CHAPTER 13
Stress Neurobiology and
Developmental Psychopathology
MEGAN R. GUNNAR and DELIA VAZQUEZ
HISTORICAL OVERVIEW 533
THE MATURE LIMBIC-HYPOTHALAMIC-PITUITARY-
ADRENOCORTICAL SYSTEM 535
The Hypothalamic-Pituitary-Adrenocortical Axis 536
Systemic versus Processive Stress and Their
Neuroanatomical Pathways 538
THE DEVELOPING LIMBIC-HYPOTHALAMIC-
PITUITARY-ADRENOCORTICAL SYSTEM 543
Key Features of the Developing Limbic-Hypothalamic-
Pituitary-Adrenocortical System 543
PSYCHOPATHOLOGY 550
Disruptive Behavior Disorders 550
Anxiety Disorders 552
Maladaptive responses to stress are components of both the
etiology and expression of many psychiatric disorders (e.g.,
Dawes et al., 1999). In addition, individuals differ in their
stress vulnerability, with some seeming to thrive despite
the odds and others succumbing to even relatively mild ad-
versity (Masten, 1999). Early adverse experiences likely
contribute to both individual differences in stress vulnera-
bility and their expression in psychiatric disorder (Heim,
Owen, Plotsky, & Nemeroff, 1997). Explicating these indi-
vidual differences and their role in psychiatric etiology is
one of the central issues in developmental psychopathology.
The goals of this chapter are to describe the current state of
our knowledge regarding the developmental neurobiology
of stress, its relation to psychiatric disorders, and the im-
pact of early adverse experiences on stress vulnerability
This work was supported by the National Institute of Mental
Health “Early Experience, Stress, and Prevention Science” re-
search network (MH60766) and a National Institute of Mental
Health Research Scientist Award (MH00946) to the first author
and National Institute of Mental Health (MH59396, MH01-
0328) and National Institute of Child Health and Human Devel-
opment (HD/ DK37431) grants to the second author.
533
Depression 554
Summary of Limbic-Hypothalamic-Pituitary-
Adrenocortical Activity in Child Clinical Disorders 555
EARLY EXPERIENCES AND STRESS REACTIVITY
AND REGULATION 555
Rodent Studies 555
Nonhuman Primate Studies 556
Summary of the Monkey Infant Studies 559
Human Studies 559
CONCLUSIONS AND FUTURE DIRECTION 565
REFERENCES 568
and resilience. We focus on studies of the limbic-hypothal-
amic-pituitary adrenocortical (LHPA) system, a critical
system fostering both resilience and vulnerability to stress
in animals and humans. Research on this neuroendocrine
system has a long history.
HISTORICAL OVERVIEW
In 1957, Levine (see review, in press) published a landmark
Science paper demonstrating that removing rat pups from
their mother for a few minutes daily during the first weeks
of life permanently reduced activity of the LHPA system.
This publication followed one from the previous year
demonstrating that this handling manipulation also reduced
fearful, anxious behavior. The half-century since these pub-
lications has seen remarkable advances in preclinical (ani-
mal) research on stress and early experience (see Levine, in
press). This work built on insights into stress physiology
that began with Cannon (1914) and Selye (1936) and took
on new meaning with Harris’s (1948) work showing that
the brain controlled endocrine activity, including that of the
534 Stress Neurobiology and Developmental Psychopathology
pituitary-adrenocortical system. More recently, McEwen’s
(e.g., McEwen, Weiss, & Schwartz, 1968) evidence that glu-
cocorticoids (GCs; cortisol in humans and nonhuman pri-
mates, corticosterone in rats), the hormonal products of the
LHPA system, both sustain normal brain function and para-
doxically endanger nerve cells, ushered in heightened
awareness of the critical role of this neuroendocrine system
in psychiatric health and disease.
Our understanding of the role of GCs in shaping the de-
veloping brain has been enriched through evidence that
they contribute to the regulation of brain-derived neu-
rotropic factors (Smith et al., 1995), up-regulate activity
of the amygdala (Makino, Gold, & Schulkin, 1994),
and target prefrontal systems involved in stress and emo-
tion (Sullivan & Gratton, 2002). Meanwhile, research by
Meaney (e.g., Weaver, Cervoni, Diorio, Szyf, & Meaney,
2001), among others, has explicated some of the molecu-
lar processes through which early experiences may pro-
duce their lifelong effects on stress neurobiology.
Critically, in rodents it appears that these molecular
processes are set into motion by maternal behavior (see
Cirulli & Alleva, 2003).
GCs are only part of the picture. A critical link to
psychopathology came with awareness that corticotropin-
releasing hormone (CRH), the major LHPA activating
hormone in the hypothalamus, also has neurostimulatory
and neuromodulatory effects in many regions of the brain
involved in stress and emotion (e.g., Heim et al., 1997).
When produced and acting outside the hypothalamus,
CRH has been shown to play a role in a variety of psychi-
atric conditions, most principally affective disorders (see
Nemeroff, 1996). CRH acts through a family of receptors
that mediate different components of behavioral and phys-
iological responses to stress. The development of this re-
ceptor system may be another key to how experience
shapes stress vulnerability and resilience (reviewed in
Heim et al., 1997). Thus, as Levine (in press) noted,
what began as a story about the role of GCs in stress regu-
lation may eventually extend into many areas of patho-
physiology that are not associated with regulation of the
LHPA system.
The history of any field rests on both conceptual and
methodological advances. Conceptually, evidence that the
stress-emotion system is highly plastic during development
and that parental care shapes its development forms
the basis for translation of the preclinical work into re-
search on developmental psychopathology. Technically, the
critical advances are legion, encompassing much of the ar-
mamentaria of modern-day neuroscience (e.g., cloning,
knock-outs, neuroimaging). All of these advances are es-
sential for understanding the processes that transduce ex-
perience into altered stress vulnerability, yet advances in
the translation of preclinical and neuroscience research
into the field of human developmental psychopathology
rests on something much more mundane: spit.
Initial measures of adrenocortical activity were indi-
rect and crude (Levine, in press). The development of com-
petitive binding assays allowed measurement of GCs in
urine and blood, thus permitting study of the adrenocorti-
cal system in humans. Soon after competitive binding as-
says were available, the first studies of GCs in children
appeared (for review, see Gunnar, 1986). However, the im-
mense challenge of collecting urine reliably and the inva-
siveness of plasma sampling limited the studies that could
be conducted on children (reviewed in Gunnar, 1986).
Then, in the early 1980s, assay techniques were refined to
allow the measurement of cortisol in small amounts
of free-flowing saliva (Riad-Fahmy, Read, Walker, &
Griffiths, 1982). Salivary cortisol reflects the unbound or
biologically active fraction of the hormone and is highly
correlated with plasma cortisol concentrations (see
Kirschbaum & Hellhammer, 1989, 1994). With the use of
salivary cortisol measures, the number of publications on
children has increased from fewer than a dozen in the
1970s to nearly that many each month in the first years of
the twenty-first century.
The emergence of a developmental systems perspective
in child psychology and developmental psychopathology
(e.g., Cicchetti & Tucker, 1994) has also encouraged hor-
mone-behavior studies in children. According to this per-
spective, development involves complex, bidirectional
flows of influence across hierarchically organized systems
and subsystems, from the level of the gene to the level of
social systems. This approach permits inclusion of biologi-
cal measures in developmental research without implying
that behavioral development can be reduced to biological
explanations. Nonetheless, these perspectives are not theo-
ries and do not provide theory-driven predictions about
how physiological measures should be related to behavioral
outcomes. Such focused predictions need to be derived
from specific models pertaining to the behavioral and bio-
logical systems under study.
Recently, investigators have called for more theory-
driven studies of cortisol-behavior relations in human de-
velopmental research (e.g., Granger & Kivlighan, 2003).
Although laudatory, attempts to predict activity of the
LHPA system in human development will be problematic
as long as most studies of children are restricted to assess-
 The Mature Limbic-Hypothalamic-Pituitary-Adrenocortical System
ing only one level (the adrenal) of this very dynamic, self-
regulating system (Gunnar & Vazquez, 2001). One goal of
this chapter is to discuss the complex, multifactorial regu-
lation of the LHPA system and to clarify why testing the-
ory-driven predictions requires probing levels of the axis
above the adrenal, up to and including corticolimbic path-
ways into the LHPA system. Because we are limited in our
ability to do this kind of research in children, theory-
driven research on the LHPA system requires close inte-
gration of human developmental research with preclinical
animal studies. Theory-driven work in human develop-
ment also benefits greatly from studies of clinical popula-
tions, including adult populations, in which it is ethical to
use pharmacological probes and more invasive sampling
techniques.
Another stumbling block to theory-driven develop-
mental psychopathology research on the LHPA system
has been the lack of criteria for defining and assessing
dysregulation. As we will see, most of the studies of
children involve assessment of GC levels and GC
change or responsivity scores. In addition, there is a
growing body of literature on the daily or circadian
pattern of GC production. If children with behavior
problems and/or adverse early histories exhibit levels,
responses, or circadian patterns in GC production that
differ from children with fewer behavior problems or
less adverse histories, the temptation has been to de-
scribe the differences as a reflection of dysregulation
of the LHPA system. However, the system might merely
be acting differently along some parameter(s), though
not be dysregulated. Derived from arguments made by
Siever and Davis (1985), dysregulation in many physio-
logical systems can be conceptualized as persistent dis-
turbances in one or more of the system’s homeostatic
regulatory mechanisms. We discuss the specific mecha-
nisms later in the chapter, but the observed phenomena
indicative of dysregulation can be listed here: (1) Basal
output is more erratic; (2) normal periodicities are dis-
rupted; (3) efficiency of feedback mechanisms is in-
creased or decreased, resulting in overly rapid or
overly slow return to set points following perturbation;
(4) adjustment to repeated stimulation fails; and (5)
clinically efficacious treatments restore efficient regu-
lation. Although few studies of children provide enough
information to determine whether the LHPA system is
dysregulated in relation to either behavior disorders or
adverse experiences, preclinical studies do allow such
assessments, as do some of the studies of adults that
we consider.
535
In what follows, we provide an overview of the neurobi-
ology of the mature LHPA system and its role in stress and
adaptation. We then describe the developmental neurobiol-
ogy of the system based on preclinical research, with links
to human development when feasible. Next, we examine
associations between LHPA activity and psychopathology
in children as a prelude to examining the role of experi-
ence in shaping the activity of the LHPA system in ani-
mals and humans.
THE MATURE LIMBIC-HYPOTHALAMIC-
PITUITARY-ADRENOCORTICAL SYSTEM
The LHPA axis is the classical stress system (Selye, 1936).
Once labeled the HPA axis, an L for limbic is now com-
monly added to indicate the critical role of the limbic sys-
tem in its activity. Two molecules at the opposite ends of
the activating pathway regulate LHPA activity: CRH and
GCs. CRH activates both hormonal and behavioral stress
responses (e.g., Heim et al., 1997). CRH-stimulated behav-
iors include heightened vigilance, defense-related learning
and memory, and context-dependent motor responses:
freezing, fighting, or fleeing. Activation of the LHPA axis
leads to increases in GCs, extremely potent steroids that
function to facilitate adaptation and to restore homeostasis
through changing internal dynamics. McEwen (1998) has
described this dynamic regulation as allostasis, or the
maintenance of stability through change. The capacity of
organisms to make allostatic adjustments is necessary for
survival. However, the biological processes set into motion
are meant for short-term adaptations and are potentially
damaging to physical and mental health if they persist too
long or occur too frequently. The cost of allostasis has been
described as allostatic load.
GCs orchestrate allostatic adjustments by operating on
the DNA to activate, enhance, or inhibit the gene transcrip-
tion in most organs and tissues of the body (Sapolsky,
Romero, & Munck, 2000). In the periphery, among other ac-
tions, GCs promote glycolysis, a process that provides glu-
cose from hepatic stores and facilitates increases in oxygen
levels. In the brain, GCs help orchestrate motivation, re-
ward, and mood (de Kloet, 1991). Brief and controllable
stress with high but controllable GC levels stimulates exhil-
arated mood and health-promoting actions. In contrast, as
first described by Seyle in 1936 (see Table 13.1), prolonged,
uncontrollable, or chronic states of distress leading to high
or prolonged GC levels can have deleterious mental and
536 Stress Neurobiology and Developmental Psychopathology
TABLE 13.1 Behavioral and Physiological Adaptations of Acute Stress
Physiological Responses: Behavioral Responses:
Redirection of Function Redirection of Behavior
Increased respiratory function Increased arousal, vigilance, and attention
Increased cardiovascular tone Increased cognition
Increased gluconeogenesis; increased Suppression of feeding
lipolysis
Inhibition of growth and reproductive Suppression of sexual behavior
hormones, inf lammatory processes,
immune system
Limitation of stress (LHPA) activation /
response
Source: Modified from “ The Concepts of Stress and Stress System Disorders: Overview of Physical and Behavioral Homeostasis,” by G. P. Chrousos
and P. W. Gold, 1992, Journal of the American Medical Association, 267(9), pp. 1244–1252.
physical consequences (Sapolsky et al., 2000). The repercus-
sion of prolonged CRH and GC activity may be especially
costly during development. High CRH levels, with or with-
out elevated GCs, inhibit physical and neural development
(Avishai-Eliner, Brunson, Sandman, & Baram, 2002). CRH
and GCs also shape brain systems involved in perception
and response to threat and, in genetically vulnerable individ-
uals, may facilitate the development of psychopathology
(Heim et al., 1997).
LHPA studies in humans are still largely confined to
measures of cortisol and, more recently, the anticortisol,
dehydroepiandrosterone (DHEA). Cortisol is a glucocorti-
coid that has catabolic effects; DHEA is an androgenic
steroid that has anabolic effects. Both are produced by the
adrenal cortex. Because measurements in humans are fo-
cused on these adrenocortical hormones, this has led to the
misleading view that the pituitary-adrenal axis is at the
center of the stress response. However, animal models and
advanced molecular techniques make it clear that CRH and
its system of receptors are as central as GCs (cortisol
and corticosterone) for stress and adaptation. This can be
demonstrated through description of stress-activating and
regulatory pathways.
The Hypothalamic-Pituitary-Adrenocortical Axis
The circuits that originate in specific brain areas and acti-
vate the LHPA system converge, directly or indirectly, on
the medial parvocellular region of the paraventricular nu-
cleus (mpPVN) of the hypothalamus that secretes CRH
Physiological Outcome:
Redirection of Energy LHPA Hormone
Oxygen shunted to CNS and critical CRH
body sites
GCs
Increased body temperature; suppressed CRH
appetite
Glucose and nutrients shunted to CNS GCs
and critical body sites
No reproduction; decreased immune CRH and GCs
function
Demands to cope with stress GCs
predominate
and arginine vasopressin (AVP; see Figure 13.1). Inputs to
the mpPVN include both activating and inhibiting path-
ways. These pathways are similar in the rat and human (re-
viewed in Lopez, Akil, & Watson, 1999). Once released,
both CRH and AVP interact with their receptors on corti-
cotropic cells of the anterior pituitary (AP), where the
proopiomelanocortin (POMC) molecule is synthesized and
processed into adrenocorticotropic hormone (ACTH).
Binding of CRH to pituitary CRH receptor 1 (CRH r1)
leads to the secretion of ACTH. AVP potentiates CRH-in-
duced ACTH release. ACTH circulates in blood and binds
to its own receptors on the adrenal cortex, leading to the
synthesis and release of GCs.
Mineralocorticoid and Glucocorticoid Receptors
GCs target many organs and tissues, including the brain.
They have their effects through two types of receptors:
mineralocorticoid (MR) and glucocorticoid (GR). Receptor
binding, particularly to GR, also accomplishes negative
feedback whereby GCs inhibit their own production. There
are three types of negative feedback: fast, intermediate,
and delayed. Fast feedback is rapid (within minutes) and
dependent on the rate rather than the absolute magnitude of
GC increase. It is achieved, at least in part, through GC
binding to receptors in the mpPVN, AP, and hippocampus
(reviewed in Dallman et al., 1992). Intermediate and de-
layed feedback, on the other hand, work over the course of
hours to days to suppress CRH and POMC gene expression
in the hypothalamus and AP, respectively, thereby decreas-
ing the ACTH secretory drive. Thus, multiple feedback
 The Mature Limbic-Hypothalamic-Pituitary-Adrenocortical System 537

Figure 13.1 The LHPA System. Panel A depicts the anatomy of the LHPA system and structures important in its regulation. AMY =
Amygdala, HC = Hippocampus, HYP = Hypothalamus, NTS = Nucleus of the tractus solitarius, PFCtx = Prefrontal cortex. Panel B
depicts the activation (+) and negative feedback inhibition (−) pathways of the HPA system. Increases in GCs are initiated by the
release of CRH/AVP from the medial parvocellular region of the paraventricular nucleus (mpPVN) in the hypothalamus. Negative
feedback inhibition operates through GCs acting at the level of the pituitary, hypothalamus and hippocampus. ACTH = Adreno-
corticotropic hormone, AVP = Arginine vasopressin, CRH = Corticotropin releasing hormone, GABA = Gamma animobutyric acid.

loops operate in different time domains and through differ-
ent processes (neuronal signaling as well as gene regula-
tion) to maintain GCs within normal basal levels.
The effects of GCs depend on which receptor is bound
(MR or GR), the ratio of MR to GR binding, and the recep-
tor’s location (de Kloet, 1991). In the rodent brain, MRs
are expressed in limbic regions (e.g., septum, hippocam-
pus) and GRs are distributed throughout the brain. How-
ever, unlike rodents, humans express MR in cortical
regions, suggesting an MR role in processive functions
538 Stress Neurobiology and Developmental Psychopathology
related to stress (see later discussion, and reviewed in
Lopez et al., 1999). MRs bind GCs readily ( high affinity),
and GRs bind GCs less readily ( lower affinity); therefore,
GCs bind first to MRs and then to GRs as MR sites satu-
rate. MRs are typically about 80% to 90% bound or occu-
pied when GCs are in basal ranges, with GRs being bound
only at the peak of the circadian cycle or when stressors
stimulate elevations over basal concentrations.
In addition to mediating negative feedback, in the brain
both MRs and GRs mediate all GC effects. Outside the
brain, MRs regulate water/salt balance through binding by
aldosterone, another steroid hormone. In the periphery,
GCs do not occupy MRs because of the presence of the en-
zyme 11 beta hydroxysteroid dehydrogenase (11ß-HSD)
that inactivates GCs. In the brain, 11ß-HSD is minimally
expressed so that MR exhibits its high affinity for GCs
(Reul & de Kloet, 1985). Through brain MRs and GRs,
GCs serve at least four critical stress functions (reviewed
in Sapolsky et al., 2000). MRs mediate permissive GC ef-
fects that sustain an organism’s ability to make adaptive
responses to changing environmental demands. For exam-
ple, MRs maintain steady and persistent electrical current,
allowing neurons to be responsive to their neurotransmit-
ters. In contrast, GRs have suppressive effects, inhibiting
ongoing behavioral and neurophysiological responses. MR
and GR, thus, tend to have opposing ef fects. Several addi-
tional examples of these opposing effects help demonstrate
this general rule. MRs facilitate cerebral glucose utiliza-
tion, and GRs inhibit glucose utilization throughout the
brain. MRs mediate forms of synaptic plasticity that under-
lie learning; GRs, operating over hours or days, disrupt the
same mechanisms of synaptic plasticity, endanger cell sur-
vival, and blunt hippocampal excitability, disrupting mem-
ory formation. The opposing effects of MR and GR
combined with the differential affinity of GCs for these
two receptors is believed to be why the relations between
GCs and adaptive functioning often takes an inverted-U
function. Thus, very low and very high GC concentrations
are associated with deleterious health and behavior conse-
quences, and moderate levels and small or well-constrained
elevations tend to support adaptive functioning.
In addition to levels and duration, the timing of the GC
response also influences its role in stress and adaptation.
Initial effects of increasing levels of GCs tend to be stimu-
latory and permissive as these operate through MR and
initial GR effects of liberating hepatic energy stores, re-
sulting in increased glucose levels and permissive GR ef-
fects on other stress-stimulated physiological systems.
Well-timed anticipatory responses to stressors thus can
place the organism in an optimal state to handle challenge
and constrain or suppress the operation of other stress-sen-
sitive systems. Therefore, not only too persistent but also
too shallow GC responses above resting basal levels can be
damaging, particularly when this permits other stress-
stimulated physiological responses to continue unchecked
(e.g., immune reactions). The final category of GC effects
is preparatory, denoting GC-induced changes in neurobiol-
ogy that affect the way organisms responds to subsequent
stressors. Thus, for example, operating through GRs, GCs
up-regulate the sensitivity of the amygdala to threat stim-
uli, resulting in heightened sensitivity to subsequent
threats (for review, see Rosen & Schulkin, 1998).
