enduring neuroanatomi-
cad and functional changes while others are significantly im-
pacted? Possible explanations can only be tentative, but we
may hypothesize that observed alterations in the morphology
and physiology of the hippocampai formation represent a
combination of both pathological change and compensatory
neuroplastic reorganization. It then follows that apparent be-
havioral normality may occur by alterations in the organiza-
tion and, presumably, function of affected brain structures.
The implications of this for future studies are that behavioral
deficits may only be revealed if the organism is challenged or
stressed in some way. In the event that normal behavioral
function is achieved by a limited "rewiring" of certain brain
areas, or by local compensatory changes in specific neuro-
transmitter systems, these might be expected to reveal them-
selves in terms of differentially disrupted behavioral perfor-
mance when probed with various drugs. For example, a
cholinergic antagonist, such as scopolamine, may impair the
learning performance of previously malnourished rats more
than that of well-nourished rats even in the face of equivalent
performance in the two nutritional groups when no drug is
administered. Thus, a behavioral deficit may be revealed that
would otherwise go undetected. Another consideration for fu-
ture studies is the need to investigate the behavioral effects of
malnutrition continuing into the early postnatal period. Such
a procedure may serve to reduce the opportunity for restora-
tion of behavioral function and, hence, may result in a greater
impact than prenatal malnutrition alone.
An important point to reinforce is that with a diffuse neu-
ronal insult we cannot be sure that a behavioral performance
deficit alone reflects dysfunction within a single, circum-
120
scribed brain area. Behavioral performance can be influenced
by many possible factors, e.g., motivational, perceptual, cog-
nitive, etc., which may be related to varying degrees with the
functioning of numerous brain areas. Furthermore, some neu-
rophysiologicai and neuroanatomicai changes do not necessar-
ily compel behavioral dysfunction. However, the application
of multidisciplinary approaches is most useful in those situa-
tions where a behavioral deficit is found to be "consistent"
with dysfunction of a particular brain area. Here again, we
reinforce the notion of careful selection of a brain model sys-
tem as an experimental approach that lends itself to multidisci-
plinary investigation. Clearly, assessment of the same rats us-
ing techniques drawn from a number of disciplines is most
likely to reveal significant anatomical or physiological changes
that correlate significantly with the behavioral changes. Al-
though few, if any, behaviors can be localized to just one
area of the brain, correlation analyses could offer support
for hypotheses of dysfunction primarily dependent upon a
particular brain area.
PROSPECTS AND CONCLUSIONS
The findings we have reviewed and discussed clearly indi-
cate that malnutrition, especially when instituted prenatally,
is a prime nongenetic factor influencing the developing central
nervous system and, ultimately, intellectual performance. Nu-
trition appears to be one of the greatest environmental influ-
ences on the developing organism. An appropriate supply of
essential nutrients is a necessity for the maintenance of growth
in all organs, as well as for the normal development of physio-
logical functions. In humans, the disruption or distortion of
prenatal brain development due to malnutrition may result,
depending on timing and severity, in various degrees of behav-
ioral dysfunction and learning disabilities. Of particular rele-
vance for society are the borderline minimal brain dysfunc-
tion-type cases, i.e., suboptimal brain development, leading
to an inability to attain the full genetic potential of the indi-
vidual.
In examining the many effects of malnutrition insult on
the central nervous system, the developmental approach is
essential in furthering our understanding of the relationships
between alterations in the brain and behavioral outcome, par-
ticularly with regard to learning and memory functions. An
adequate understanding of these complex relationships cannot
result from any single approach but requires the integration
of various approaches in interdisciplinary projects. Obviously,
behavioral outcome is the result of the functional integration
of the anatomy, biochemistry, and electrophysiology of the
central nervous system. The brain processes underlying intelli-
gence, learning, and memory receive, integrate, and respond
to both endogenous and exogenous stimulation. These pro-
cesses, including the various neurotransmitter interactions
that facilitate their operations, derive from numerous bio-
chemical and metabolic systems in the various interacting
parts of the brain. All of these interrelated processes are af-
fected both by nutrition and by external stimulation. Accord-
ingly, studies of the effects of deficits of nutrients and/or
stimulation on them are providing new and important insights
into their impact on the behavior of the organism. Addition-
ally, these various approaches are serving as research tools for
exploring the basic mechanisms underlying various aspects of
learning, memory, and behavior. These many studies are not
only contributing to the understanding of the central nervous
system but also to the amelioration and prevention of its dys-
function resulting from malnutrition. They are also of value
MORGANE ET AL.
in developing approaches to maximize the ultimate potential
of the brain.