The effects of GCs depend not only on their level and
timing, but also on the ratio of MRs to GRs that are occu-
pied in different brain regions (de Kloet, 1991). This is
demonstrated using transgenic preparations (reviewed in
Korte, 2001). Transgenic mice that underexpress GR pro-
duce high and prolonged GC responses (impaired feed-
back), are hyperactive, and have great difficulty with
spatial learning, presumably because of secondary hip-
pocampal effects. Transgenic mice that overexpress GR
have increased anxiety-related behavior with increased
CRH expression in the amygdala, unaltered CRH levels in
the PVN, and normal basal GC levels. MR knock-outs have
impaired neurogenesis presumably secondary to chronic el-
evations in basal GC levels.
Systemic versus Processive Stress and Their
Neuroanatomical Pathways
A number of seemingly disparate stimuli activate the
LHPA axis. Some stimuli activate the axis through systemic
paths; others require interpretation by the organism and
thus are termed processive or collative (reviewed in Herman
& Cullinan, 1997). Systemic stressors are physical and
context-independent. Thus, they stimulate the LHPA axis
even in an unconscious animal. Examples are hypotension
(decreases in blood pressure), hypoxia (decreases in oxy-
gen levels), and infection. In contrast, processive stressors
are context-dependent, requiring the comparison of current
information with past experience and the assignment of
emotional meaning. Systemic and processive stressors op-
erate through diffuse networks of neurons that converge at
the level of the mpPVN. For systemic stressors, the infor-
mation stimulating activation of the LHPA is relayed to the
mpPVN via the brain stem through afferents originating in
the dorsal roots of the autonomic system and the glossopha-
ryngeal and vagus cranial nerves. For example, the nucleus
 The Mature Limbic-Hypothalamic-Pituitary-Adrenocortical System
of the tractus solitarius (NTS, a key parasympathetic relay
station) receives afferent fibers that indicate hypotension
as a result of hemorrhage. In turn, ef ferent fibers travel
away from NTS into the CRH-rich mpPVN area to activate
an LHPA response. Some of these autonomic connections
are direct into the mpPVN, but most are indirect via other
limbic structures.
Limbic centers transmit processive stressor information
to the mpPVN via corticolimbic ef ferent pathways involv-
ing the BNST ( bed nucleus of the stria terminalis), preop-
tic nucleus, lateral and medial septum, the amygdala,
prefrontal cortex, and ventral subiculum (reviewed in Her-
man et al., 2003). Efferent pathways converge in the fim-
bria /fornix, a collection of nerve bundles emanating from
the hippocampus, subiculum, and cortical regions. Some of
these pathways provide activating and others inhibiting
input to the mpPVN. The medial amygdala and lateral
BNST are centrally involved in activating pathways. The
various nuclei of the amygdala mediate behavioral and also
autonomic cardiovascular responses to stress indirectly
using the BNST or NTS as relay stations, with the fibers
terminating two or more steps removed from the mpPVN
(de Olmos, Alheid, & Beltramino, 1985). Whether or not
cardiovascular effects are induced along with LHPA acti-
vation depends on the nuclei excited by stress-induced
amygdala activation (e.g., basolateral versus basomedial;
M. Davis, 1992).
For processive stressors, inhibitory inf luences are also
mediated through limbic structures: the ventral subiculum,
preoptic area, medial BNST, and cingulate/prefrontal cor-
tex (Herman, Muller, & Figueiredo, 2004). Damage to the
subiculum, its major outflow pathway (the fimbria /fornix),
or the medial frontal cortex increases CRH mpPVN expres-
sion and enhances GC production to processive stressors.
However, some overlap in excitatory and inhibitory inputs
exists as medial prefrontal cortex damage has been shown
to both increase and decrease GC production and auto-
nomic activation (reviewed in Herman et al., 2003; C. D.
Walker, Welberg, & Plotsky, 2002).
Neurotransmitters and the Limbic-Hypothalamic-
Pituitary-Adrenocortical Axis
Neurotransmitters are the mediators of LHPA activity that
operate along the neuroanatomical pathways outlined ear-
lier (see Figure 13.2). Many neurotransmitter systems exist
in the brain, but noradrenaline (NA), serotonin (5-HT),
glutamate, and gamma-aminobutyric-acid (GABA) are par-
ticularly important in modulation of the LHPA system. All
of these molecules have excitatory effects on the LHPA
539
axis through their innervation of limbic structures that, in
turn, have direct access to the mpPVN (reviewed in Her-
man et al., 2003). This excitatory stimulation operates
through ascending monoaminergic input from the pons
area, where presynaptic neurons containing NA ( locus
coeruleus) and 5-HT (raphe nuclei) originate. Direct acti-
vation of the LHPA system, however, involves short circuits
arising from glutamate neurons within the mpPVN that act
on glutaminergic receptors (NMDA, kainate, or AMPA)
and through inhibition of inhibitory GABA-ergic afferents
( because double negative equals positive; see later discus-
sion and Figure 13.2).
Inhibition of the LHPA axis at the level of the mpPVN
is mediated through GABA-ergic neurons. The inhibitory
structures (e.g., septum, prefrontal cortex, medial BNST,
and suprachiasmatic nucleus) are rich in GABA neurons
(Herman et al., 2004). GABA modulation is how GRs in
the hippocampus mediate negative feedback. GR-acti-
vated electrical currents travel through hippocampal ef-
ferents to interact with GABA-containing neurons in the
BNST, preoptic area, and hypothalamus, achieving a net
inhibition on the mpPVN. Activation of the LHPA system
also can involve GABA activity. In this case, GABA inter-
acts with adjacent GABA neurons and results in a de-
crease of the downstream inhibition (first GABA neuron
inhibits the release of a second GABA neuron). Amygdala
innervation of the mpPVN is a good example of this
double negative. Enhanced amygdaloid activity leads
to disinhibition of disynaptic GABA-ergic input to the
mpPVN and therefore enhances LHPA activity (see Fig-
ure 13.2). GABA-ergic tone is also the main factor influ-
encing resting basal state of the LHPA axis, characterized
by circadian oscillations of its key hormones (see later
discussion).
Critical Features of Processive Stressors in Humans
Although the neuroanatomical pathways of processive
stressors are beginning to be worked out in studies of ani-
mals, in human research the key features of processive
stimuli capable of activating an LHPA response are still
uncertain. These features have variously been described as
novelty, uncertainty, unpredictability, uncontrollability,
and potential for harm or loss (see reviews by Kirschbaum
& Hellhammer, 1989, 1994). Distressingly, however, de-
spite arguments that the LHPA axis is highly sensitive to
processive stressors, it is often difficult to provoke GC re-
sponses in the laboratory (see review by Biondi & Picardi,
1999). Indeed, in many laboratory studies of children
and adolescents, rather than an increase in cortisol to the
* Glucocorticoids provide positive stimulation to the amygdala for the synthesis and release of CRH, but negative to the pituitary, hypothalamus and
hippocampus.
† Interaction is through glutamate outf low from these regions that synapse on local GABAergic neurons, producing inhibition of mpPVN.

Figure 13.2 Schematic representation of the activating (right side) and and inhibiting ( left side) circuits that contribute to regula-
tion of the LHPA system. Catecholamines, norepinephrine (NE) and epinephrine (E) arising from medullary nuclei of the brainstem
are the primary neurotransmitters providing activation of CRH synthesis and release from the mpPVN. Serotonin originating from
dorsal raphe is weakly activating; it act both directly on mpPVN and indirectly through excitatory glutamate neurons or inhibitory
gamma animobutyric acid (GABA) inputs. Paradoxically, the inhibitory GABA neurotransmitter activates the mpPVN to secrete CRH
since two GABA neurons activated in series leads to excitation and not inhibition. Extra-hypothalamic CRH also acts as a neurotrans-
mitter to initiate autonomic and behavioral responses to stress. The activation of the extra-hypothalamic CRH system is initiated by
rising glucocorticoids levels that operate on the amygdala to secrete CRH that, in turn, impacts on the locus cerouleus (LC). Through
the activation of catecholaminergic brainstem nuclei there is also stimulation of descending pathways leading to NE/ E release form
the adrenal medulla that facilitates cardiovascular autonomic responses to stress. Inhibition of the LHPA axis seen in the left side is
provided by glucocorticoids acting on glucocorticoids receptors (GR) in the hypothalamus and pituitary where CRH and ACTH release
is halted. The hippocampus serves to inhibit the stress response via multiple circuits, some of which are direct inhibitory GABA in-
puts; others are indirect through glutamate excitatory inputs to GABA neurons converging in the mpPVN. GABA neurons located in
each of the structures further modify the stress reactivity and inhibition from other brain regions such as the thalamus, association
cortex, cortical and limbic afferents.

540
 The Mature Limbic-Hypothalamic-Pituitary-Adrenocortical System
intended stressor, cortisol levels for most subjects fall over
the testing period! This may reflect anticipatory increases
in GCs and inadequate periods for adaptation to the labora-
tory, but it may also reflect our lack of understanding of
the stimuli capable of activating the LHPA axis in humans.
In a recent meta-analytic review of 208 adult studies,
Dickerson and Kemeny (2004) provide an empirical test of
the core features of potent GC-stimulating tasks. They ar-
gued that for an event to activate the LHPA system, goals
that are central to the individual must be threatened. Self-
preservation is a central goal of all living organisms. Cer-
tainly, physical threats to self-preservation are likely to
produce marked increases in cortisol, as evidenced by stud-
ies of individuals in emergency rooms assessed shortly after
life-threatening events (see Yehuda, 2000). These authors
argue, however, that the preservation of the social self is also
a central goal for humans. The results of their meta-analysis
showed that paradigms that threaten the social self (e.g., so-
cial evaluation by an audience of judges) are potent stimula-
tors of the LHPA axis. Tasks that require mental effort but
where poor performance poses little threat to the social self
are not very effective in elevating cortisol. Tasks that pro-
duce negative emotions but do not threaten the social self
are not effective in elevating GCs. Novelty, unpredictability,
and uncontrollability act synergistically with threats to cen-
tral goals to produce the most marked GC responses.
No one has conducted a similar meta-analysis of stressors
in children. However, in early childhood, separation acti-
vates the LHPA axis, an event that has an evolutionary his-
tory of threatening self-preservation. Later in childhood,
Flinn and England (1995) have reported, serious family con-
flicts and threats of rejection /abandonment are highly po-
tent in elevating cortisol in their study of Caribbean
children. In addition, even very early in life, social rejection
by peers is associated with high GC levels among preschool
children (Gunnar, Tout, Donzella, & van Dulmen, 2003).
Thus, it seems likely that the Dickerson and Kemeny (2004)
analysis will apply to children. Critically, because it is rare
to find a laboratory study beyond the infancy period in
which the stressor elevates cortisol in most children, it is
challenging to interpret the result of most laboratory-based,
GC human developmental stress studies.
A System with a Rhythm
The LHPA axis is not just an alarm system waiting to be ac-
tivated. It is also a system that exhibits a circadian or 24-
hour rhythm. GC levels reach their peak approximately 30
minutes after waking, although they begin to rise in the last
hours of sleep. From peak levels, GCs fall throughout the
day, with transient increases about 45 minutes following
541
protein-rich meals (postprandial surge), until levels reach
their lowest point or nadir at the end of the activity phase
(e.g., Kwak, Morano, Young, Watson, & Akil, 1993). GCs
measured in saliva follow the same pattern, offset by ap-
proximately 2 minutes, from GCs measured in plasma. The
circadian rhythm not only alters basal levels of the hor-
mones along the LHPA axis, but also changes in its sensi-
tivity to stressful stimuli. At the nadir of the rhythm
(roughly 10 to 12 P.M. in humans on a regular day/night
schedule), the LHPA system is more responsive to both ac-
tivating and inhibiting (negative feedback) signals (Dall-
man et al., 1992). Thus, time of day is a critical factor in
interpreting LHPA effects and designing LHPA studies.
The need to control for diurnal rhythm in assessing activ-
ity of the LHPA system places critical restrictions on
researchers studying this system. However, in human devel-
opmental research, time of day often is not controlled. It is
not clear how much of a problem this poses. In their review,
Dickerson and Kemeny (2004) were not able to detect differ-
ences in the magnitude of the adult GC response depending
on whether assessments were made in the morning or after-
noon. Although this would seem to stand in marked contrast
to the animal studies, the human studies deal with a smaller
time of day variation. Rather than contrasting LHPA activity
at the peak and nadir of the cycle (wake-up and bedtime),
testing times in human studies typically vary across the mid-
portion of the day. This is especially true for studies with
children, for whom testing schedules are constrained by such
factors as when the child typically naps and when other fam-
ily members get home from work or school. It may be that
when testing occurs between 9 A.M. and 5 P.M., variations in
testing times introduce little variability in the responsiveness
of the system. On the other hand, until the appropriate meta-
analysis of the child development literature on cortisol reac-
tivity is conducted, it is probably better to avoid testing times
that vary uncontrollably between subjects.
The LHPA circadian rhythm is one of many physiological
(e.g., body temperature) and behavioral (e.g., sleep, feeding)
rhythms in mammals. The suprachiasmatic nucleus (SCN) is
the master pacemaker controlling circadian rhythms (re-
viewed in van Esseveldt, Lehman, & Boer, 2000). It oscil-
lates with a near 24-hour period under constant light
conditions but is entrained to day/night rhythm by periodic-
ity of light exposure and nonphotic modulators, including
patterns of locomotor activity, feeding and sleep (Follenius,
Brandenberger, Bandesapt, Libert, & Ehrhart, 1992; Folle-
nius, Brandenberger, Hietter, Simeoni, & Reinhardt, 1982).
In humans, the SCN is also entrained by our expectations
about when we are to wake up in the morning (Born, Hansen,
Marshall, Molle, & Fehm, 1999). For example, if we expect
542 Stress Neurobiology and Developmental Psychopathology
to wake up at 6 A.M., activity of the LHPA system will reflect
that expectation, regardless of when we actually wake up.
Thus, not only the time since waking but also whether the in-
dividual is waking at his or her usual time is important to
consider in assessing early morning cortisol concentrations.
Unfortunately, we do not know what happens to the circadian
rhythm when bedtimes and wake-up times are unpredictable,
as they may be in more chaotic households. Sleep restriction
also modifies LHPA axis activity, leading to elevated resting
GC levels (Leproult, Copinschi, Buxton, & Van Cauter,
1997). Thus, sleep-wake rhythms are important to consider
in studies of LHPA activity.
The pathways that integrate the perception of light into
the SCN are the retinohypothalamic tract (GABA-ergic),
the geniculohypothalamic tract (neuropeptide Y or NPY),
and raphe (5-HT). The nonphotic input is received from
the cortex, basal forebrain, and hypothalamus using
NPY, 5-HT, and possibly GCs (Liu & Reppert, 2000). A
myriad of neuropeptides are expressed in the SCN; how-
ever, it is likely that the GABA-ergic system plays a more
important role than these neuropeptides in transmitting
the rhythmic function of the SCN to other brain regions
(Liu & Reppert, 2000). Once a circadian rhythm is gener-
ated in the SCN, it is imposed on the mpPVN primarily
via projections into the brain stem autonomic centers
(Herman et al., 2003). But there is also an ACTH indepen-
dent multisynaptic projection to the adrenal that results in
a fast decrease of circulating GCs at the beginning of
sleep (Buijs et al., 1999). This pathway helps explain why
activation of the axis at the nadir of the LHPA rhythm
around the onset of nighttime sleep in humans results in
greater GC responses than activation at the peak of
the circadian rhythm (Kaneko, Hiroshige, Shinsako, &
Dallman, 1980).
In the LHPA system, MRs play a critical role in main-
taining the LHPA basal rhythm. This relationship has been
demonstrated by using antagonists that are specific to MR
or GR (reviewed in de Kloet, 1991). Selective MR antago-
nists produce elevated basal GCs near the nadir of the
rhythm without affecting levels near the circadian peak or
responses to a processive stressor. Conversely, selective
GR antagonists have no ef fect on resting basal or restraint
stress GC levels at any time of day. Combining MR and GR
antagonists, however, produces elevated GCs over the en-
tire day as well as increased elevations to stressors. In hu-
mans, pharmacological MR antagonists also increase
cortisol levels in the evening, near the nadir of the LHPA
rhythm (e.g., Grottoli et al., 2002; but see Michelson,
Chrousos, & Gold, 1994). In sum, MR activation is neces-
sary and sufficient to maintain low basal GC levels during
the nadir of the circadian rhythm, whereas MR and GR ac-
tivation is necessary to constrain GC secretion during the
circadian peak and during acute stress.
The circadian rhythm of cortisol can be observed in hu-
mans as early as 6 to 12 weeks of life (Larson, White,
Cochran, Donzella, & Gunnar, 1998). However, throughout
infancy and early childhood, cortisol production from mid-
morning through late afternoon fluctuates markedly with
naps and feedings (for review, see Gunnar & Donzella,
2002). As children give up their daytime naps, the typical
adult diurnal pattern of cortisol production emerges (Wata-
mura, Donzella, Kertes, & Gunnar, 2004). Ambulatory
studies of salivary cortisol have shown that not all individ-
uals reliably produce this diurnal pattern of cortisol pro-
duction. Even in the absence of frank physical or mental
disorders, about 10% to 15% of the adult population ap-
pears to have days when early morning levels are not high
and/or evening levels are not low (Smyth et al., 1997). The
etiology and significance of these atypical patterns is not
well understood (see Heim, Ehlert, & Hellhammer, 2000),
although they have been associated with emotional and
mental exhaustion (e.g., burnout), chronic pain disorders
(e.g., fibromyalgia), and, in infants and young children,
conditions of chronic neglect (Gunnar & Vazquez, 2001).
In addition to the classic diurnal pattern, recently it has
been noted that there is about a 60% increase in salivary
cortisol concentration in the 30 to 40 minutes after waking.
Termed the cortisol awakening response (CAR), this in-
crease is stable within individuals and reflects genetic as
well as other factors (Wüst, Federenko, Hellhammer, &
Kirschbaum, 2000). Individuals with constant work over-
load and chronic worries tend to exhibit larger increases in
cortisol upon waking (Schlotz, Hellhammer, Schultz, &
Stone, 2004).
Stability and Heritability
Studies of the LHPA axis often focus on individual differ-
ences, thus making it essential that we understand the reli-
ability of these differences. Regarding basal cortisol, using
latent state-trait modeling it has been shown that the bal-
ance of state and trait components varies over the day, with
generally larger trait components obtained soon after
morning awakening and larger state components obtained
later in the day (Kirschbaum et al., 1990; Shirtcliff,
Granger, Booth, & Johnson, in press). In fact, it has been
difficult to fit state-trait models to cortisol measures ob-
tained later in the day because state variability is so large
that stable trait components are difficult to discern (e.g.,
Kirschbaum et al., 1990). Assessing the stability of the
LHPA stress response is more complicated because the
 The Developing Limbic-Hypothalamic-Pituitary-Adrenocortical System
same stressor repeated a second time is not as novel and
thus would be expected to produce less activation. Further-
more, different stressors operate over different neural
pathways and thus produce different responses. However,
several researchers have recently reported modest test-
retest stability (e.g., rs of around .5) in cortisol responses
to stressors in infancy (e.g., Goldberg et al., 2003; Lewis &
Ramsay, 1995). E. F. Walker, Walder, and Reynolds (2001)
also noted rank-order stability of a similar magnitude in
their study of adolescents assessed over a 2-year period. As
in other psychometric measurement, aggregation tends to
produce more reliable indices. For example, in one study
using the Trier Social Stress Test (TSST) administered re-
peatedly over several days, the GC response to the first
TSST did not correlate with responses to the later TSSTs,
but GC responses to the later TSSTs were correlated
(Pruessner et al., 1997). In addition, although the GC re-
sponse to the first TSST was not correlated with personal-
ity traits, responses to the later TSSTs were, particularly
when these responses were averaged. Unfortunately, there
is little information on how many GC values need to be ag-
gregated before reliable individual difference estimates are
obtained. And, given the state-trait modeling results, the
number needed likely varies with time of day. Another reli-
ability-reducing factor may be individual differences in
the rise time of the response. Ramsay and Lewis (2003)
showed that when cortisol measures were taken repeatedly
following an inoculation stressor in infants, the average
peak response was at approximately 25 minutes poststimu-
lation. However, there was a substantial number of infants
who reached their peak cortisol levels at other time points.
When only two measures (pre- and post-) are taken, it is
very possible that delta cortisol underestimates cortisol re-
activity for many individuals.
Stable individual differences in LHPA activity may
partly reflect genetic influences. A recent review of human
twin studies concluded that these influences were only
modest (Bartels, van den Berg, Sluyter, Boomsma, & de
Geus, 2003). However, in most of the reviewed studies,
only a single, potentially unreliable, cortisol measure was
obtained. To address the problem of limited measurement,
these authors analyzed salivary cortisol data collected at
four points over the day on 2 school days in 209 12-year-old
twin pairs. Heritability estimates varied over the daytime
cycle. They were weak (h2 = .32) at the moment of waking,
highest 30 to 40 minutes postwaking (h2 = .71), modest late
in the morning (h2 = .43), and absent or undetectable in the
evening before bed. These data fit with the latent state-trait
modeling results, suggesting that cortisol levels 30 to 40
minutes after morning awakening exhibit a heritable trait
543
component, whereas those taken at other points in the day
reflect stronger, unique, and possibly transient environ-
mental or internal state effects. Basal levels later in the day
are likely to exhibit heritable influences, but it may take
many more than 2 days of cortisol assessment to obtain
measures that are reliable enough to yield significant heri-
tability estimates.
THE DEVELOPING LIMBIC-
HYPOTHALAMIC-PITUITARY-
ADRENOCORTICAL SYSTEM
The rules governing activity of the LHPA axis in adults dif-
fer from those for the neonate. Most of this evidence comes
from studies of rats. We describe preclinical or animal re-
search and then consider how this information applies to
human development. In rats, during the first 2 postnatal
weeks (postnatal days 4 to 14), ACTH and GC responses to
many stressors are markedly diminished. This period has
been termed the relative stress-hyporesponsive period
(SHRP). Although GC responses are low during the SHRP,
marked stressor responses can be observed in the hypothal-
amus and other brain regions in a species- and developmen-
tally specific manner (Smith, Kim, Van Oers, & Levine,
1997; C. D. Walker, Scribner, Cascio, & Dallman, 1991).
Structural and functional brain immaturity and marked de-
velopmental changes in the adrenal gland appear to under-
lie these developmental differences.
Key Features of the Developing Limbic-
Hypothalamic-Pituitary-Adrenocortical System
The LHPA system undergoes marked changes during devel-
opment. Here we briefly review the changes most pertinent
to our understanding of developmental psychopathology in
relation to LHPA functioning.
The Adrenal and Glucocortical Secretion
In the mature animal, the adrenal glands consist of two di-
visions: a cortex that produces three types of steroids and
a medulla that produces catecholamines. The three
steroids produced by the cortex are derived from morpho-
logically and functionally distinct zones (Parker & Schim-
mer, 2001). The zona glomerulosa (ZG) synthesizes GCs;
the zona fasciculata (ZF) produces mineralocorticoids
(MCs), and the zona reticulata (ZR) produces sex steroids.
Prenatally, the maternal placenta and the fetal adrenal
cortex constitute a fetal-placental unit. The fetal adrenal
cortex consists primarily of one zone (fetal zone), which
544 Stress Neurobiology and Developmental Psychopathology
is equivalent to the reticulata and is geared to the produc-
tion of estrogen precursors. These molecules are trans-
ferred from the fetus to the placenta, where they are
metabolized to estrogen, essential for the maintenance of
pregnancy. As gestation progresses, a primitive ZG devel-
ops and begins producing GCs. At birth, GC levels are ele-
vated as a consequence of the stress of parturition. In the
rat, due to hepatic immaturity, it takes a long time for the
pup to clear GCs from circulation. However, by postnatal
day (PND) 4, basal GC levels are low in the rat pup and re-
main low through PND 14, the period of the SHRP (C. D.
Walker et al., 1991). Parturition also initiates an ischemic
process resulting in involution (developmental removal) of
the fetal zone.
For ACTH to stimulate GC production, cell-specific
gene expression leading to specific enzyme synthesis is re-
quired in the ZG. However, the development of chromaffin
cells in the adrenal medulla also plays a role (reviewed in
Vazquez & Levine, in press). Isolated adrenal cells lose the
capacity to produce GCs unless chromaffin cells from the
medulla are added to cell culture preparations. In the rat,
the adrenal medulla does not become a well-defined region
until the end of the first postnatal week, corresponding to a
period of rapid development of the GC- and MC-producing
zones of the adrenal cortex. Thus, the adrenal is quite im-
mature in the rat during the first 2 weeks postnatal, a fact
consistent with the SHRP.
Yet, there are environmental events that can stimulate
marked GC responses in the neonatal rat. Among these is re-
moval of the mother for 24 hours. Maternal absence for this
duration raises GC levels and permits the adrenal to respond
to both stressors and ACTH (see Vazquez & Levine, in
press). Feeding (milk into the gut) and stroking help to block
this effect, suggesting that it may be partially mediated
by feedback through autonomically mediated pathways from
the gut and skin. Recent studies suggest that the nutritive
components in milk can also modulate the response of the
neuroendocrine system to stress and, possibly, influence
some aspects of brain development (see review, C. D. Walker
et al., 2004). In particular, fat and leptin, a protein produced
in the adipose tissue and present in maternal milk in both
humans and rodents, reduces responses to stress in the in-
fant. This is different in the adult, in which high-fat feedings
exacerbates the LHPA response to stress. Although the
mechanisms involved in this phenomenon are not clear,
Walker and colleagues suggest that leptin acts on both cen-
tral ( hypothalamus and hippocampus) and peripheral (pitu-
itary, adrenal gland) systems in the infant to reduce
exposure to GCs and enhance hippocampal development
during a sensitive period of brain development. Table 13.2
lists genes that are modulated by endogenous and synthetic
GCs in developing mammals.
The Anterior Pituitary
Consistent with the immaturity of the adrenal and the rele-
vance of the adrenal fetal zone during gestation, the ante-
rior pituitary (AP) also displays marked morphological and
functional changes during fetal and early postnatal devel-
opment in the rat pup. The development of the AP from epi-
dermal precursor cells is under the control of transcription
factors that are expressed early in embryogenesis (reviewed
in Swanson, 1992). Hypothalamic hormones help differen-
tiate specific AP cells that produce specific hormones. In
the late fetal /early postnatal period, the AP contains more
ACTH-producing cells (corticotrophs) than in the mature
rat. Corticotrophs are among the first AP cells to mature,
being functional by gestational day 16. However, the ability
to process POMC into mature ACTH develops only gradu-
ally (for review, see Vazquez, 1998; C. D. Walker et al.,
2002). During gestation, high molecular ACTH forms pre-
dominate that may be important for fostering development
of the adrenal and for maintaining the fetal-placental unit
but stimulate minimal GC production. During the late fetal
and early postnatal period, the ACTH molecules produced
by corticotroph cells gradually change to increasingly yield
GC-stimulating forms. Corticotroph cells express recep-
tors for CRH (i.e., CRHr1) by late gestation, with numbers
declining from PND 5 to adulthood. AVP receptors follow
a similar developmental pattern but are not functionally
coupled to second messenger systems until PND 10. Thus,
consistent with the waning of the SHRP, after PND 10, the
AP exhibits increasing capacity to produce mature ACTH
and mature responses to both CRH and AVP by the end of
the 2nd week.
Hypothalamic Corticotropin-Releasing Hormone
In the rat, by gestational day (GD) 17, neurosecretory neu-
rons appear in the mpPVN along with evidence of CRH
mRNA. One day later, AVP mRNA can be observed. Soon
after, CRH and AVP levels begin to increase. At approxi-
mately this same time, axon terminals containing granular
vesicles can be seen in the external layer of the median em-
inence. However, the mpPVN is not completely mature
until PND 28, the time of typical weaning in the wild
(Bugnon, Fellmann, Gouget, & Cardot, 1982). CRH recep-
tors herald the emergence of a functional role for CRH in
the brain and pituitary. The distribution of these CRH re-
ceptors (1 and 2) is very similar in both the developing and
mature rodent. However, several brain regions have high
expression of both CRHr1 and r2 early in life, followed by a
TABLE 13.2 Partial Listing of Genes Modulated by Endogenous and Synthetic Glucocorticoids in the Developing Mammal
Brain Cell Lineages and Dif ferentiation
Bax induces apoptosis. Low GCs increase bax mRNA levels in adult rats (Cardenas et al., 2002).
Bcl-2 prevents apoptosis and neutralizes effects of bax. Low GCs increase bcl-2 mRNA levels (Cardenas et al., 2002).
Ay controls epidermal cell lineage migration. GCs decrease birthweights and increase rates of cleft palate, as examined using prenatal rats (E18;
Teramoto, Hatakenaka, & Shirasu, 1991).
Myelin basic protein functions in the development of myelin. GCs increase mRNA levels of this gene in oligodendrocytes, as detected in the neona-
tal rat (Kumar, Cole, Chiappelli, & de Vellis, 1989).
GPDH plays a role in oligodendrocyte differentiation and myelinogenesis. GCs increase its tRNA levels in oligodendrocyte nuclei (Kumar et al., 1989).
GFAP is an astrocytic marker. In postnatal (2 wks) rats, GCs increase GFAP expression (O’Callaghan, Brinton, & McEwen, 1991).
NGF is required for the development of sympathetic neurons and some sensory neurons. In postnatal (PND11) rats, GCs increase prefrontal cortex
( both sexes) and hippocampal (males) NGF; low GCs cause changes in distribution and expression of NGF receptors in the hippocampus (Scac-
cianoce, Catalani, Lombardo, Consoli, & Angelucci, 2001).
Glutamine synthetase catalyzes recycling of glutamine in glial cells. In embryonic rats, inhibition of GC receptors suppresses the expression of
this gene (Vardimon, Ben-Dror, Avisar, Oren, & Shiftan, 1999).
Glucocorticoid receptor plays a critical role in regulation of the HPA axis. In sheep and rats late in gestation, GCs increase GR mRNA levels
(Holloway, Whittle, & Challis, 2001).
Anterior Pituitary
POMC is the pro-hormone that is cleaved to yield ACTH. GCs reduce POMC mRNA levels, as noted in late-gestation sheep (Holloway et al., 2001).
Vasopressin receptor (V1b) regulates AP response to vasopressin. GCs decrease V1b receptor expression in the AP, as shown in sheep (E100;
Young, Smith, Figueroa, & Rose, 2003).
Prolactin is critical in lactation and delivery. GCs in sheep (E130-E140) increase prolactin mRNA in the AP (Phillips, Fielke, Young, & McMillen, 1996).
FTB controls maturation of glycoprotein galactosylation and fucosylation processes important for hormonal bioactivity. In neonatal rats, GCs in-
crease its transcriptional regulation (Biol-N’gargba, Niepceron, Mathian, & Louisot, 2003).
Peripheral Catecholaminergic System
PNMT is the rate-limiting enzyme in adrenaline synthesis. GCs regulate PNMT activity throughout life, and GR appears to be necessary for the de-
velopmental appearance of PNMT expression (Anderson & Michelsohn, 1989).
PENK gene regulates enkephaline-related petide in chromaffin cells. GCs reduce adrenal PENK mRNA levels, as shown in fetal sheep (E124;
Fraser, Matthews, Braems, Jeffray, & Challis, 1997).
Inf lammation
HLA-G plays a critical role in inf lammation. In cells cultured from humans (E7-9), GCs enhance transcription of HLA-G in trophoblasts (Moreau
et al., 2001).
Proteolipid protein may trigger the immune system to attack myelin. GCs increase mRNA levels in oligodendrocytes, as shown in neonatal rat brain
tissue (Kumar et al., 1989).
Physical Growth
IGF-II regulates cell proliferation and metabolism. Low GCs increase hepatic transcription rate of IGF-II, as shown in late-gestation sheep gene
culture (Kumar et al., 1989).
CTGF/ IGFBP rP2 plays a role in bone formation. In E22 rats, GCs cause a time- and dose-dependent increase in mRNA levels in bone cells and in-
crease transcription rate (Pereira, Durant, & Canalis, 2000).
GHR-1A, GHR-2, and GHR-3 regulate this family of growth hormone receptors, which directly stimulate the production of IGF-1 in the liver and
other tissues. As shown in fetal sheep, increases in GCs during fetal development stimulate increases in GRH mRNA (Li, Gilmour, Saunders,
Dauncey, & Fowden, 1999).
IGF binding protein-1 regulates IGF availability in postnatal life and contributes to somatic growth in utero. In fetal rat livers, GCs increase syn-
thesis of IGFBP-1 (Menuelle, Babajko, & Plas, 1999).
Tryptophan oxygenase is an enzyme required for normal growth and development. In neonatal rat livers, GCs increase trypotophan oxygenase
mRNA (Nagao, Nakamura, & Ichihara, 1986).
Alpha 1 (I) procollagen plays a role in proper osteoblast proliferation. In rats (calvaria cultures), GCs decrease alpha I (I ) procollagen transcripts
(Delany, Gabbitas, & Canalis, 1995).
Respiratory System
TTF-1 regulates thyroid transcription factor-1, which plays a role in lung morphogenesis and is involved in the transcription of surfactant proteins.
Synthetic GCs increase mRNA expression of TTF-1 in rats with congenital hypoplasia.
LGL2 is coordinated with key transcription factors that regulate signal transduction pathways in the fetal lung. In rats (E14) and humans (gesta-
tional week 16), GCs induce LGL2 (Zhang et al., 2000).
SP-A, SP-B, and SP-C (surfactant proteins) are critical for respiration. Three variants of these proteins (A, B, and C) work in coordination to re-
duce alveolar surface tension at the air-liquid interface in lung tissue. In humans, GCs induce surfactant proteins (Losada, Tovar, Xia, Diez-
Pardo, & Santisteban, 2000). In rats with congenital hypoplasia, synthetic GCs increase SP-B mRNA levels (Losada et al., 2000). In human lung
tissue, synthetic GCs in coordination with DBcAMP reduce SP-A mRNA levels (McCormick & Mendelson, 1994; though see contradictory re-
sults in rabbit fetal lung tissue, Durham, Wohlford-Lenane, & Snyder, 1993). In fetal rabbit lung tissue, synthetic GCs, as well as GCs increased
due to maternal stress, increase SP-C mRNA levels (Durham et al., 1993).

545
546 Stress Neurobiology and Developmental Psychopathology
decline. For example, in the pyramidal region of the hip-
pocampus and frontal cortex, CRHr1 are two- to fourfold
higher during the 1st week of life and then decline to adult
levels by postnatal day 12 (Avishai-Eliner, Yi, & Baram,
1996). CRHr2 are detected in the frontal cortex only dur-
ing fetal and early postnatal life (Eghbal-Ahmadi et al.,
1998). These distinct developmental profiles indicate a pre-
cise regulation of these receptors during development and
the possibility that alteration of these profiles in areas crit-
ical to physiological and behavioral responses to stressors
may be targets of early experience effects.
Monoamines
During the postnatal period in the rat, monoamine cir-
cuitry is being laid down and fine-tuned. The pattern of
overproliferation and pruning of the major neurotransmit-
ter systems in the rat (discussed in Vazquez et al., 1998)
and human (summarized in Spinelli, 1987) have been de-
scribed elsewhere. For example, in the rat, 5-HT-contain-
ing cell bodies are present at birth, reach maximum density
by PND 14, and then decline to the adult innervation pat-
tern by puberty (i.e., PND 35 to 45). The 5-HT receptor
system follows a similar developmental course. Notably,
development of the serotonin system corresponds to devel-
opment of the LHPA stress response. Thus, increased levels
of brain 5-HT metabolites following stress are observed
around PND 12 in the rat pup, which is also when the
LHPA response to stress becomes more predictable
(Mitchell, Iny, & Meaney, 1990). GCs also influence the
development of the 5-HT system, affecting the expression
of the rate-limiting enzymes for 5-HT production (Singh,
Corley, Phan, & Boadle-Bider, 1990). A similar develop-
mental pattern is observed for the central NA system that
again is linked closely to the ability of the NA system to ac-
tivate the LHPA system (Herrenkohl, Ribary, Schlumpf, &
Lichtensteiger, 1988). Here, too, GCs influence activity of
the adrenergic system, in part through regulating levels of
its rate-limiting enzymes (Markey, Towle, & Sze, 1982).
Thus, there are bidirectional interactions between the abil-
ity to mount an LHPA response and the development of the
brain monoaminergic systems.
Glucocorticoid Receptors
Both MR and GR receptors develop quite early in gesta-
tion in the rat and presumably also the human brain.
Overall, MR density is greater during the early postnatal
period than later in life in the rat (Vazquez et al., 1998;
Vazquez, Morano, Lopez, Watson, & Akil, 1993). GRs
are observed as early as GD 13 and are evident between
GD 15 and 22 in the hippocampus, mpPVN, and locus
coeruleus. Different regions of the hippocampus display
different developmental progressions. MR and GR are most
abundant around PND 10 in the rat, with GR mRNA reach-
ing adult levels after this time. In contrast, MR mRNA in
the hippocampal dentate gyrus does not reach adult distri-
butions until around PND 28 (for review, see Vazquez,
Morano, et al., 1993). Overall, the hippocampal GR system
matures long before the animal is capable of feedback inhi-
bition. This likely reflects continued immaturity of connec-
tions of the hippocampus to the mpPVN (Vazquez & Akil,
1993). As noted earlier, emotional reactivity appears to be
linked to hippocampal corticoid receptor expression, with
an optimal balance of MR to GR argued to be critical to a
number of functions (de Kloet, 1991). The MR and GR sys-
tems exhibit developmental changes and are highly sensi-
tive to early experiences in the rat.
Circulating Glucocorticoids and Their Role
in Development
Low GC levels in young organisms do not necessarily mean
low levels of biologically active hormone. Most GCs in ma-
ture animals are bound to corticosteroid-binding globulin
(CBG) and other proteins. Only the unbound or free GC can
bind to receptors and thus have biological effects. In neona-
tal rats and humans, CBG and other binding globulins are
low, and thus most circulating GCs have biological activity
(Hadjian, Chedin, Cochet, & Chambaz, 1975; Henning,
1978). Furthermore, the clearance of GCs from the circula-
tion is significantly slower in the pup and in the human
newborn than in the adult. Thus, despite low plasma levels
of GCs during the SHRP in rats and during the newborn pe-
riod in humans, the levels of biologically active GCs are
more than sufficient to have marked physiological effects.
GCs are steroid hormones, and as such, their receptors
do not lie on the surface of cells (as is the case for neuro-
transmitters) but in the cytosol inside the cell (Lombroso &
Sapolsky, 1998). In the cytosol of brain cells, GCs bind
with either MR or GR to form GC-receptor complexes that
then move into the cell nucleus and bind to the promoter re-
gions of many different genes, initiating or inhibiting syn-
thesis of different cell proteins. Rapid brain growth,
accelerated synaptogenesis, myelinization, astrocyte pro-
liferation, and the organization of multiple neurotransmit-
ter systems characteristic of the developing organism
definitely require GCs. Thus, changes in GC activity can
be significant to the developing brain. However, for GCs to
affect brain development, they must get into the brain and
the cell must express MR or GR. In addition, having the re-
ceptors is not enough; GCs must also be able to engage the
necessary intracellular mechanisms following receptor
 The Developing Limbic-Hypothalamic-Pituitary-Adrenocortical System
binding (reviewed in C. D. Walker et al., 2002). Finally, the
impact of GCs on the developing brain also depends on the
maturity of other signaling systems and their capacity to
interact with GCs. Given that signaling systems have their
own development time courses that vary with brain region,
the effects of GCs on the developing brain cannot be simply
stated. The effects depend on brain region, maturity and
activity of other signaling systems, and developmental tim-
ing. The interaction of these factors, in addition, varies
with species in ways that are not well understood. This
poses a major challenge in translating preclinical studies to
predictions about GC effects on human development.
As noted earlier, most circulating GCs in the fetus and
neonate are unbound and thus potentially biologically ac-
tive. Furthermore, circulating levels of maternal GCs in-
crease in late pregnancy. This might seem to place the fetal
brain at high risk of GC exposure. However, under normal
circumstances, in rats and primates high placental levels of
the enzyme 11ß-HSD rapidly convert GCs to biologically
inert forms (Seckl, Cleasby, & Nyirenda, 2000). There are
two known isoforms of 11ß-HSD. Type 1 is bidirectional,
capable of converting GCs to inactive forms and then back
again, whereas the Type 2 form converts GCs only to less
active forms. The expression of the Type 2 form increases
with gestation but varies in efficiency among species. In
humans, the Type 2 form is the most important, but does
not inactivate dexamethasone (DEX) or betamethasone,
two GCs used medically to expedite fetal lung maturation
in women at risk for premature delivery (Seckl et al.,
2000). Thus, in humans, these two medically prescribed
GCs pass readily from mother to fetus.