Continuing studies in animals and man are needed to iden-
tify and document more clearly the interacting effects between
nutrition and other environmental factors in terms of brain
development and behavior. The subtleties of these interrela-
tionships and their consequences for the individual and the
community need to be more precisely defined. In particular,
future studies will aim at identifying underlying mechanisms
and improving means of prevention as well as treatment of
the derangements. The concept of a deprived environment
must continue to enter into the equation. Thus, it is now well
accepted that malnutrition and sociocultural factors clearly
act synergistically to depress both growth and development.
Since malnourished animals are also frequently understimu-
lated, intellectual and social stimulation from the environment
are essential aspects of long-term treatment. Developmental
changes in brain potential are related not only to the structural
maturation of the various neural elements and their connec-
tions within the brain, but also to the emergence of organiza-
tional patterns that depend to a large extent on the individual's
interaction with the environment. It is thus clear that malnu-
trition does not occur in isolation but rather in a context of
social deprivation. As emphasized, it is clear there are numer-
ous similarities between nutritional and environmental depri-
vation (145). Further, malnutrition produces behavior that is
incompatible with the incorporation of environmental infor-
mation necessary for optimal cognitive growth.
When malnutrition insult affects the developing brain, the
growth program of the organism can be seriously and perma-
nently upset, with its dimensions being replanned in a perma-
nently altered, somewhat distorted form. As we have empha-
sized, the pathology of malnutrition insult does not involve
focal tissue destruction or gross deformation of the brain, but
rather various irrecoverable distortions of pattern and alter-
ations in normal age-related sequences of brain development.
Thus, distortions in the normal pattern of brain development
are detectable and affect coordinated development of cell
types and neurotransmitter systems. In view of the importance
of chronological programming in development in relation to
the ultimate integrative organization of the brain, such organi-
zational distortions rather than overt permanent lesions con-
tribute significantly to the functional impairment. In relation
to monoamine development in the organizing nervous system,
there is reason to suspect that, even prior to synapse forma-
tion, monoamines may be released as hormones, which then
act as morphogenetic or developmental signals that play a
role in cell proliferation, migration, differentiation (including
synaptogenesis), and cell death (47,48,181,211,302). From this
it seems the nutritionally deficient fetal brain may undergo
profound developmental alterations resulting from changes,
in particular, over-abundances of neurotransmitters, well
before synapses have even begun to develop (48). As to
long-term prognoses, it is important to stress that, although
it is possible that distortions in the coordinated maturation
of various differentiating functions may have lasting effects
on behavior, the plasticity of the central nervous system ap-
pears to offer various possibilities for the implementation of
rehabilitation programs. It appears likely, based on a variety
of studies, that differentiation of brain processes, both den-
drites and axons, may represent a more plastic phenomenon
than cell genesis or migration, and thus could provide one
basis for partial restoration of function following neuronal
insult.
A few basic concepts need some reiteration regarding in-
PROTEIN MALNUTRITION AND DEVELOPING BRAIN
sults to the developing brain and their ultimate effects. In
general, the nearer the period of fastest growth, the smaller
will be the stress or insult required to produce a given ultimate
defect in the adult. Further, malnutrition insult is likely to
affect only those developmental processes that are contempo-
raneous with it, as well as some that follow it. There appears
to be only limited, diffuse lesions or distortions of structures
that are already in place, though the function of these may be
partially compromised. It is clear that malnutrition has
marked effects in the immature brain that are not achievable
in the mature animal. Thus, it is known that the adult brain is
resistant to changes, for example, in its total weight, even in
the most severe malnutrition and undernutrition, provided it
has developed normally to a mature size before the insult
begins. The later in development that brain growth is retarded
before reaching adult size and weight, the more completely is
"catch-up" growth possible when animals are rehabilitated. It
is also clear that malnutrition imposes a definite limitation on
the complexity of neuronal circuits. In particular, malnutri-
tion insult, both pre- and postnatal, appears to affect largely
postnatally developing microneurons in the brain and,
thereby, alter local circuit formation and function. These lat-
ter are essential for establishing the delicate balances between
inhibitory and excitatory activities in critical brain structures,
such as the dentate gyrus of the hippocampal formation, the
cerebellum, and olfactory bulb. Microneuronal hypoplasia,
without any evidence of pathological effects at the light or
electron microscopic levels, leads inexorably to learning and
attentional disorders often mimicking the syndrome of mini-
mal brain dysfunction in man (7,8). Though we do not have
adequate measures for psychological processes such as atten-
tion, cognition, information processing, etc., nor any local
anatomical correlates of these higher-order brain processes,
other than distributed-type lesions in several interacting brain
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