Studies in which endogenous GC was administered to
fetal rats show that high levels preferentially affect matu-
ration of neurons in the cerebral cortex, reticular forma-
tion, limbic system, and spinal cord. They also globally
inhibit neurogenesis, gliogenesis, cell division, and myelin-
ization throughout the brain by altering patterns of gene
expression (see Table 13.1). GC-induced cell death and cell
survival mechanisms are also normally involved in remod-
eling of neurocircuit structure and function (e.g., Duman,
Malberg, & Thome, 1999). The effects of GCs on fetal
brain development have been examined in many studies
using DEX, because, as noted, this synthetic hormone is
used medically in premature infants (reviewed in
Matthews, 2000) and bypasses the placental 11ß-HSD sys-
tem. These studies indicate that DEX can have profound ef-
fects on brain development. DEX exposure in the last week
of gestation results in adult rats with reduced NA content in
the hippocampus and cortex and reduced NA turnover in
the forebrain. However, maturation of the adrenergic sys-
547
tem is actually advanced in the brain stem and forebrain
and there is an overexpression of NA transporter, allowing
increased NA recycling. No effect has been detected on
adrenergic receptor expression. Interestingly, animals ex-
posed to DEX in utero have increased LHPA function and
faulty negative feedback. It is unclear whether DEX-
induced modifications of the NA system are involved in
producing these changes in LHPA function. Indeed, these
changes actually may be due to serotonin. In rats, DEX in
the last week of gestation leads to male offspring with in-
creased hypothalamus, hippocampal, and brain stem 5-HT
concentrations, probably due to an increase in 5-HT trans-
porter synthesis in the raphe. The combination of increased
hypothalamic expression of 5-HT transporter and in-
creased availability of 5-HT are, in part, responsible for
the elevated LHPA activity observed in DEX-exposed male
offspring. Additionally, impairment in negative feedback
in the DEX-exposed fetus may be due to reduced NA levels
and 5-HT turnover that may reduce hippocampal GR recep-
tor numbers (reviewed in Matthews, 2000).
Behavioral effects of developmental DEX exposure have
also been observed in rodents (reviewed in Matthews,
2000). In mice, a single DEX injection on PND 14 affects
fear behavior, memory, and social interactions but not
sensory, motor, motivation, and learning performance. In
primates, DEX and cortisol administered during fetal de-
velopment have profound effects on the morphology of the
hippocampus. Although behavioral effects have not been
reported in rhesus monkeys, in humans DEX exposure be-
ginning before the 10th week of gestation and continued
until delivery results in heightened emotionality, unsocia-
bility, avoidance, and behavioral problems in children 6
months to 6 years of age (Trautman, Meyer-Bahlburg,
Postelnek, & New, 1995). Although many older studies of
the effects of antenatal steroids in humans have yielded lit-
tle evidence of long-term effects, recent studies have
demonstrated reduced brain volumes at birth (Modi et al.,
2001; Murphy, 2001). Thus, it is clear that GCs have a crit-
ical role in brain development.
Development and the Limbic-Hypothalamic-
Pituitary-Adrenocortical System in Humans
The developmental neurobiology and physiology of the
LHPA system in humans are not well understood. Pri-
mates, including humans, do possess a fetal adrenal zone
that produces estrogen precursors during gestation
(Mesiano & Jaffe, 1997). In humans, this zone involutes
over the first 6 postnatal months (Reynolds, 1981). CBG
levels are low in the human neonate, increasing over the
same age period (Hadjian et al., 1975). Consequently, free
548 Stress Neurobiology and Developmental Psychopathology
or unbound cortisol levels decrease slightly, and plasma or
total cortisol production increases over the first 4 to 6
months after birth. There is also evidence that the adrenal
becomes less sensitive to ACTH over the 1st year (Lashan-
sky et al., 1991). Thus, as in the rodent, the pituitary-
adrenocortical system continues to develop postnatally.
Whether there is a human or primate equivalent of the
SHRP is highly uncertain; however, numerous studies
show developmental changes in GC responses from the
prenatal through the adolescent period. By 18 to 20 weeks
of gestation, the fetal LHPA system produces increases in
cortisol to aversive stimulation (Giannakoulpoulous,
Sepulveda, Kourtis, Glover, & Fisk, 1994). With increased
gestational age, basal levels of plasma cortisol and ACTH
increase. Healthy, term babies are capable of mounting
cortisol and ACTH reactions to the types of perturbations
routinely encountered in the newborn period (i.e., physical
examinations, heel stick blood draws, and for boys, cir-
cumcision; reviewed in Gunnar, 1992).
Cortisol stress responses decrease over the infancy pe-
riod (e.g., Gunnar, Brodersen, Krueger, & Rigatuso, 1996;
Lewis & Ramsay, 1995). On average, it becomes difficult
to produce elevations in cortisol to both laboratory and
mild medical stressors by the end of the 1st year (see Gun-
nar & Donzella, 2002), although the absolute change (irre-
spective of increase or decrease) is similar in the 2nd year
as compared to that observed around 6 months of age
(Lewis & Ramsay, 1995). It is unlikely that this diminution
of cortisol responses is equivalent to the rodent SHRP be-
cause it is highly dependent on the immediate presence and
support of the caregiver. This was clearly demonstrated in
a recent study of toddlers entering child care (Ahnert,
Gunnar, Lamb, & Barthel, 2004). During an adaptation pe-
riod of several days with the mother present, infants in se-
cure attachment relationships showed only very small
increases in cortisol over home baseline levels. However, on
the 1st day at child care without the mother and continuing
for at least the first 9 days, large increases in cortisol were
noted for these same infants. Over the course of the pre-
school years, children become more capable of maintaining
basal cortisol levels in the absence of their attachment fig-
ures (for review, see Gunnar & Donzella, 2002). A number
of mechanisms likely account for the developmental
changes in GC responsivity over the infancy and preschool
years, including the development of the attachment system
and the child’s developing social and emotional regulatory
competence.
Whereas GC responses tend to decrease over the early
childhood years, it has been argued that, with the transition
to adolescence, children become more hormonally respon-
sive and thus perhaps more vulnerable to stressors (e.g.,
Spear, 2000). Several studies have shown both between-
subject correlations of age with basal cortisol levels and
within-subject increases in basal GCs in children 6 to 17
years of age (e.g., Kiess et al., 1995; Legro, Lin, Demers, &
Lloyd, 2003; Netherton, Goodyer, Tamplin, & Herbert,
2004; Shirtcliff et al., in press). Three studies suggest that
the most marked increase in basal GCs occurs between 10
and 14 years (Elmlinger, Kuhnel, & Ranke, 2002; Lupien,
S. King, Meaney, & McEwen, 2001; Tornhage, 2002); oth-
ers yield evidence of more gradual, linear increases
(Jonetz-Mentzel & Wiedemann, 1993; Lashansky et al.,
1991; E. F. Walker et al., 2001). Studies using Tanner stag-
ing have suggested that increases in basal levels are ob-
served around Tanner stage 3 (Halligan, Herbert, Goodyer,
& Murray, 2004; Netherton et al., 2004). Thus, although
basal GC levels do seem to increase from childhood
through adolescence, the precise timing and certainly the
mechanisms underlying this effect are not well understood.
All of these studies examined salivary or plasma corti-
sol levels, which do not provide an integrated measure of
total cortisol production over the day. Studies using inte-
grated urinary cortisol sampling tend to be at odds with the
plasma /salivary studies. Specifically, although 24-hour
urinary free cortisol concentrations do increase across the
adolescent years, this finding is no longer significant if
urine volume (creatine) and body mass are taken into ac-
count (Dimitriou, Maser-Gluth, & Remer, 2003; Gomez,
Malozowski, Winterer, Vamvoakopoulos, & Chrousos,
1991; Honour, Kelnar, & Brooks, 1991; Legro, Lin, De-
mers, & Lloyd, 2003). Lack of evidence for increases in in-
tegrated 24-hour cortisol production raises the possibility
that increases in basal cortisol with age and/or pubertal
status may reflect changes in diurnal patterning of cortisol
production rather than overall increases in basal cortisol
levels. In this regard, it is noteworthy that most of the stud-
ies showing increases in basal salivary or plasma cortisol
have collected samples in the morning hours. Furthermore,
when both morning and evening samples were obtained,
only the morning values were found to correlate positively
with pubertal status (Halligan et al., 2004; Netherton et al.,
2004). Thus, it is conceivable that changes in basal cortisol
may actually reflect the continued maturation of the adult
diurnal rhythm that may not become fully mature until
around the midpoint of pubertal development.
The vulnerability hypothesis (e.g., Spear, 2000), of
course, depends not on basal levels, but on reactivity of the
LHPA system. Basal levels may increase with age and/or pu-
bertal status, but it is not clear whether reactivity of the
LHPA system also increases. There are simply too few stud-
 The Developing Limbic-Hypothalamic-Pituitary-Adrenocortical System
ies that actually have produced elevations in cortisol to ad-
dress this question. E. F. Walker et al. (2001), however, have
interpreted their findings as evidence of increased reactivity
to the experience of coming to the lab for testing. Studying
children 11 to 18 years of age, they took four salivary corti-
sol measures during a laboratory stressor paradigm. Cortisol
levels decreased across the stressor period, suggesting that
the initial levels were in fact elevations over baseline. It was
these initial levels that were positively correlated with age.
Furthermore, several years later, when they brought the
children in again for testing, only the initial levels were
higher than the child’s own initial levels several years be-
fore. However, two studies using the TSST modified for
children failed to note significant effects of age when chil-
dren 9 to 16 years were studied (Klimes-Dougan, Hastings,
Granger, Usher, & Zahn-Waxler, 2001; Kudielka, Buske-
Kirschbaum, Hellhammer, & Kirschbaum, 2004). There is
also no evidence that the adrenal becomes more sensitive to
ACTH (Dahl et al., 1992; Lashansky et al., 1991) or that
CRH infusion elicits overall more cortisol and ACTH with
age for children between 6 and 16 years (Stroud, Papando-
natos, Williamson, & Dahl, 2004). Thus, if the LHPA axis
becomes more reactive during adolescence, the effect is
likely to be subtle.
All but one of these studies involved typically develop-
ing children. Notably, when risk populations are used,
there is no evidence of age changes in cortisol activity,
basal or response values, over the transition to adolescence
(Dahl et al., 1991; Dorn et al., 2003; Goodyer, Park,
Netherton, & Herbert, 2001; Granger, Weisz, & Kauneckis,
1994; McBurnett et al., 1991; McBurnett, Lahey, Rathouz,
& Loeber, 2000; Scerbo & Kolko, 1994). It seems possible
that if changes in cortisol levels and reactivity with age are
generally subtle, these age changes may be readily over-
whelmed by the stress of emotional problems or life events
in high-risk populations.
Sex Differences and Sex Steroids
Subtle changes in basal levels and/or reactivity of the
LHPA system during adolescence, if they exist, may be re-
lated to changing sex steroid activity. In rats and in hu-
mans, sex differences are apparent at many levels of the
LHPA axis. In rats, estrogens have excitatory effects and
androgens have inhibitory effects on LHPA function. Thus,
in rats, females are the more stress-reactive sex (Critchlow,
Liebelt, Bar-Sela, Mountcastle, & Lipscomb, 1963; Le
Mevel, Abitbol, Beraud, & Maniey, 1979), and both basal
and stress levels of ACTH and GC are high when estrogen
levels are elevated (e.g., Bohler et al., 1990). Ovariectomy
reduces plasma ACTH and GCs, and replacement of estra-
549
diol returns ACTH and GCs to normal levels (Burgess &
Handa, 1992; Viau & Meaney, 1991). Females also exhibit
high levels of CBG (Gala & Westphal, 1965) and CRH (Hi-
roshige & Wada-Okada, 1973). In addition, gonadal hor-
mones modulate GC metabolism (Grant, Forsham, &
DiRaimondo, 1965) and hippocampal MR and GR protein
and mRNA levels (e.g., Kerr, Beck, & Handa, 1996; Viau
& Meaney, 1996).
In humans, the evidence is fairly consistently the re-
verse. Men show larger GC and ACTH responses than
women to most stressors administered in the laboratory,
but are not more reactive than females to systemic or
pharmacological stressors (for reviews, see Kirschbaum,
Kudielka, Gaab, Schommer, & Hellhammer, 1999;
Kirschbaum, Wüst, & Hellhammer, 1992). Men also show
larger catecholamine responses than women to stressors
such as IQ testing, cognitive-conflict tasks, major examina-
tions, and speech tasks (e.g., Frankenhaeuser, 1983). The
greater male vulnerability to processive stressors has been
related to their greater vulnerability to cardiovascular dis-
ease and stroke, disorders related to hyperstress reactivity.
In contrast, the blunted response of women to stressors has
been related to their greater vulnerability to hyporespon-
sivity disorders, most notably autoimmune and chronic
pain disorders (McEwen, 1998). Depression stands in
marked contrast to these patterns of sex difference, being
associated with hypercortisolism (Nemeroff, 1996) and
being more prevalent in women, beginning about midado-
lescence (Piccinelli & Wilkinson, 2000).
The central gender stress question always has been
whether these male-female differences in humans are psy-
chological or physiological. Those arguing for psychologi-
cal differences note that men and women likely process and
cope with stress differently (e.g., Taylor et al., 2000). In
addition, they note that most of the stressors examined in
human studies have been instrumental or achievement-ori-
ented and that these types of stressors likely threaten the
social self in ways that are more central to male self-
construals (see Stroud, Salovey, & Epel, 2002). Consistent
with this argument is evidence that males compared to fe-
males excreted more adrenaline during major university
entrance exams. But, among the young women, those who
were adrenaline increasers were more likely to have
adopted male achievement values, whereas adrenaline de-
creasers were more likely to place a high value on marriage
and parenting and to perceive themselves as fulfilling tra-
ditional feminine social roles (Rauste-von Wright, von
Wright & Frankenhauser, 1981). More pertinent is evi-
dence that stressors that threaten affiliation are more
provocative among women than men. Using participants
550 Stress Neurobiology and Developmental Psychopathology
age 17 to 23, Stroud and colleagues recently showed that
men elevated cortisol to a speech/math stressor, and
women elevated cortisol to a social rejection stressor. If
replicable, these data suggest serious limitations in the lit-
erature on LHPA responses to processive or psychological
stressors in human adults—specifically, that the literature
is limited to examination of male-biased stressors. We lack
adequate evidence of sensitivity to processive stressors in
women because we have failed to identify stressors that are
ideally designed to threaten core female self-construals.
In contrast, for those taking a physiological perspective
on adult sex differences in LHPA reactivity, there is evi-
dence that the sex differences in response to achievement-
type stressors varies with the woman’s menstrual cycle.
Thus, cortisol responses to the TSST do not differ between
men and women in the follicular phase of their cycles
(when estrogen levels rise to their monthly peak), but men
respond more than women in the luteal phase of the cycle
(when estrogen levels are falling; Kirschbaum et al., 1999).
These data suggest that failure to control for stage of men-
strual cycle may impede our understanding of sex differ-
ences in reactivity of the LHPA system in human adults.
According to the physiological argument, we would not ex-
pect to observe sex differences in basal cortisol at any point
in childhood, but we would expect to observe sex differ-
ences in responses to processive stressors beginning around
the midpoint in puberty. The child data are relatively con-
sistent with these predictions. Of over 20 studies of basal
cortisol that included boys and girls, we found only three
that reported significant sex differences, one showing
higher levels for girls (Smider et al., 2002) and two for boys
(Elmlinger et al., 2002; Jonetz-Mentzel & Wiedemann,
1993). Studies of GC responsivity to stressors similarly re-
port few sex differences in infancy and early childhood.
One study of newborns did report larger increases for boys
(M. Davis & Emory, 1995), and a study of peer entry stress
in preschoolers reported larger increases for girls (Sethre-
Hofstand, Stansbury, & Rice, 2002). There is some sugges-
tion, however, that studies involving competitive challenges
might produce larger GC responses in prepubertal boys
than girls (Donzella, Gunnar, Krueger, & Alwin, 2000;
Jansen et al., 1999; Kertes & Gunnar, 2004). Furthermore,
one study noted that prepubertal boys showed larger GC re-
sponses to CRH than did girls (Dahl et al., 1992). However,
overall, prior to adolescence, sex differences in GC activ-
ity tend to be infrequently reported.
Sex differences in response to processive stressors ap-
pear to emerge in adolescence. Male adolescents taking
university entrance exams exhibit cortisol increases, but fe-
males typically do not (Frankenhaeuser et al., 1978). Boys
between 14 and 16 years of age produce larger cortisol re-
sponses than girls to a psychosocial stressor, but this was
not the case among younger, 10- to 13-year-old children in
the same study (Klimes-Dougan et al., 2001). Among 13-
to 17-year-old adolescents, boys have also been observed to
exhibit a larger ACTH response to CRH administration
(Dorn et al., 1996; although see Stroud et al., 2004). Thus,
in a number of studies of typically developing children,
boys begin to exhibit larger LHPA responses than girls
around the midpoint in puberty. As noted, however, the
types of stressors examined to date may be male-biased, in-
volving threats to instrumental competence. We simply do
not know whether girls respond more to threats to affilia-
tive competence (i.e., social rejection) and, if so, whether
such sex differences in stress reactivity, if they exist,
emerge or change from childhood through adolescence.
PSYCHOPATHOLOGY
Adverse early experiences in human and nonhuman pri-
mates increase the variability of LHPA functioning, in
some individuals producing hyperreactivity and in others,
seemingly, hyporesponsivity. This may partly reflect inter-
actions between genetic dispositions to particular disorders
and experience. Prior to reviewing the early experience lit-
erature, we provide a brief overview of patterns of LHPA
activity in three types of behavior disorders associated
with adverse early rearing environments: disruptive behav-
ior disorder, anxiety disorders, and depression.
Disruptive Behavior Disorders
Although there are several models of the processes under-
lying Oppositional Defiant Disorder (ODD) and Conduct
Disorder, referred to collectively as disruptive behavior
disorders (DBD), they all revolve around constructs of un-
derarousal and underreactivity (Burke, Loeber, & Birma-
her, 2002). The general idea is that children at risk for
DBD are underreactive to threat stimuli, resulting in im-
paired avoidance learning and underarousal, which may en-
courage sensation seeking as a means of increasing arousal.
None of the models is complete, and most assume that they
may apply only to subgroups of children with DBD (Burke
et al., 2002; McBurnett & Lahey, 1994).
Consistent with models of underarousal, low basal or
pretest cortisol has been reported in a number of studies,
most using variants of the following protocol. Children ar-
rive for testing in the morning, often around 9 A.M. A

saliva sample is taken, after which the child is subjected
to a mild stressor task, and a second saliva sample is taken
at the end of testing 30 to 40 minutes later. Typically de-
veloping children exhibit high cortisol levels at pretest
with decreasing levels over the testing period. In contrast,
DBD children or those at high risk for DBD have low cor-
tisol levels at pretest that do not change over the testing
period (Hardie, Moss, Vanyukov, Yao, & Kirillovac, 2002;
King, 1998; Moss, Vanyukov, & Martin, 1995; Moss,
Vanyukov, Yao, & Kirillovac, 1999). This pattern has
been observed in both boys and girls (Hardie et al., 2002;
Pajer, Garner, Rubin, Perel, & Neal, 2001). Even in stud-
ies that have failed to obtain group differences in cortisol
between DBD and control children, disruptive behavior
symptom counts have correlated with lower early morning
cortisol levels (Pajer, Gardner, Kirillovac, & Vanyukov,
2001; Scerbo & Kolko, 1994; Vanyukov et al., 1993). Al-
though most cortisol studies of DBD children have sam-
pled cortisol in the early morning, not all have. McBurnett
and colleagues (McBurnett, Lahey, Frick, et al., 1991;
McBurnett, Lahey, & Rathouz, et al., 2000) have reported
lower cortisol levels for DBD children, despite not con-
trolling for time of day.
Perhaps more significant, several studies have demon-
strated that low cortisol levels predict later disruptive be-
havior problems. Granger, Weisz, and McCracken (1996)
reported that clinic-referred 9- to 16-year-olds who had
low cortisol levels at the beginning of a late-afternoon lab-
oratory test displayed more delinquency at follow-up test-
ing several months later. Similarly, in another study (van
de Wiel, van Goozen, Matthys, Snoek, & van Engeland,
2004), 8- to 13-year-old boys with DBD who had cortisol
levels below the median had higher externalizing, ODD,
and overt aggression scores at a 9-month follow-up assess-
ment than did boys with cortisol levels above the median.
Finally, in a study of several hundred adolescent boys,
resting cortisol levels assessed when the boys were be-
tween 10 and 12 years of age were negatively correlated
with self-reported aggression and positively correlated
with constraint, control, and harm avoidance 5 years later
(Shoal, Giancola, & Kirrilova, 2003). Thus, DBD children
and those who develop DBD symptoms appear to have
lower cortisol levels than do nondisordered children. Low
basal or pretest cortisol would be consistent with an un-
derarousal hypothesis, and through impairment of permis-
sive actions of GCs might reduce responsiveness of other
stress-sensitive systems (e.g., brain monoamines).
What is not as well established is whether low reactivity
of the LHPA system is associated with DBD. To examine
the underreactivity hypothesis requires that experimentally
Psychopathology 551
imposed stressors elevate cortisol in the control or compar-
ison group. To date, only two studies have met this crite-
rion (van Goozen, Matthys, Cohen-Kettenis, Buittelaar, &
van Engeland, 2000; van Goozen et al., 1998). Van Goozen
and colleagues exposed severely disordered DBD children
and controls to a task during which a peer (on audiotape)
denigrated their performance. When the paradigm was
conducted during the morning hours, the task prevented the
normal decrease in cortisol among the typically developing
and DBD children. When performed in the afternoon
hours, typically developing but not DBD children exhibited
significant cortisol increases. In addition, in both the
morning and afternoon studies, DBD children exhibited
lower heart rate and skin conductance than did the typi-
cally developing children. Of particular importance, in a
recent follow-up study, these researchers (van del Wiel
et al., 2004) examined the effectiveness of treatment for
DBD children as a function of cortisol reactivity to their
paradigm. Treatment included medication and individual
and family therapy. Children with DBD who exhibited no
cortisol response to their stressor paradigm also failed to
show improvement in DBD symptoms 9 months after the
onset of treatment, whereas those who did show a cortisol
response did show improvement. Thus, there is some evi-
dence from at least this research group that DBD, and per-
haps especially DBD that is resistant to treatment, is
associated with low GC responsivity.
However, not all DBD children exhibit low cortisol lev-
els. In fact, several researchers have noted that the combi-
nation of DBD and anxiety disorders is associated with
high rather than low cortisol activity relative to controls.
Thus, McBurnett and colleagues (1991) found higher levels
of cortisol among comorbid (DBD + anxiety) children than
in DBD-only children. In both of their studies, van
Goozen and colleagues (1998, 2000) found that DBD chil-
dren, but not normal controls, who showed a cortisol re-
sponse to the stressor task were more anxious and
depressed than those who did not respond. Finally, Scerbo
and Kolko (1994) found that parent reports of internalizing
problems were positively correlated with cortisol levels in
their DBD children. Thus, across all of the studies that in-
cluded measures of anxiety, low cortisol levels were typi-
cally observed only for those disruptive children who were
not clinically anxious. McBurnett and Lahey (1994) cau-
tion that to clarify the relations between cortisol and dis-
ruptive behavior problems, we may need to distinguish
between aggressive (undersocialized Conduct Disorder)
and nonaggressive forms of the disorder. The 10- to 12-
year-old boys studied by McBurnett and colleagues (1991)
who had the lowest cortisol levels were also described by
552 Stress Neurobiology and Developmental Psychopathology
peers as the meanest and by adults as having the most ag-
gressive symptoms. Pajer and colleagues (2001) noted a
similar, but nonsignificant, difference between the aggres-
sive and nonaggressive DBD girls.
It is unclear why at least some subtypes of DBD chil-
dren might exhibit low basal cortisol levels and possibly
hyporeactivity of the HPA axis. Studies of DBD children’s
response to serotonergic challenges have shown that these
children exhibit blunted cortisol reactions to fenfluramine
(Soloff, Lynch, & Moss, 2000) and sumatriptan (Snoek
et al., 2002), suggesting that serotonergic regulation of the
LHPA system is compromised. Monkeys carrying the short
version of the serotonin transporter allele are especially
vulnerable to disturbances in the early caregiving environ-
ments that elevate cortisol (Bennett et al., 2002). Studying
children of substance-abusing fathers, investigators have
hypothesized that low cortisol levels in these children may
be the product of disturbances in their family environment
(Hardie et al., 2002; Pajer, Gardner, et al., 2001). Thus,
low cortisol levels may be the result of down-regulation of
the axis following periods of elevated cortisol. Unfortu-
nately, none of the studies of DBD children or those at risk
for DBD problems has started assessing children’s neu-
roendocrine and autonomic reactivity during the first
years of life.
In this regard, it is useful to examine relations between
disruptive behavior and cortisol activity in community sam-
ples of primarily nondisordered children. For school-age
and older children, the evidence fairly consistently points to
significant associations between low cortisol and more ex-
ternalizing-type behaviors (e.g., Flinn & England, 1995;
Spangler, 1995; Tennes & Kreye, 1985; Tennes, Kreye,
Avitable, & Wells, 1986). Using a very large sample of 6- to
16-year-old children, Shirtcliff and colleagues (in press)
applied latent state-trait modeling techniques to identify
the trait component of early morning cortisol production.
Then they examined whether the trait component was asso-
ciated with either internalizing or externalizing problem
behavior in the nonclinical range. For boys, but not girls,
they found a significant negative relation between trait cor-
tisol shortly after waking and externalizing problems.
Studies of typically developing children under the age of
7, when oppositional and aggressive behaviors are more
normative, have yielded weaker and more mixed results.
Lower cortisol levels for more aggressive children have
been noted in two studies (de Haan, Gunnar, Tout, Hart, &
Stansbury, 1998; Hart, Gunnar, & Cicchetti, 1995). Larger,
rather than smaller, cortisol reactivity among more aggres-
sive children to new peer situations was noted in three
studies (de Haan et al., 1998; Dettling, Gunnar, &
Donzella, 1999) but not in other similar studies (Bruce,
Davis, & Gunnar, 2002; Dettling, Parker, Lane, Sebanc, &
Gunnar, 2000; Gunnar, Tout, de Haan, Pierce, & Stans-
bury, 1997). Thus, during the toddler and preschool years,
when aggression and oppositional behavior is observed at
relatively high frequencies, the relationship between these
behaviors and cortisol is unreliable in typically developing
children. However, children who persist in more aggres-
sive, oppositional behavior during the school-age and ado-
lescent years, even though their behavior is within the
nondisordered range, do appear to have lower basal and
perhaps less reactive LHPA systems.
Anxiety Disorders
Pathological anxiety in animals and humans may reflect an
exaggeration of normal anticipatory LHPA responses
(Rosen & Schulkin, 1998). This argument builds on related
models of the LHPA system’s role in the pathophysiology of
affective disorders (e.g., Gold, Goodwin, & Chrousos,
1988). Anticipatory anxiety involves phasic immobility or
freezing, autonomic changes (e.g., increased sympathetic
discharge), increased neuroendocrine activity (e.g., ele-
vated levels of GCs), heightened reflexive responses to
sensory stimuli (e.g., fear-potentiated startle), hypoanalge-
sia, and increased urination and defecation (Rosen &
Schulkin, 1998). In the presence of elevated GCs and CRH,
a cascade of biomolecular events that include increased ex-
pression of immediate-early genes (Makino, Gold, &
Schulkin, 1994) increases the sensitivity of central fear cir-
cuits, heightens anxiety to distal danger cues, and supports
the transition from normal to pathological anxiety.
A number of studies of anxiety-disordered children re-
veal elevations in basal GC levels (Carrion et al., 2002;
DeBellis, Baum, et al., 1999; Klimes-Dougan et al., 2001;
however, see Martel et al., 1999 for a counterexample). In
addition, there is also growing evidence that clinically anx-
ious children are hyper-GC-responsive to some types of
stressors, including air puff startle (Ashman, Dawson,
Panagiotides, Yamada, & Wilkinson, 2002) and mother-
child conflict discussions (Granger, Weisz, et al., 1994), al-
though such associations have not always been found (Dorn
et al., 2003; Martel et al., 1999). Cortisol reactivity has
also been shown to predict subsequent anxiety symptoms
among clinic-referred adolescents (Granger et al., 1996).
Finally, as discussed earlier, anxiety plus DBD is associ-
ated with higher cortisol activity than observed among
control and nonanxious DBD children.

If hyperresponsivity of the LHPA axis constitutes a
diathesis for the development of anxiety disorders, then we
should find evidence of heightened LHPA axis activity in
extremely temperamentally inhibited and/or socially reti-
cent children who are at risk for the development of these
disorders (Rosenbaum et al., 1993). However, because
novel situations should elevate cortisol in nonanxious as
well as anxious individuals, this may be difficult to demon-
strate. Many studies of adults have shown that GC eleva-
tions are larger the first time individuals confront a
challenging or threatening event and diminish markedly
with repetition (as reviewed in Kirschbaum & Hellhammer,
1989). Elevating GCs in anticipation of challenge thus is
not pathological but instead is an adaptive function of the
LHPA axis (Rosen & Schulkin, 1998; Sapolsky et al.,
2000). In studies of adults and animals, individuals who ex-
hibit the largest increases in cortisol under normative con-
ditions of uncertainty are sometimes more dominant and
assertive, rather than shy or socially reticent (e.g., Golub,
Sassenrath, & Goo, 1979; Hellhammer, Buchtal, Ingmar, &
Kirschbaum, 1997). Thus, whereas anxious individuals
may exhibit anticipatory cortisol reactions, so may compe-
tent, assertive individuals. However, as most individuals
should show habituation of the LHPA axis response upon
repetition of the same stressor, we might find evidence that
failure to habituate is associated with risk for developing
anxiety and depression.
Indeed, this conclusion appears to be the thrust of work
by Pruessner and colleagues (1997). Personality differ-
ences among adult subjects did not predict elevations in
cortisol to the first stressor trial. However, although most
subjects habituated on subsequent trials, responses on
these trials were significantly and highly correlated with
two risk factors in the etiology of affective disorders: low
self-esteem and external locus of control. In a similar
vein, van Eck and colleagues (van Eck, Berkhof, Nicolson,
& Sulon, 1996; van Eck, Nicolson, Berkhof, & Sulon,
1996) demonstrated that high trait-anxious individuals
were more likely to exhibit cortisol elevations to familiar
daily hassles (i.e., being late for the bus, a disagreement
with a coworker). This was the case even though high trait
anxiety was not associated with larger GC responses the
first time individuals were subjected to a laboratory stres-
sor (i.e., the TSST). Taken together, these adult findings
suggest that continuing to activate the LHPA axis to fa-
miliar stressors, rather than exaggerated activation to a
novel stressor, may form part of the diathesis for the de-
velopment of pathological anxiety. If so, then during de-
velopment, cortisol activity might correlate less often
Psychopathology 553
with inhibited, socially reticent temperament when chil-
dren are tested responding to novel, unfamiliar stressors
than when they are tested reacting to familiar challenges
at home or school.
There is some support for this hypothesis. Young chil-
dren who exhibited more facial expressions of fear to emo-
tion-eliciting laboratory events did not have higher cortisol
levels in the laboratory, but did at home (Buss et al., 2003).
Likewise, in another study, shy/anxious temperament pre-
dicted home but not school levels of cortisol during the ini-
tial weeks of preschool (de Haan et al., 1998). Similarly,
Schmidt and colleagues (Schmidt & Fox, 1998; Schmidt
et al., 1997) did not find that extremely shy, inhibited chil-
dren produced higher GC responses to a laboratory stressor,
but did have higher home early morning GC levels. Kagan,
Reznick, and Snidman (1987) reported similar results.
Using new peer group settings, several researchers have
noted higher cortisol levels among more anxious, intro-
verted children, but only once the play group setting was
familiar (e.g., Granger, Stansbury, & Henker, 1994). In one
study of children entering a new playgroup, anxious chil-
dren did have high cortisol levels, but so did the most so-
cially outgoing children (Legendre & Trudel, 1996). In
several studies of children starting new school years, we
(Bruce et al., 2002; E. Davis, Donzella, Krueger, & Gun-
nar, 1999) noted that by the 5th day of school, a time when
the situation should still be novel especially for first-grade
children, exuberant or extroverted children had higher cor-
tisol levels relative to weekend baseline days earlier in the
day prior to and during school. In contrast, shy, inhibited
children exhibited elevated bedtime cortisol levels hours
after they had returned home. Thus, increasing cortisol lev-
els to novel, uncertain stressors may not be unique to shy,
inhibited children, but, shy, inhibited children may be less
likely to habituate cortisol responses as the situation be-
comes more familiar, and they may be less able to turn off
the cortisol response once they have left the novel situation.
Studies examining relations between fearful tempera-
ment and physiological reactivity typically have used labo-
ratory challenges to elicit wary or fearful behavior (e.g.,
Kagan et al., 1987). Fussing, crying, seeking proximity
with attachment figures, and withdrawing from or avoiding
contact with the threat stimuli are then used to measure
fear. One reason associations between fearful behavior and
cortisol may be low is that these behaviors also reflect ac-
tive attempts to manage or control threat. As long as the in-
dividual possesses responses to cope with threat, we would
not expect to see elevations in GCs. This may be why sev-
eral researchers now report that freezing may be the fear
554 Stress Neurobiology and Developmental Psychopathology
behavior that is the most closely correlated with increases
in GCs and in sympathetic reactivity (Buss, Davidson,
Kalin, & Goldsmith, 2004; Gunnar & Nelson, 1994). Freez-
ing in situations where the child has many avenues avail-
able to exert control over the threat may be particularly
noteworthy (Buss et al., 2004).
We cannot tell from these studies whether cortisol activ-
ity also predicts the development of anxious, internalizing
problems. Some evidence suggests that it may. Essex and
colleagues (Essex, Klein, Eunsuk, & Kalin, 2002; Smider
et al., 2002) obtained salivary cortisol samples at home
when the children were 4.5 years of age, and these mea-
sures predicted internalizing problems in kindergarten a
year later and both internalizing and externalizing problem
behavior in first grade. Somewhat similar findings have
been reported in studies of young adolescents (Granger
et al., 1996; Susman, Dorn, & Chrousos, 1991; Susman,
Dorn, Inoff-Germain, Nottelman, & Chrousos, 1997). No-
tably, however, when behavior and cortisol are both used to
predict later behavior problems, behavior is the better pre-
dictor (Granger et al., 1996; Susman et al., 1991). However,
because cortisol levels are often based on only one or two
assessments, the greater predictive value of behavioral
measures may reflect their greater statistical reliability.
Finally, when family process variables are included in
studies of cortisol and internalizing problems, the family
measures predict both cortisol levels and internalizing
symptoms (Granger et al., 1998).
Depression
There is typically high comorbidity between anxiety and
depression. We consider depression here separately from
anxiety, however, because so much of the LHPA research
on psychiatric disorders has focused on Major Depressive
Disorder (MDD), often using anxiety-disordered individu-
als as a comparison group. Indeed, disturbance in LHPA
regulation in adults with MDD is among the most reliable
findings in biological psychiatry (see review, Pariante,
2003). Alterations are observed in both the diurnal rhythm
and negative feedback regulation of the axis. In many adult
MDD patients, basal levels of cortisol and ACTH remain
high throughout the day, resulting in hypercortisolemia and
a loss of the normal diurnal rhythm. Fast-feedback regula-
tion of the axis is impaired, resulting in larger and more
prolonged elevations to stressors and failure to suppress
cortisol to DEX. Chronic CRH drive and high cortisol lev-
els produce down-regulation of pituitary ACTH and thus
blunted ACTH responses to CRH challenge. Blunted
growth hormone responses to a variety of challenges, in-
cluding stimulation with growth hormone releasing hor-
mone (GHRH), are also frequently observed, perhaps re-
flecting chronic CRH drive at the level of the pituitary
(Ryan, 1998). These results meet several of the criteria for
dysregulation outlined earlier in this chapter. Indeed, sev-
eral prominent theories of the etiology of depression focus
on dysregulation of the LHPA axis and/or of CRH (e.g.,
Holsboer, 2000; Nemeroff, 1996).
There have been a number of excellent reviews of LHPA
axis functioning in child and adolescent depression (e.g.,
Goodyer, Park, & Herbert, 2001; Ryan, 1998). Thus, we
only briefly review this area of research. Studies using am-
bulatory measures of salivary cortisol in depressed adoles-
cents often yield evidence of elevated GC levels, similar to
results obtained for adults (Goodyer, Herbert, & Altham,
1998; Goodyer, Herbert, Moor, & Altham, 1991). Higher
cortisol levels and higher cortisol: DHEA ratios may also
predict longer illness duration (Goodyer et al., 1998).
Nonetheless, fewer MDD adolescents than MDD adults ex-
hibit evening distortions of the LHPA rhythm (Dahl et al.,
1991; Forbes, Williamson, Ryan, & Dahl, 2004; Goodyer
et al., 1998). In addition, evidence for hypersecretion of
GC in early-onset depression is sparse when depressed
children and adolescents are studied under controlled labo-
ratory conditions. Under highly controlled sleep-laboratory
conditions, most MDD school-age and adolescent children
exhibit normal diurnal patterns of cortisol and ACTH pro-
duction relative to carefully screened, supernormal con-
trols (e.g., Dahl et al., 1991).
There are many other indications that MDD in children
and adolescents is less associated with endogenous dys-
regulation of the LHPA axis than it is in adults. Nonsup-
pression of cortisol to DEX is rare in MDD children and
adolescents, and ACTH and cortisol responses to CRH
are also typically normal, at least in depressed children
who have not also been physically abused (for review,
see Ryan, 1998). Perhaps important, however, MDD in
children, as in adults, is associated with blunted growth
hormone responses to provocative challenges as well as
to stimulation by GHRH, and this blunted response ap-
pears to continue after recovery from the illness (reviewed
in Ryan, 1998). As noted, this may reflect the impact
of chronic CRH drive. Failure to observe alterations in
the axis at the level of the pituitary and adrenal in de-
pressed children and adolescents may reflect the re-
silience of the axis to endogenous dysregulation during
childhood. Furthermore, this would suggest that such en-
dogenous dysregulation might become more apparent by
late adolescence or with more chronic, recurrent illness
(see also Kutcher et al., 1991).

Given that abnormalities in LHPA axis functioning are
less characteristic of early-onset depression than adult-
onset depression, it would be surprising to find that pre-
morbid activity of the axis predicts the development of
depression in high-risk samples. Nonetheless, there is
some evidence to this effect. Luby and colleagues (2003)
found that anhedonic preschoolers exhibited increases in
cortisol to mild laboratory challenges that were not noted
among children with more typical affective dispositions.
Among adolescents, highly variable early morning basal
cortisol levels across days predicted which adolescents
would experience their first bout of clinical depression
over the next months (Goodyer, Herbert, Tamplin, &
Altham, 2000). Specifically, when 4 days of 8 A.M. and
8 P.M. cortisol were obtained, adolescents who spiked
(exhibited at least one value in the upper 10% of the dis-
tribution of values) were 7 times as likely to develop de-
pression over the next few months. This effect was
specific to the morning cortisol samples (see also Halli-
gan, Herbert, Goodyer, & Murray, 2004). Loneliness has
also been shown to be a correlate of higher ambulatory
cortisol levels in typically developing, low-risk adoles-
cents, and for adolescents at school, being alone at the
time of sampling rather than being with friends has been
associated with increasing cortisol levels (Adam, 2002).
Thus, although an endogenous dysregulation of the LHPA
axis does not appear to be present in most children with
depression, heightened cortisol reactivity and higher lev-
els associated with loneliness suggest that activity of the
LHPA system may contribute to the etiology of depression
in children and adolescents. However, in many of these
studies of children at risk for depression, the role of anxi-
ety in potentially disturbing activity of the LHPA system
was not clearly ruled out. Thus, in some of these studies of
at-risk children, high anxiety and LHPA axis disturbances
may be indicating risk for the development of depression.
Summary of Limbic-Hypothalamic-Pituitary-
Adrenocortical Activity in Child Clinical Disorders
This brief review of the literature on LHPA associations
with clinical disorders and subclinical behavior problems
in children yields evidence of association with both hypo-
(e.g., DBD without anxiety) and hypercortisol activity
(anxiety and, to some extent, depression). Because in hu-
mans early adverse experiences tend to increase rates of
both disruptive and anxious/depressed symptoms, we
might expect the impact of early experiences on LHPA
functioning to be filtered through individual vulnerabili-
ties to different patterns of disordered functioning. There
Early Experiences and Stress Reactivity and Regulation 555
is little evidence for this in the rodent research that
spawned much of this field of inquiry. However, because
the preclinical studies have been conducted on highly in-
bred strains, we might expect less evidence of the multifi-
nality (different end points associated with similar
experiences) typically observed in human studies of early
experiences.
EARLY EXPERIENCES AND STRESS
REACTIVITY AND REGULATION
Studies of the impact of early experience on the LHPA sys-
tem have important implications for developmental psycho-
pathology. Here we first review the detailed work conducted
in rodents and then examine the implications of this work for
our understanding of primate development, including the de-
velopment of young children.
Rodent Studies
The long history of early experience stress studies in rats
provides the basis for attempts to understand how variations
in care early in life may impact individual differences in
stress resilience and vulnerability to psychiatric disorders.
There have been many recent and excellent reviews of
this literature (e.g., Cirulli & Alleva, 2003; Graham, Heim,
Goodman, Miller, & Nemeroff, 1999; Sanchez, Ladd, &
Plotsky, 2001); thus, we only very briefly outline the gen-
eral findings. A number of early experience paradigms have
been used in the rat. Most involve some form of maternal
separation. Handling involves very brief (3 to 5 minutes)
daily removal of the pup from the mother and nest. Separa-
tion involves longer periods, ranging from 3 hours daily to 6
to 24 hours one or more times prior to weaning. Over 20
years ago, Levine (1975) argued that these manipulations
produce their effects, at least in part, by altering mother-pup
interaction ( but see also Denenberg, 1999). Current evi-
dence suggests that the critical components of maternal be-
havior affected by early experience manipulations center on
feeding interactions. Specifically, they center on how much
the mother licks and grooms her pups (typically occurring
in conjunction with nursing bouts) and how well she assumes
the nursing posture (arched back). Handling seems to stimu-
late increased licking/grooming and arched-back nursing
(LG-ABN), although in some strains ( but not in others),
separation reduces these aspects of maternal behavior. In
strains where these aspects of maternal behavior are not re-
duced by separation, hyperresponsivity of the LHPA axis is
not observed. Similarly, spontaneous variations in LG-ABN
among mother rats have also been associated with long-term
556 Stress Neurobiology and Developmental Psychopathology
effects on offspring similar to those observed in handling
( high LG-ABN) and separation ( low LG-ABN). The effects
of handling are greater in the 1st than in the 2nd postnatal
week. In addition, 24 hours of maternal separation has op-
posite effects when performed at the end of the 1st com-
pared to the 2nd postnatal week. Both of these findings
suggest sensitive periods for effects on the LHPA system
early in postnatal development in the rat.
Handling and high LG-ABN produce offspring who
later in life are less fearful and exhibit lower CRH activity
in the hypothalamus and amygdala, increased GR in the
hippocampus, and better containment of the GC response.
They also evidence enhanced functioning of the noradren-
ergic, dopamine, and serotonin system and increased
synaptogenesis in various regions of the brain (see review
articles cited earlier). Maternal separation and low LG-
ABN produce the opposite: high fearful offspring with
heightened CRH and LHPA reactivity. Of particular im-
portance, the early experience manipulations in rodents
appear to affect responses to processive stressors more
than systemic stressors, thus implicating corticolimbic
components of the stress-emotion system. GCs may not me-
diate the effects of maternal separation, as suppression of
GCs has no ameliorating effects on pups separated for 24
hours. Instead, there is evidence that CRH operating
through CRHr1 may contribute to the effects of these early
experience manipulations. In addition, although the effects
of early experience manipulations on GR may be perma-
nent, exposing juvenile rats to environmental enrichment
can reverse many of the other behavioral and neurobiologi-
cal effects of maternal separation. This suggests that in the
rat, postweaning manipulations continue to shape neural
components of the stress-emotion system (Francis, Diorio,
Plotsky, & Meaney, 2002).
Because so much of the data on early experiences and
later stress reactivity and regulation comes from rat mod-
els, we face a number of challenges in translating this liter-
ature to human or, for that matter, primate development.
Critically, gestational time varies such that birth occurs at
a different point of neurobiological development: 3 weeks
for the rat, 7 months for the monkey, and 9 months for the
human. Rats are weaned at 21 to 22 days of age in the labo-
ratory (28 days in the wild), monkeys at 1 year, and 2 to 3
years of nursing is not uncommon for human infants in
hunter-gatherer cultures. Based on first signs of reproduc-
tive ability, rats are juvenile between 35 and 45 days of age,
nonhuman primates around 3 to 4 years of age, and humans
until the 2nd decade of life. The central nervous system is
much more immature in the rat at birth than in the human
and nonhuman primate, although at this time, there is no
agreed upon method of identifying corresponding develop-
mental periods of neural plasticity among species.
Nonetheless, there is reason to believe that the 1st postnatal
week in the rat likely corresponds to prenatal development
in humans (Dobbing, 1981; Vazquez, 1998). Postnatal ex-
periences should operate primarily on brain regions that
develop after birth. Because at birth the HPA system is
more mature in the primate than in the rat, we might expect
that postnatal effects on the stress-emotion system in pri-
mates would be less apparent in the HPA system than in the
corticolimbic pathways influencing stress reactivity and
regulation.
Nonhuman Primate Studies
Early experience research in nonhuman primates has
largely progressed through the lens of attachment theory.
Accordingly, the mother-infant relationship has been con-
ceptualized as a social buffer against stress (Bowlby, 1969;
Suomi, 1995). Maternal buffering describes situations in
which the infant may be behaviorally distressed but physio-
logical stress responses are attenuated. For example, cap-
ture and handling elicits intense behavioral distress, but
when mother and infant are immediately reunited, cortisol
responses are minimal (Levine & Wiener, 1988). Although
there is as yet no clear evidence of an SHRP in the monkey
infant, maternal buffering may serve the same function, re-
ducing the likelihood of high GC exposure during periods
of rapid brain development. In primates, physical contact is
critical for the development of the attachment relationship
(Harlow, Harlow, & Suomi, 1971). However, monkey in-
fants can also use distal modes of communication with
mother to buffer LHPA and NA responses to separation
(e.g., Bayart, Hayashi, Faull, Barchas, & Levine, 1990).
The natural ecology of most primate species is social.
Mother and infant live in troupes that include other moth-
ers and infants, adult males, and the infant’s older siblings.
Species differ in how often the infant is parented by other
members of the troupe, a phenomenon termed alloparenting
or aunting. Although the availability of alloparents does
not prevent elevations in cortisol to maternal separation, it
does result in a more rapid termination of the GC response
and a reduction in the likelihood of despair reactions (I. C.
Kaufman & Rosenblum, 1967; Levine & Wiener, 1988).
Thus, in primate infants, maternal buffering is not re-
stricted to physical contact between mother and infant and
is a function that can be provided to some extent by other
nurturing conspecifics.
Separation confronts the infant with two conflicting
challenges: Reestablish contact and avoid attack or preda-

tion. Two stages of separation response have been de-
scribed: protest, or the period of active searching, and de-
spair, a period of passivity and withdrawal (Bowlby,
1973). Although once described as a necessary biphasic
process following maternal loss (Bowlby, 1973), infant be-
havior during both phases is affected by the context of
separation. Protest appears to be related to preseparation
factors (e.g., Gunnar, Gonzalez, Goodlin, & Levine, 1981)
and cues signifying predation /attack (Kalin, Shelton,
Rickman, & Davidson, 1998). Despair appears to be af-
fected by the presence of alloparents and the degree of so-
cial threat confronting the infant in the mother’s absence
(Hinde & Davies, 1972). Separation thus is not a unitary
phenomenon (Kraemer, Ebert, Schmidt, & McKinney,
1991). Its complexity may help explain why protest behav-
iors at times are inversely related to separation-induced
LHPA activity and why during prolonged and/or repeated
separations protest behaviors are sometimes reduced,
while LHPA continues or sometimes increases (e.g., Coe,
Glass, Wiener, & Levine, 1983). Levine and Wiener
(1988) have argued that protest reflects the active at-
tempts to cope with separation, and LHPA responses re-
flect failed coping. An alternative, but not mutually
exclusive, explanation is that protest reflects operation of
the attachment system, and LHPA activity reflects actions
of central fear/anxiety systems.
Support for this latter argument comes from studies by
Kalin and his colleagues. In rhesus infants, administering
exogenous opiates during separation reduces protest behav-
ior but not GCs or defensive behaviors (e.g., freezing and
threatening experimenters; Kalin, Shelton, & Barksdale,
1988). In contrast, intraventricular CRH administration
during separation increases GCs and defensive behavior
but not protest (Kalin, Shelton, & Barksdale, 1989; Kalin,
Shelton, & Turner, 1992). In rhesus infants, there is a close
correspondence between defensive behaviors (e.g., freez-
ing) and LHPA activity. LHPA activity in rhesus infants is
also associated with greater right frontal EEG asymmetry,
suggesting corticolimbic organization of defensive, with-
drawal behaviors (Kalin et al., 1998). Combined, these
data argue that separation can produce fear in infant pri-
mates, and that when it does, we see marked elevations in
LHPA activity and defensive behaviors, perhaps orches-
trated, at least in part, by extrahypothalamic CRH. How
much the infant protests, however, is often uncorrelated
with activity of the LHPA axis and may be organized along
different neurobiological pathways. There is evidence of a
similar dissociation between separation-induced crying
and cortisol elevations in human infants (e.g., Spangler &
Schieche, 1998).
Early Experiences and Stress Reactivity and Regulation 557
Whereas repeated separation in rats produces hypercor-
tisolism, in monkeys it may reduce LHPA activity. Infant
squirrel monkeys do not exhibit habituation of the LHPA
response to repeated 6-hour separations, even with as many
as 80 trials (Hennessy, 1986). However, several years later,
previously separated juveniles demonstrate blunted GC and
NA responses to social isolation stress (Levine, Lyons, &
Schatzberg, 1997). Repeated, daily separations of several
hours in marmosets during the 1st month of life lower basal
cortisol near the peak of the circadian cycle while increas-
ing fearful, anxious behaviors (Dettling, Feldon, & Price,
2002). In contrast, in squirrel monkeys, 1 hour of separa-
tion weekly for 10 weeks had the opposite effect, reducing
clinging and increasing exploration of a novel environment
and blunting LHPA responsivity (Lyons et al., 1999;
Parker, Buckmaster, Schatzberg, & Lyons, 2004). Lyons
and colleagues (e.g., 1999) suggest that brief separations in
monkeys may function like handling in rats. However, a re-
cent study of repeated, unpredictable separations in rhesus
infants questions this conclusion (Sanchez et al., in press).
Monkey infants typically spend less time in close proximity
to mother with age; however, infants who experienced the
series of unpredictable separations showed the reverse: in-
creases in proximity with age. Evidence of heightened
stress reactivity was noted, although this was more appar-
ent for females in cortisol reactivity and males in height-
ened acoustic startle reactions. Of importance for
comparison with human studies of early trauma and depri-
vation, both males and females exhibited disturbances in
diurnal cortisol production, although the disturbances var-
ied by sex, with males showing a shallower rhythm over the
morning hours and females exhibiting a flatter rhythm
from morning to evening. Thus, repeated separation para-
digms produce alterations in fear/anxiety and LHPA activ-
ity in monkey infants. Nonetheless, these changes do not
always suggest heightened stress reactivity. In some cases,
they appear to produce blunted responsivity to subsequent
stressors. Whether these differences reflect species differ-
ences, differences in developmental timing, and/or differ-
ences in the supportiveness of the separation environment
(e.g., presence of alloparents) is not yet clear. These find-
ings do, however, raise critical questions about how best to
translate the rodent findings to primates, including humans.
The challenge of translating the rodent findings is also
demonstrated by work on prolonged separations in in-
fant monkeys. Separations of several days or weeks in-
crease fearful, anxious behavior in monkey infants. Tested
months and years after the separation experience, juvenile
rhesus who were separated once or twice in infancy were
found to be more neophobic and behaviorally reactive than
558 Stress Neurobiology and Developmental Psychopathology
never-separated animals (Spencer-Booth & Hinde, 1971).
These effects were greater when the infant was left in the
social group than when it was removed and the mother
remained in her familiar surroundings (Hinde, Leighton-
Shapiro, & McGinnis, 1978). Continuing the theme of ma-
ternal mediation of separation effects, Hinde argued that
removing the mother from the social group was more devas-
tating than removing the infant because, in the former case,
the mother had to ignore the infant and reestablish her social
position in the troupe upon her return. However, there is also
evidence that leaving the infant in the social group, if it is not
adopted by another female, also results in prolonged GC in-
creases and other neurobiological disturbances (Lau-
denslager et al., 1995; Reite, Harbeck, & Hoffman, 1981). In
contrast, if infant rhesus monkeys are placed alone in single
cages during separation, GC levels return to near baseline
within 24 hours (Gunnar et al., 1981). These findings are
consistent with Kalin’s (Kalin, Shelton, & Barksdale, 1988)
argument that LHPA activity during separation reflects the
degree of ongoing threat in the separation environment. De-
spite evidence of continued elevations in GCs during pro-
longed separations, however, there is little evidence of
long-term changes in the LHPA axis (Capitanio, Rasmussen,
Snyder, Laudenslager, & Reite, 1986).
In addition to separation, the effects of several aberrant
rearing environments have been studied, including total
and partial social isolation rearing, surrogate-peer rearing,
and peer-only rearing. Sensitive periods have been demon-
strated for behavioral abnormalities with deprivation dur-
ing the first 6 months, resulting in more drastic effects
than similar deprivation periods later in life (for review,
see Kraemer, 1992). As in the research on separation, de-
spite marked increases in bizarre behavior and anxious,
neophobic reactions, long-term effects on the LHPA axis
rarely have been observed. Considering social isolation
rearing first, although there is evidence of marked aberra-
tions in the coordination of central monoamine systems
(Kraemer, Ebert, Schmidt, & McKinney, 1989), isolate-
reared monkeys do not show increased LHPA reactivity
(Meyer & Bowman, 1972; Sanchez, Young, Mathys, Plot-
sky, & Insel, 1999). Indeed, normalizing experience in
these paradigms tends, if anything, to increase basal GCs
and autonomic responsivity to stressors (Hill, McCormack,
& Mason, 1972; Wood, Mason, & Kenney, 1979). Struc-
tural and neurochemical studies also have failed to reveal
effects on the LHPA system (Sanchez et al., 2001).
Changes have not been detected in BNST, in the number of
hypothalamic dopaminergic neurons, the density of CRH
neurons in the PVN or in CRH or AVP or their receptors in
the hypothalamus, including the mpPVN (Sanchez et al.,
1999). Failure to find effects on the LHPA axis may reflect
the greater maturity of this system at birth in the primate
as compared to the rodent. Nonetheless, corticolimbic
areas important in the regulation of fear/anxiety and LHPA
activity are affected by isolation rearing (see Sanchez
et al., 2001). Isolate-reared animals exhibit increased in-
tensity of neurofilament protein immunoreactivity in the
hippocampus (Siegel et al., 1993), as well as increased den-
sity of CRF1 receptors in the prefrontal cortex and down-
regulation of CRF2 receptors in the amygdala (Sanchez
et al., 1999).
Allowing surrogate-reared infants daily peer experience
supports reasonably normal social development (Suomi &
Harlow, 1972). LHPA effects, however, are variable (Cham-
poux, Coe, Schanberg, Kuhn, & Suomi, 1989; Clarke, 1993;
Shannon, Champoux, & Suomi, 1998). Similar to repeat-
edly separated infants, smaller LHPA responses to brief pe-
riods of social isolation have been observed (Shannon et al.,
1998), along with atypically low GC levels early in the
morning, near the peak of the rhythm (Boyce, Champoux,
Suomi, & Gunnar, 1995). Diurnal temperature and rest-
activity rhythms have also been found to be dysregulated
in surrogate-reared animals (Lubach, Kittrell, & Coe,
1992). Peer-only rearing, in which infants are reared with
only other infants as attachment figures, produces more
markedly deviant stress responsivity and behavior than sur-
rogate-peer rearing (Suomi, 1997). Studies of these animals
indicate larger and more prolonged LHPA responses to sep-
aration, increased alcohol consumption as juveniles that
correlates with separation-induced cortisol reactions, and a
combination of both neophobia and heightened reactive ag-
gression (Higley, Suomi, & Linnoila, 1992). Unfortunately,
there have been no published anatomical analyses of surro-
gate-peer and peer-only reared animals. Consequently, we
do not know whether their LHPA axis anomalies reflect
changes in the LHPA system proper or in corticolimbic in-
puts to the LHPA system.
In addition to separations and various deprivation rear-
ing manipulations, researchers have manipulated demands
on the mother to disturb maternal behavior. For example,
they have altered maternal foraging demands. When forag-
ing demands are unpredictable (i.e., variable foraging de-
mand or VFD paradigm), marked alterations in the social
organization of female bonnet macaques have been noted
(Rosenblum & Andrews, 1994). Specifically, VFD reduces
the maternal responsiveness to infant signals, resulting in
insecure patterns of attachment behavior and long-term ef-
fects on offspring development (Andrews & Rosenblum,
1994). As juveniles and young adults, VFD-reared animals
tend to be fearful of new social situations and low in dom-

inance. They also have elevated CSF levels of CRH and
monoamine abnormalities, suggesting that their early ex-
periences sensitized the hypothalamic and/or extrahypo-
thalamic CRH system (Coplan et al., 1996; Rosenblum
et al., 1994).
Finally, monkey mothers do spontaneously abuse their
infants. In rhesus, approximately 2% of infants are physi-
cally abused by mothers who were likely to have been
abused themselves as infants (Maestripieri, 1999). Abusive
behavior includes violent behaviors such as dragging the in-
fant on the ground, crushing, throwing, or stepping/sitting
on it. These abusive behaviors are observed most frequently
in the first 3 postnatal months. In recent work, McCormack
and colleagues (2003) noted significant alterations in
LHPA functioning among abused infants. Specifically,
compared to nonabused controls, abused infants exhibited
higher basal morning levels of cortisol during the 1st post-
natal month when abuse rates were the highest, but begin-
ning in the 2nd postnatal month and continuing at least
through the 6th postnatal month, their basal cortisol levels
were suppressed, especially in the early morning hours. At
2 months of age, abused infants were unable to use maternal
contact to buffer elevations in cortisol when mother and in-
fant were captured together and singly caged for 30 min-
utes; in contrast, nonabused infants showed no increase in
cortisol to these procedures. Of particular importance, dis-
turbances in the LHPA system were also noted using phar-
macological challenges. Specifically, the abused relative to
the nonabused infants exhibited blunted ACTH responses to
CRH, suggesting that some of their alterations in HPA func-
tion could be due to CRF receptor down-regulation at the
level of the pituitary. Notably, down-regulation of ACTH to
CRH was detected at both 6 and 12 months, many months
after the infants were no longer highly dependent on mater-
nal care. Similar but less dramatic effects of harsh treat-
ment have also been noted in Goeldi’s monkey infants
(Dettling, Pryce, Martin, & Döbeli, 1998), where larger
cortisol responses and behavioral distress to brief separa-
tions were obtained from infants whose parents engaged in
more aggressive weaning behavior.
Summary of the Monkey Infant Studies
As in rodents, monkey mothers provide an external regula-
tor of the LHPA and other stress-sensitive systems. In pri-
mates, separation protest (crying and searching) does not
appear to be associated with LHPA activation, but defen-
sive behaviors do, perhaps coordinated by extrahypothala-
mic CRH. The separation context influences the magnitude
of the LHPA response. When the infant is nurtured by other
Early Experiences and Stress Reactivity and Regulation 559
conspecifics in the mother’s absence, small GC responses
are obtained that resolve quickly. When the separation en-
vironment is more threatening, high GC responses are pro-
duced that may not habituate over trials. The long-term
impact of the various separation and rearing environment
paradigms that have been studied in monkeys are difficult
to summarize. Overall, however, as in the rodent research,
the clearest impacts on infant behavior and stress neurobiol-
ogy appear to arise in those paradigms that produce distur-
bances in mother-infant interaction or, in the case of
peer-only rearing, in the supportiveness of the infant’s at-
tachment figures. Thus, as in the rodent research, the be-
havior of the infant’s caregivers may be the most important
factor in determining the long-term impact of early ad-
verse experiences on the development of the LHPA system.
Furthermore, unlike in the rodent, more of the effects of
postnatal adversity in monkeys appear to be evident on lim-
bic circuits impacting activity of the HPA axis than on the
HPA axis proper. This is consistent with the greater matu-
rity of the HPA axis at birth in primates than in rodents.
Human Studies
In this section, we cover studies of caregiving and LHPA
activity in human infants and children. These studies have
been conducted through two major theoretical lenses. First,
as in the nonhuman primate research, concepts and as-
sumptions of attachment theory have guided much of the
work. Second, in work on childhood maltreatment, re-
search on adult Posttraumatic Stress Disorder (PTSD) has
shaped the questions asked as well as interpretation of the
findings. We first examine LHPA associations with varia-
tions in caregiving in the normative or nonabusive range
and then consider LHPA studies of maltreated children.
Normative Variations in Caregiving
Consistent with attachment theory, in human infants insen-
sitive, intrusive maternal care is associated with height-
ened GC activity. Insensitive or intrusive caregiving has
been associated with GC increases during mother-infant
play bouts (Spangler, Schieche, Ilg, Maier, & Ackermann,
1994), higher presession GC levels during well-child health
checkups (Gunnar, Brodersen, Nachmias, Buss, & Riga-
tuso, 1996), and larger GC responses to strange and poten-
tially threatening events (Nachmias, Gunnar, Mangelsdorf,
Parritz, & Buss, 1996). In these studies, the sensitivity
rather than the amount of maternal behavior seems to be
the critical factor. Thus, mothers who engaged in both
more and more varied, but not necessarily more sensitive,
soothing activities have not been found to have infants who
560 Stress Neurobiology and Developmental Psychopathology
exhibited smaller GC response to an inoculation stressor
(Lewis & Ramsay, 1999). Similarly, mothers who engaged
in more, but not more sensitive, interactions with fearful
toddlers did not reduce their toddlers’ GC responses to po-
tentially threatening events (Nachmias et al., 1996).
Maternal sensitivity associated with reduced GC re-
sponses has also been shown to predict whether the mother-
infant relationship would be classified as secure based
on Ainsworth and Wittag’s Strange Situation assessment
(Gunnar, Brodersen, Nachmias, et al., 1996; Nachmias
et al., 1996). When cortisol is not sampled too early follow-
ing the stressor to detect the effect (e.g., Gunnar, Mangels-
dorf, Larson, & Hertsgaard, 1989), the evidence fairly
consistently supports the hypothesis that by the end of the
1st year, attachment security provides a powerful stress
buffer in humans. There is evidence for this conclusion
using stressors ranging from the Strange Situation (Span-
gler & Grossmann, 1993; Spangler & Schieche, 1998), to
novel, arousing stimuli (Nachmias et al., 1996), to inocula-
tions (Gunnar, Brodersen, Krueger, et al., 1996). However,
as yet there is no evidence that a history of secure attach-
ment reduces infant or toddler GC reactivity when the at-
tachment figure is not available or when separations last
more than a few minutes (see Ahnert et al., 2004). Secure
attachment may foster better stress regulation, but this has
been observed only in the presence of the attachment figure
during the infant and toddler periods.
The hypothesis that sensitive and responsive caregiving
is critical in regulating stress hormones in young children
is also supported by research on nonparental caregivers. In
a study in which 9-month-olds were separated from their
mother for 30 minutes, infants who were randomly as-
signed to a sensitive and supportive babysitter condition
did not produce elevations in cortisol, but those who were
assigned to an unresponsive and inattentive babysitter
condition did (Gunnar, Larson, Hertsgaard, Harris, &
Brodersen, 1992). In a study of home-based child care,
preschoolers produced increases in cortisol over the child
care day if their care providers were less attentive and pro-
vided less supportive stimulation (Dettling et al., 2000).
Given evidence that unresponsive, insensitive, and/or in-
trusive care fails to provide an adequate stress buffer, it is
not surprising that maternal depression has been associated
with higher GC levels in young children. Depressed moth-
ers are often more insensitive, erratically responsive,
and/or intrusive with their children (Dawson & Ashman,
2000). Dawson and Ashman reported that right frontal
EEG asymmetry in toddlers of depressed mothers was
largely mediated by these aspects of maternal care. Fur-
thermore, they found that at 3 years of age, morning GC
levels could be predicted by how clinically depressed the
mother had been during the child’s 1st year of life. At 7
years, these children’s laboratory pretest GC levels were
higher if their mother had been clinically depressed during
their first 2 years (Ashman et al., 2002). Similarly, adoles-
cents whose mother had been depressed in their 1st postna-
tal years were more likely to produce one or more days of
elevated cortisol in the early morning, a pattern predictive
of depression in previous work (Halligan et al., 2004). Fur-
thermore, Halligan and colleagues found this effect of early
maternal depression while controlling for the total number
of months the mother had been depressed during the child’s
life and the mother’s current depression status. These find-
ings are important for two reasons. First, they suggest that
higher GC levels in offspring of depressed mothers are not
due merely to shared genetics. Second, they support the
possibility that insensitive, unresponsive care in the early
years of the child’s life may shape LHPA activity.
This latter point is one implication of another study that
also examined maternal depression. Essex and colleagues
(2002) found that home GC levels in 4.5-year-old children
were higher if the mother scored high on depressive symp-
toms both during the prior year and during the children’s
infancy. Mothers with high depression symptoms only
when their children were 4 or only during infancy did not
have children with higher GC levels than mothers who
never had high symptom counts. Unfortunately, the re-
searchers did not examine whether the effects of maternal
depressive symptoms were mediated by intrusive, unre-
sponsive, or insensitive treatment of the child. Further-
more, these data do not allow us to untangle the impact of
chronic maternal depression from organizational effects of
early maternal care that then heightens vulnerability to
stressors later in the child’s life.
Unfortunately, few of the studies of postnatal maternal
behavior permit us to rule out prenatal influences on the
development of the child’s LHPA system. This is a serious
shortcoming as there is increasing evidence of fetal pro-
gramming of the stress neuraxis. Several recent studies
suggest that exposure to elevated levels of CRH and/or cor-
tisol prenatally may influence the behavioral and physio-
logical reactivity of the child, at least shortly after birth
(see Wadhwa, Sandman, & Garite, 2001). For example, de-
pressed mothers tend to produce higher urinary cortisol
levels around midpregnancy than do nondepressed mothers,
and these levels predict the newborn’s urinary cortisol lev-
els (Diego et al., 2004). Of particular importance, it was
prenatal, not concurrent postnatal, maternal cortisol values
that were predictive of infant cortisol activity. We do not
know how long such effects persist.

Finally, as in so many other areas, we have very little ev-
idence that maternal depression and associated insensitive,
intrusive care increase cortisol reactivity. In all of the stud-
ies described, maternal depression was either associated
with home baseline GC or laboratory pretest GC levels. In
the one study describing GC responses to a stressor, Ash-
man and colleagues (2002) did not find effects of depres-
sion on the GC stress response, even though associations
with pretest GC levels were obtained. They did, however,
find an interaction between maternal depression during the
child’s infancy and the child’s current internalizing prob-
lems. Children with internalizing problems who had been
cared for by a mother who was clinically depressed during
their first 2 years had the largest GC responses to the stres-
sor. As we will see, activity of the LHPA axis in a number
of studies of maltreated children also is a function of the
interaction between the individual’s history of care and his
or her current mental health status.
Maltreatment
Because most maltreatment occurs at the hands of parents
or guardians, childhood maltreatment typically represents
a profound failure of the caregiving system (Cicchetti &
Rogosch, 2001a). It is also associated with host of patho-
logical outcomes, including both affective (anxiety, depres-
sion) and disruptive behavior disorders (for review, see
Pine, & Cohen, 2002). Recently, there has been increased
interest in identifying the various neurobiological mecha-
nisms through which maltreatment produces its deleterious
effects (e.g., Bremner & Vermetten, 2001; Cicchetti &
Tucker, 1994; J. Kaufman & Charney, 2001; Teicher, An-
dersen, Polcarri, Anderson, & Navalta, 2002). Most of this
work has started with the assumption that maltreatment is
stressful and/or traumatic and that it produces its effects,
at least in part, through episodic or chronic stress. No in-
vestigator expects the effects of maltreatment to be medi-
ated by any one neurobiological system, but most include
GCs and CRH in their models. This means that there have
been a relatively large number of studies of the LHPA sys-
tem in relation to childhood maltreatment (see recent re-
views by Bremner & Vermetten, 2001; Glaser, 2000; Heim,
Ehlert, Hanker, & Hellhammer, 1998; J. Kaufman & Char-
ney, 2001).
Childhood maltreatment, however, is a multifaceted
phenomena (Glaser, 2000). It differs in type (physical, sex-
ual, emotional, neglectful), severity, and duration, and
often several types are inflicted on the same child, some-
times at different points in his or her development (Bar-
nett, Manly, & Cicchetti, 1993). Furthermore, it often
occurs in the context of family conflict, parental psycho-
Early Experiences and Stress Reactivity and Regulation 561
pathology, economic adversity, and other factors that may
also impact the child’s developing stress system. As is the
case for other stressors, the impact of child maltreatment
on the neurobiology of stress and emotion likely depends
on the age of the child, the child’s gender, his or her pre-
morbid functioning, and genetic factors. Finally, its effects
on the developing stress system will depend on the individ-
ual’s age when these effects are assessed, current life
stressors, social support, mental health status, time since
maltreatment, and the measure(s) chosen for assessment.
Given all of these factors, it is no surprise that the litera-
ture on maltreatment and stress neurobiology includes con-
tradictory findings.
As noted earlier, much of the research on childhood
maltreatment has been generated from a trauma frame-
work. As such, many of the questions have been derived
from adult work on PTSD. In adults, PTSD in response to
traumas experienced in adulthood has been sometimes
(Yehuda, 2000), but not always (Bremner et al., 2003), as-
sociated with low basal GCs (particularly late in the day),
prolonged GC suppression to DEX, increased GR as mea-
sured on lymphocytes, and possibly heightened negative
feedback regulation of the axis. One central question in
this literature is whether these LHPA abnormalities pre-
date rather than follow from the trauma (see Yehuda,
2000). Childhood maltreatment is one of the risk factors
for developing PTSD in response to a traumatic event expe-
rienced in adulthood. Thus, researchers have been inter-
ested in whether adults who were maltreated as children
exhibit prolonged suppression of the LHPA axis in response
to a low dose of DEX regardless of their current PTSD sta-
tus. If so, this might indicate that early maltreatment pro-
duces LHPA alterations that serve as a risk factor for the
development of PTSD to traumas encountered in adult-
hood. In one small study, adult women who had been sexu-
ally abused as children were given a low-dose DEX test
(Stein, Yehuda, Koverola, & Hanna, 1987). Compared to
controls, the maltreated women did show prolonged corti-
sol suppression, regardless of their current PTSD status.
We do not know whether this would also be true of men or
of individuals who experienced other forms of childhood
maltreatment.
Other studies of adults maltreated as children provide
evidence of hypofunctioning at the adrenal but not neces-
sarily at other levels of the LHPA system. These results
were obtained in individuals without current psychiatric di-
agnoses. Using pharmacological probes, Heim, Newport,
Bonsall, Miller, and Nemeroff (2001) found elevated
ACTH production in response to CRH in women who had
been abused prior to puberty. At the level of the adrenal,
562 Stress Neurobiology and Developmental Psychopathology
however, they obtained evidence of relative adrenal insuffi-
ciency. In response to both CRH and ACTH challenge
tests, these women showed blunted GC responses relative
to control women who had not experienced childhood
abuse. Similarly, when a processive stressor rather than a
pharmacological probe was used, they obtained evidence of
elevated ACTH responses and normal GC reactions (Heim,
Newport, et al., 2000). When the data from Heim et al.
were subjected to a regression analysis to examine the ef-
fects of childhood abuse after controlling for current life
events and psychopathology, peak GC and ACTH responses
were both found to correlate positively with childhood
abuse scores (Heim & Nemeroff, 2001). Finally, several
studies of adults (male and female) who experienced phys-
ical and emotional abuse as children have reported low 24-
hour urinary free cortisol (UFC) production (Roy, 2002;
Yehuda, Halligan, & Grossman, 2001), although interest-
ingly, not in women who were sexually maltreated as chil-
dren (Yehuda et al., 2001).
Current psychiatric diagnosis interacts with childhood
abuse in studies of the LHPA axis in adults abused as chil-
dren. Thus, among depressed women maltreated as chil-
dren, Heim and colleagues (Heim & Nemeroff, 2001; Heim,
Newport, et al., 2000) noted elevated GC as well as ACTH
responses to the TSST relative to nonabused, depressed
controls. Indeed, the combination of childhood abuse and
adult depression accounted for 21% of the variance in
LHPA responses to the TSST. However, when a pharmaco-
logic probe of the axis was used (i.e., CRH challenge test),
similar to other depressed adults, these women exhibited a
blunted ACTH reaction. In contrast, women with borderline
personality diagnoses who had been maltreated as children
showed augmented ACTH responses to CRH relative to
similarly diagnosed women without a history of childhood
maltreatment (Rinne et al., 2002). There is also evidence
that adults with PTSD pursuant to childhood abuse may ex-
hibit hyper- rather than hypo-LHPA functioning to proces-
sive stressors. Bremner and colleagues (2003) recently
reported markedly elevated salivary GCs during an hour-
long anticipatory period in a small sample of PTSD adults
(male and female) maltreated in various ways as children.
For women, no GC differences from nonpsychiatric, non-
maltreated controls were obtained in response to a cognitive
stressor in this study, although PTSD males abused as chil-
dren showed significantly higher GC reactions.
In summary, there is some evidence that childhood mal-
treatment is associated with low 24-hour GC production
in adulthood, consistent with other work on adults with
PTSD. However, there is little evidence that childhood
maltreatment decreases LHPA reactivity at levels of the
axis above the adrenal. Furthermore, there is little evi-
dence, so far, that GC and ACTH responses to processive
stressors are blunted in adults who were maltreated as chil-
dren. Indeed, increased ACTH and GC reactivity to such
stressors has been obtained. The LHPA effects of child-
hood maltreatment, however, appear to be influenced by
the individual’s current mental health status. Maltreated,
depressed patients exhibit LHPA activity characteristic
of MDD adults. Specifically, they show blunted ACTH re-
sponses to CRH challenge. Adults with PTSD who are not
also clinically depressed may show exaggerated LHPA re-
sponses to psychological or processive stressors, similar
to other studies of LHPA reactivity in clinical anxiety-
disordered subjects.
Of course, despite the work just described, there is a
great deal that is not yet known about the impact of child-
hood maltreatment on the activity of the mature stress sys-
tem. Most of the studies have included only women. Few of
the studies controlled for current life stressors, social sup-
ports, and/or recent trauma experiences. Most have had
small samples that did not allow researchers to examine the
timing, duration, or combinations of types of abuse to
which the individual was exposed in childhood. Nonethe-
less, even given these limitations, the studies of adults mal-
treated as children tend to support the hypothesis that
childhood maltreatment sensitizes the LHPA system, per-
haps rendering individuals more vulnerable to stressors and
increasing their risk for stress-related disorders.
This research on adults maltreated in childhood should
caution us against assuming that childhood maltreatment
will produce evidence of heightened activity at all levels of
the axis and for all individuals. Increased pituitary respon-
sivity to ACTH and to psychosocial stressors may exist in
concert with low cortisol baselines. Responses to pharma-
cological probes specific for different levels of the axis
may reveal patterns of response that are markedly different
from, or even the opposite of, those obtained when proces-
sive stressors that involve corticolimbic circuitry are used.
Given the complexity of the findings for adults, we should
anticipate that the data on children, obtained while the
stress system is still maturing, might be even more complex
and challenging to interpret.
As in adults, whether maltreatment is associated with
increased or suppressed LHPA activity in children appears
to depend, at least in part, on the child’s current psychi-
atric diagnoses and/or behavioral disposition. Internalizing
problems and/or affective disorders (PTSD or depression)
interact with abuse history to produce evidence of height-
ened reactivity of the LHPA system. In contrast, external-
izing problems in the absence of concurrent anxiety

disorders appear to reduce evidence of altered LHPA activ-
ity, at least when maltreated children are compared to nor-
mal controls. As discussed subsequently, ongoing adversity
in the home may also stimulate higher LHPA activity in
previously maltreated children. As current adversity is
often unanalyzed, this makes interpretation of many of the
studies difficult.
First we consider studies of maltreated children with
PTSD. Here, unlike in adults, there is evidence of elevated
rather than suppressed basal cortisol levels. Thus, DeBellis,
Baum, and colleagues (1999) reported that, compared to
normal controls, 24-hour UFC and urinary catecholamine
concentrations were elevated in children (Tanner stage <
III ) with chronic PTSD pursuant to early, severe, and pro-
longed maltreatment. UFC and urinary catecholamine lev-
els were also correlated positively with the duration of
abuse and the number and severity of PTSD symptoms.
These children’s UFC concentrations, however, were only
marginally higher than nonabused, clinically anxious chil-
dren. Some of the children from this study were part of
a larger neuroimaging study that revealed smaller brain
volumes and smaller corpus callosum size but not smaller
hippocampal volumes in maltreated children with PTSD
relative to normal controls (DeBellis, Keshavan, et al.,
1999). In a very similar set of studies, Carrion and col-
leagues (2001, 2002) found elevated salivary GC levels
over the day in maltreated, PTSD children relative to nor-
mal controls. Using the subjects from the cortisol study
plus others, they also reported reduced brain volumes and
corpus callosum size, lack of hippocampal volume differ-
ences, and less asymmetry of the prefrontal cortex.
There is also evidence that depression interacts with
maltreatment history to produce heightened LHPA activity.
Thus, in a study of 7- to 15-year-old sexually abused girls
studied within 3 to 4 years of abuse reporting, DeBellis and
colleagues (1994) found marginally higher 24-hour GC lev-
els (averaged over 3 days). Consistent with adult depres-
sion, they also found blunted ACTH but not GC responses
to CRH challenge. Most of the girls in the study were dys-
thymic, and nearly half had attempted suicide. In contrast,
J. Kaufman and colleagues (1997) found that depressed,
maltreated 7- to 13-year-old children exhibited larger
ACTH responses to CRH than did nondepressed maltreated
children. However, this was true only of the girls who were
still living under conditions of ongoing economic and social
adversity. Similarly, in a study of DEX suppression, adoles-
cent girls with PTSD pursuant to early abuse showed simi-
lar suppression of the HPA axis to the low-dose DEX test
compared to a trauma /no PTSD and normal control group.
Only the traumatized girls who met criteria for clinical de-
Early Experiences and Stress Reactivity and Regulation 563
pression differed, exhibiting less DEX suppression than the
other girls. Finally, there are two studies of depressed,
maltreated children that have used Cicchetti’s day camp
paradigm to collect salivary cortisol measures (Hart, Gun-
nar, & Cicchetti, 1996; J. Kaufman, 1991). In both of these
studies, depressed, maltreated children exhibited increas-
ing GC levels over the camp day, but this was not observed
among the nondepressed maltreated children or among the
comparison children. As in the J. Kaufman et al. (1997)
study, many of these depressed children were living under
conditions of ongoing adversity. This was also true of mal-
treated children with internalizing behavior disorders stud-
ied by Cicchetti and Rogosch (2001a, 2001b). These
children had higher 9 A.M. and 4 P.M. salivary GC levels av-
eraged over 5 days of summer camp than did nonabused
children with clinical levels of internalizing problems.
As noted earlier, children with externalizing problems
in the absence of comorbid anxiety disorders and those
with aggressive, oppositional dispositions in the nonclini-
cal range tend to exhibit low levels of LHPA activity. If
maltreatment increases LHPA activity in these children,
their basal cortisol following maltreatment may not appear
elevated compared to nondisordered controls, but they may
still be elevated relative to nonmaltreated children with ex-
ternalizing problems. Indeed, Hart and colleagues (1995)
found that median cortisol levels at school did not differ
among maltreated preschoolers relative to age-, sex-, and
economically matched controls. However, maltreated chil-
dren did exhibit less day-to-day cortisol variability, espe-
cially if they were less socially competent, or rather, more
aggressive and undercontrolled. In addition, among the
maltreated children who were so out of control that they
had to be gently restrained, cortisol levels on the days re-
quiring restraint were lower than on days when the chil-
dren’s behavior was more appropriate. Cicchetti and
Rogosch (2001b) also found that maltreated boys, but not
girls, with clinical levels of externalizing problems had
higher cortisol levels at summer camp relative to nonmal-
treated boys with externalizing problems. For the mal-
treated, externalizing boys, however, cortisol levels were
not elevated over levels exhibited by nondisordered, non-
maltreated controls.
Not all maltreated children exhibit these effects. Chil-
dren without behavior disorders have not been shown to
have elevated basal cortisol levels (see Cicchetti & Ro-
gosch, 2001b). These children, however, may have experi-
enced less prolonged, varied, and/or severe maltreatment
(Cicchetti & Rogosch, 2001a) and/or may be less geneti-
cally vulnerable (J. Kaufman et al., 1998). And, as noted,
the presence of ongoing adversity and family dysfunction
564 Stress Neurobiology and Developmental Psychopathology
may contribute to heightened LHPA activity in maltreated
children. Despite all that remains to be understood regard-
ing maltreatment and LHPA activity during childhood, we
can at least conclude that there is little evidence of hypo-
functioning or lowered basal GC levels in maltreated chil-
dren, unlike the results described in studies of adults who
were maltreated as children.
This difference between hypercortisolism in studies of
children and hypocortisolism in studies of adults has led
some to argue that hypofunctioning at the level of the adre-
nal, at least, emerges as a function of living for a prolonged
period of time with the sequelae of trauma exposure
(Yehuda et al., 2001). A longitudinal study of sexually
abused girls by Frank Putnam and colleagues is cited as
the best evidence for a transition from hyper- to hypofunc-
tioning. Unfortunately, as yet, most of the results of this
study are available only as conference presentations. Put-
nam and colleagues (Frank Putnam, personal communica-
tion, June 10, 2003) collected a group of sexually abused
girls (averaging 11 years of age) who were first studied
within 6 months of the abuse reporting. At this point, ele-
vated cortisol levels were reported for girls tested in the
morning hours; suppressed levels were noted for a small
group of girls who self-selected into an early afternoon
testing slot. DeBellis and colleagues (1994), in the study
described earlier, examined a small subsample of these
girls several years after abuse reporting. At that time, mea-
sures of cortisol (24-hour UFC, late afternoon baseline,
response to CRH) did not differ from nonmaltreated con-
trols. Nonetheless, low baseline ACTH and blunted ACTH
responses to CRH were noted. This pattern of hypopitu-
itary ACTH and normal cortisol activity suggests that the
adrenal may have been hyperresponsive to ACTH in these
girls at this point in their development. That is, the adrenal
was able to sustain normal GC levels despite reduced
ACTH drive. Finally, at a 7-year follow-up visit, when
most of the girls were in their teens, evidence of hypocorti-
solism was obtained. Specifically, the previously mal-
treated girls had lower plasma GC levels than controls both
at the beginning of a laboratory cognitive task and in serial
samples obtained over the 30-minute cognitive challenge.
Thus, this study suggests a transition from hyper- to
hypocortisolism with time or pubertal development.
Clearly, more longitudinal work is needed to examine dy-
namic changes in LHPA activity pursuant to early mal-
treatment and family adversity.
In this respect, an ongoing study of response to the
trauma of a natural disaster may provide some useful in-
sights. Children exposed at around age 8 to the 1988 Ar-
menian earthquake have been examined at 5 and 6.5 years
following the disaster (Goenjian et al., 1996, 2003). The
children were recruited from two towns, one near the epi-
center that experienced incredible destruction and loss of
life, and one farther away that was much less devastated.
Children living near the epicenter exhibited more evidence
of PTSD and depression than did those farther from the
epicenter. Using one sample of children studied 5 years
posttrauma, the researchers reported prolonged suppres-
sion to low-dose DEX of the adrenocortical axis in the
more traumatized children, along with lower early morn-
ing, but not late afternoon, basal cortisol levels. Drawing
another sample from these cities, 6.5 years posttrauma
lower basal ACTH, but not cortisol, levels were noted.
However, in this study, no differences in ACTH or cortisol
responses to a mild exercise stressor were obtained. In both
cases, measures of LHPA functioning were negatively cor-
related with PTSD and depression scores. These children,
who were in their early adolescent years when tested,
tended to exhibit the suppression of LHPA axis functioning
that has been more characteristic of the adult PTSD than
child PTSD literature. Although this could be because they
were tested many years after the trauma exposure, several
of the studies described earlier (e.g., Carrion et al., 2002;
DeBellis, Baum, et al., 1999) that examined younger chil-
dren were also conducted after at least this many years had
transpired since the onset of maltreatment. This would
seem to leave concurrent adversity and age at assessment
(childhood versus adolescence) as critical variables need-
ing further exploration. Of course, the neurobiological se-
quelae of trauma due to maltreatment and trauma due to
natural disaster may well be different.
Another factor that confounds our understanding of the
impact of abuse on the developing LHPA system is that
many maltreated children also experience significant neg-
lect. Neglect, while it may characterize deficient or depriv-
ing care, needs to be considered separately from abuse, as
it may not, by itself, be traumatic or frightening to chil-
dren. Furthermore, neglect or deprivation of care is more
similar to the primate deprivation rearing paradigms (i.e.,
isolation, surrogate-peer rearing), which, as noted, tend to
be associated with few long-term effects on the LHPA sys-
tem. In most studies of maltreatment, it is extremely diffi-
cult to isolate the effects of neglect from other types of
abuse, as children who are physically, sexually, and/or
emotionally abused are also likely to be neglected. Indeed,
in a study of nearly 400 maltreated children, after careful
background checks, Cicchetti and Rogosch (2001a) were
able to identify only 8% of their summer camp population

as having been subject solely to neglect. Furthermore, neg-
lect-only histories were not associated with altered GC lev-
els among the children studied in the day camp paradigm.
Most of the work on LHPA effects of severe neglect
comes from studies of institutionally reared children. Here
there is evidence of marked abnormalities in diurnal GC
patterns for infants and toddlers living in institutions in
Russia and Eastern Europe (as reviewed in Gunnar &
Vazquez, 2001). Specifically, low early morning GC levels
and a general lack of diurnal variation have been found.
These results mimic those reported for rhesus infants in
surrogate-peer rearing paradigms (Boyce et al., 1995). It is
unlikely that the lack of what has been called the short or
daytime rhythm (wake-up to bedtime) is permanent, as sev-
eral years after adoption, most institutionally reared chil-
dren exhibit fairly normal decreases in cortisol from
wake-up to bedtime (Gunnar, Morison, Chisholm, &
Schuder, 2001). There is some evidence that those from the
most deprived preadoption environments, however, may ex-
hibit slightly elevated salivary GC levels at least at some
times during the day (Gunnar et al., 2001). In a recent
study of postinstitutionalized children adopted from
around the world, slightly higher early morning GC levels
were noted only for those children who also were physically
stunted at adoption and/or had parents who reported that
the children experienced extreme privation prior to adop-
tion (Kertes, Madsen, & Gunnar, 2005). Given these data,
it seems likely that basal GC levels may not be strongly af-
fected by early deprivation in human children. If so, this
conclusion would be consistent with much of the animal
data we reviewed earlier. What has not been studied yet is
whether reactivity of the LHPA system is altered in chil-
dren pursuant to early deprivation.
CONCLUSIONS AND FUTURE DIRECTION
In the nearly 50 years since Levine (1957) published the
paper in Science that launched this field of inquiry, we have
learned a great deal about how regulation of the LHPA sys-
tem in early development influences the development of
stress vulnerability and resilience. In rats, monkeys, and
children, it is now well established that caregivers are po-
tent regulators of this neuroendocrine system. In primates,
sensitive, responsive care from mothers and other nurtur-
ing figures buffers the system from being activated when
youngsters confront strange or threatening events. In rats,
maternal behaviors surrounding feeding bouts keep this
Conclusions and Future Direction 565
system operating in low ranges characteristic of the stress-
hyporesponsive period. In rats, monkeys, and children,
separation can provoke large increases in the activity of
this system, especially when separation results in a reduc-
tion in nurturance from the mother and other nurturing fig-
ures and confronts the youngster with conditions that are
frightening.
We are still a long way, though, from understanding how
disturbances in early care may affect the development of
this system. Tremendous advances have been made in un-
derstanding these processes at the molecular level in rats.
In rats, we also have a fairly detailed understanding of the
developmental neurobiology of this system in relation to
other neural systems involved in stress reactivity and regu-
lation. However, as we have seen, translating the rodent
findings to primates and humans is not trivial. One unan-
swered problem is how best to map developmental periods
across species. There is no agreed upon metric. The 1st
week of life in the rat is often mapped to the last trimester
of gestation in humans. Each month of development in
monkeys is often mapped to 4 months in humans. However,
this mapping is largely based on motor maturity. Clearly, a
better metric would be based on the development of the
specific neural systems, in this case, the components of the
LHPA system and its corticolimbic regulatory pathways.
However, we lack so much information about the normative
developmental neurobiology of the relevant systems in the
monkey and the human that any attempt at mapping the
timing from rats to monkeys to humans is crude at best.
And of course, in making this mapping we cannot consider
all monkeys together. The developmental neurobiology of
the marmoset, squirrel monkey, and rhesus will be differ-
ent. Thus, even translating across monkey species is
fraught with questions about developmental neurobiology
that cannot yet be answered. Yet another consideration is
the fact that cortical development and therefore executive
function differ among rodents, monkeys, and humans.
Thus, we might expect that aspects of early experience,
stress, and emotion regulation that depend on the develop-
ment of corticolimbic circuits will be difficult to map
across species.
One solution to this problem might seem to be to aban-
don preclinical models. If we are interested in human de-
velopment, concentrate on studies of human development.
However, there are good reasons this is not the best solu-
tion. In human infants and children, we can measure the
production of GCs noninvasively using salivary measures
of cortisol. We can also measure DHEA, an androgenic
steroid hormone that is believed to have anticortisol effects
566 Stress Neurobiology and Developmental Psychopathology
(see, e.g., Granger & Kivlighan, 2003). However, as this re-
view has repeatedly demonstrated, activity of the LHPA
system at the level of the adrenal (cortisol and DHEA) is
only part of the story. The impact of early experiences on
activity of the LHPA system can be seen at higher levels of
the system (e.g., pituitary), even when it is not readily ap-
parent at the adrenal level. Currently, blood sampling is
needed to assess ACTH. Unless techniques are developed
that allow noninvasive assessment of ACTH in infants and
children, those of us conducting developmental studies
with children will be limited in what we can understand
when only humans are a part of the developmental analysis.
More critical, the long history of research that was re-
viewed in this chapter demonstrates that many of the im-
portant impacts of early experiences are likely operating at
levels above the pituitary. Recall that in rats, early experi-
ence manipulations during the SHRP appear to produce no
responses at the pituitary and adrenal level. However, mea-
sures of early genes in the hypothalamus indicate that the
mpPVN may be responsive to these manipulations (see re-
view by Cirulli & Alleva, 2003). Thus, CRH may increase
in the hypothalamus and likely also in extrahypothalamic
regions during the period when it is difficult to elevate
ACTH and GCs in the rat. CRH in rats has been shown to
have effects on the developing hippocampus and likely on
other neural systems involved in stress reactivity and regu-
lation. We do not know whether this is true in human devel-
opment or, if it is true, when during development these
effects might be observed. However, findings such as these
should make us very wary of relying only on measures of
cortisol and ACTH to understand whether activity of the
LHPA system may mediate the effects of early perturba-
tions. Thus, we need the preclinical models because of the
opportunity they provide to perform the highly invasive
procedures that allow measures of central CRH activity
and explication of the molecular processes through which
the activity of stress-sensitive systems may affect brain de-
velopment. However, we must be very cautious in translat-
ing these models directly to human development.
The monkey studies provide an important lesson in this
regard. These studies, along with some of the recent work
with rats, challenge us to understand and integrate the im-
pact of early experiences on corticolimbic stress and emo-
tion regulatory pathways into the neuroendocrine early
experience story. They also point to the possibility that ac-
tivity of the LHPA system may be significant in shaping
postnatal brain development in primates, without at the
same time affecting the neurobiology of the HPA system
proper. It seems very likely that some of the molecular
events that permanently alter activity of the HPA system in
rodents during the first 2 postnatal weeks are events,
which, if they operate in primates (including humans), typ-
ically occur prior to birth. Although this still needs to be
determined, it is clear that corticolimbic pathways that are
involved in regulating emotional, behavioral, and physio-
logical responses to perturbations are immature at birth in
primates and that these systems exhibit very protracted
postnatal development. In humans, it is likely that these
systems are not fully mature until late adolescence. The
molecular evidence obtained in preclinical studies of rats
and mice clearly indicates that GCs are involved in regulat-
ing genes that affect the development of relevant corticol-
imbic pathways and their neurotransmitter systems. Thus,
it is likely that postnatal experiences that affect concurrent
GC activity may affect the development frontal systems
underlying stress vulnerability and resilience, without at
the same time affecting the neuroanatomy and physiology
of the HPA system. As noted, in primates, including hu-
mans, the time when GCs might affect developing corticol-
imbic pathways without altering the neurobiology of the
HPA system probably covers much of the postnatal devel-
opmental period.
If true, this alters the end points we need to be examin-
ing in studies of monkeys and humans. Unlike in the rat, we
are probably not looking for evidence of endogenous dys-
regulation of the HPA system. And, if we find this evi-
dence, as has been shown in studies of adults who were
maltreated as children, it probably means that the mecha-
nisms are not the same as those uncovered in preweaning
studies of rodents. Instead, we need to be examining mea-
sures of the relevant stress- and emotion-regulating corti-
colimbic systems. And, for those of us interested in
whether disturbances in regulation of the LHPA system in
early life may have altered development of these later-ma-
turing systems, we need to be assessing activity of the
LHPA system during the time when we expect these effects
may be occurring.
Here we are in relatively uncharted waters; however, as
demonstrated in this review, there are a number of indica-
tors that this approach will be fruitful. First, there are sev-
eral indications that activity of the LHPA system, as
measured using salivary cortisol, predicts the emergence of
emotional problems even when those problems are not ap-
parent at the time of cortisol assessment. Thus, in the work
by Essex and colleagues (Essex et al., 2002; Smider et al.,
2002), higher later afternoon cortisol levels when children
were 4.5 years of age were not correlated with behavior
problems at that time, but they predicted the emergence of

behavior problems 1 and 2 years later. Second, in adoles-
cents at risk for depression, spiking cortisol one or more
times over the course of basal assessment predicted the
onset of depression within the next few months (Goodyer
et al., 2000). This was the case even though studies of
early-onset depression under highly controlled conditions
indicate that the LHPA system functions normally in chil-
dren and adolescents with MDD (Ryan, 1998). Third, stud-
ies of children reared in orphanages reveal very atypical
patterns of basal cortisol production while the children are
in these orphanages. However, once adopted into families,
most of the children display normal patterns of diurnal cor-
tisol activity (Kertes et al., 2005). This is true despite that
fact that many of the postinstitutionalized children with
typical patterns of cortisol production exhibit elevated be-
havioral problems. Certainly, the studies just described do
not prove the point. However, they do raise the possibility
that longitudinal studies tracing relationships between
LHPA regulation and adverse patterns of care during peri-
ods of rapid corticolimbic development are needed, rather
than focusing our attention so narrowly on dysregulation of
the LHPA system as the critical end point.
The primate studies and the presumed maturity of
the LHPA system early in infancy in humans should
also caution us against interpreting altered patterns of GC
activity in children from adverse life conditions as evi-
dence of dysregulation of the LHPA system. Rather, it is
possible that differences in GC production under basal or
response conditions may reflect ongoing adversity in
the child’s life. Overall, we have probably paid too little
attention to children’s current life circumstances in
assessing LHPA activity in high-risk populations. As
mentioned, sleep disturbances, unpredictable bedtimes
and wake-up times, family conflict, and other circum-
stances of children’s daily lives can affect basal GC lev-
els, diurnal patterns of GC production, and reactivity to
stressors. Assessing the impact of experiences earlier in
life on current activity of the LHPA system should be
greatly enhanced by careful attention to such factors in
the child’s life at the time of assessment. Again, as
demonstrated in the work by Essex and colleagues (2002),
neither early nor concurrent circumstances may be associ-
ated with GC levels in children; however, when combined,
the interaction may be highly predictive. Also, as demon-
strated by J. Kaufman and colleagues (1997), what ap-
pears to be dysregulation of the LHPA system may on
closer inspection be evidence that when children are liv-
ing in highly stressful families, their system is more sensi-
tive to stimulation.
Conclusions and Future Direction 567
A call to include better assessments of the stressfulness
of children’s current life circumstances, however, presumes
that we have a good understanding of the characteristics of
situations that activate the LHPA system in children of dif-
ferent ages. We don’t. There is good evidence in infants and
young children that the security of the parent-child attach-
ment system predicts the power of the parent’s presence to
buffer increases in GCs to novel, arousing, or distress-elic-
iting events (see review by Gunnar & Donzella, 2002).
Work by Flinn and England (1995) clearly suggest that sig-
nificant family conflicts can provoke elevations in GCs in
children that may last several days. However, we have no
clear theory of the psychosocial processes that impact the
activity of the LHPA system in children. The meta-analysis
of laboratory stressors used in studies of adults (Dickerson
& Kemeny, 2004) may provide some guidelines for develop-
ing such a theory in developmental research. Indeed, the
time may be ripe for a similar meta-analytic synthesis
of the research on children. Certainly, we need clear guide-
lines so that we can develop laboratory paradigms that
actually produce elevations in GCs in children and adoles-
cents if we are to move beyond primarily basing our re-
search on measures of basal and/or pretest GC levels.
In addition, we need to better understand the interaction
between early experiences and individual differences in
predispositions to different disorders. As demonstrated in
this review, there does not seem to be a sharp or qualitative
difference in the association of LHPA activity for children
with behavior problems and those with temperaments or
emotional dispositions that place them at risk for develop-
ing these disorders. Children with anxiety disorders tend to
be more stress-reactive, but those with shy or inhibited
temperament also tend to exhibit higher GC activity, at
least under certain conditions. Children with disruptive be-
havior disorders (in the absence of comorbid anxiety) tend
to exhibit low GC activity. However, school-age and older
children with externalizing behaviors within the normal or
nonclinical range also show negative correlations between
cortisol levels and externalizing behaviors. We need a bet-
ter understanding of the neurobiology of these individual
differences. We also need models that will help us predict
the impact of adverse experiences on the developing stress
system of children with these different dispositions. These
models will likely include an understanding of the role of
experience in gene expression (e.g., Bennett et al., 2002).
With regard to studies of LHPA activity, this will likely
mean understanding how GCs and CRH interact with genes
regulating the activity of monoamine systems involved in
anxiety and disruptive behaviors. However, as research in
568 Stress Neurobiology and Developmental Psychopathology
developmental psychopathology has amply demonstrated,
transactional models are needed to understand develop-
ment, and these models need to include how individuals in-
fluence and increasingly select their environments. Thus,
research on early experiences, stress neurobiology, and de-
velopmental psychopathology will likely benefit from ad-
vances in molecular genetics, particularly if studies can be
conducted in which gene and environment interactions are
investigated and with a focus on how individuals both in-
fluence and are influenced by their experiences over time.
As this review demonstrates, research on early experi-
ence and stress has been fruitful. Despite this, as we at-
tempt to adequately translate the preclinical research into
its appropriate implications for human development, we
have our work cut out for us. Based on the human research
that has already been conducted, there is ample reason to
believe that there are insights to be gained by pursuing such
translational research. As the work thus far indicates, how-
ever, the translation is not likely to be direct. Nonetheless,
animal models are needed to help explicate the mecha-
nisms that transduce experience into altered neurobiologi-
cal development. Adequate translation and integration of
early experience stress research, therefore, will require re-
searchers to be familiar with not only the findings but also
the challenges of translating preclinical studies. This forms
the basis for our final argument about future directions for
this research area: Much more attention is needed in train-
ing human and animal researchers in translation and inte-
gration. To move this field forward most fruitfully,
researchers studying human development need to under-
stand both the promises and pitfalls of using the early ex-
perience animal studies as the basis for predictions about
human development. Similarly, researchers doing preclini-
cal work need to go beyond suggesting that their findings
have implications for human development. Somehow, the
bridge between those doing human developmental and
those doing animal developmental research on early expe-
riences and stress vulnerability needs to be built so that we
can make real progress in understanding how adverse early
experiences increase the risk for psychopathology.
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