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dietary constraint (Langley-Evans et al., 1996b; Bertram et al., 2001).
Further evidence for the importance of the details of exposure in determining the
long-term effects of glucocorticoid programming comes from recent studies in guinea
pigs. These animals are relatively glucocorticoid resistant because of a mutant GR gene
(Keightley and Fuller, 1994). Perhaps in consequence, prenatal glucocorticoid expos-
ure has smaller effects on the HPA axis in guinea pig offspring (Dean et al., 2001; Liu
et al., 2001). The duration of exposure and the sex of the offspring also have an
impact in this species as in others. In males, short-term exposure to dexamethasone
(2 days) leads to signi¬cantly elevated basal plasma cortisol levels; whereas repeated
doses reduce basal and stimulated plasma cortisol levels in adults. In contrast, juven-
ile females exposed for 2 days have reduced HPA responses to stress, whereas adult
females exposed to repeated antenatal doses of dexamethasone have higher plasma
cortisol levels in the follicular and early luteal phases. Similar sex-speci¬c program-
ming of the HPA axis have been reported for prenatal stress in rats (Weinstock et al.,
1992; McCormick et al., 1995).
A study of the effects of glucocorticoid programming in primates showed that the
offspring of mothers treated with dexamethasone during late pregnancy had elevated
basal and stress-stimulated cortisol levels and a 30% reduction in hippocampal size
(Uno et al., 1994). These studies in rodents, guinea pigs, sheep and primates indicate
that exposure to excess glucocorticoids in utero can programme HPA axis function.
The data addressing HPA programming in humans are discussed at the end of this
review.

Programming behaviour
Overexposure to glucocorticoids in utero, as a result of either prenatal dexamethasone
administration or 11 -HSD inhibition leads to alterations in adult behaviour.
Administration of dexamethasone to rats for all 3 weeks of gestation or only in the last
week reduces ambulation and rearing in the open ¬eld in adult animals (Figure 5.6;
Welberg et al., 2001), although another study did not ¬nd this (Holson et al., 1995).
These studies employed subtly different timings of exposure, again suggesting that
very speci¬c time windows exist for the effects of prenatal treatments (Figure 5.5).
Additionally, late gestation administration of dexamethasone alters exploration
on an elevated plus maze and reduces immobility both in the acquisition and the
160 J. R. Seckl et al.


120 30
Ambulation (grid crossings)




100 25




Number of rears
80 20


60 15

——
40 10

20 5

0 0
(a) Control DEX1-3 DEX3 (b) Control DEX1-3 DEX3
Figure 5.6 Administering dexamethasone to pregnant rats reduced ambulation and rearing of their
offspring in the open ¬eld. Adapted from Welberg et al. (2001)



retrieval phase of a forced-swim test, implying impaired coping and a reduced cap-
acity for acquisition, consolidation and/or retrieval of information under stressful cir-
cumstances (Welberg et al., 2001). This suggests that fetal glucocorticoid exposure,
especially during the last week of gestation, may programme ˜behavioural inhibition™
and reduced coping in aversive situations later in life. Intriguingly, inhibition of
11 -HSD, which is most highly expressed in midgestation, produces a phenotype
intermediate between continuous and ¬nal week dexamethasone exposure (Welberg
et al., 2000). Prenatal glucocorticoid exposure also affects the developing dopamin-
ergic system (Diaz et al., 1995; 1997) with clear implications for proposed devel-
opmental contributions to schizo-affective, attention-de¬cit hyperactivity and
extrapyramidal disorders. Indeed, stressful events in the second trimester of
human pregnancy associate with an increased incidence of schizophrenia in the
offspring (Koenig et al., 2002).


Structural effects of antenatal glucocorticoids on the CNS
Exposure to glucocorticoids in utero has widespread acute effects upon neuronal
structure and synapse formation (Antonow-Schlorke et al., 2003), and may per-
manently alter brain structure (Matthews, 2000). Studies in young and aged ani-
mals and humans have demonstrated that stress and increased glucocorticoid
concentrations can lead to changes in hippocampal structure (Bremner et al., 1995;
Sheline et al., 1996; Stein et al., 1997; Sapolsky, 1999). In rhesus monkeys, treatment
with antenatal dexamethasone caused a dose-dependent neuronal degeneration of
hippocampal neurones and reduced hippocampal volume in the fetuses, which per-
sisted at 20 months of age (Uno et al., 1990). Fetuses receiving multiple lower-dose
161 Prenatal glucocorticoids and the programming of adult disease


injections showed more severe damage than those receiving a single-large injection.
Human and animal studies have demonstrated that altered hippocampal structure
may be associated with a number of consequences for memory and behaviour
(Bremner et al., 1995; Sheline et al., 1996; Stein et al., 1997).
In mice, prenatal treatment with prednisolone appears to lead to delayed motor
development, offspring have delayed eye opening and delayed development of lifting,
walking and gripping skills (Gandelman and Rosenthal, 1981). In rhesus monkeys,
prenatal dexamethasone was not associated with delayed motor development (Uno
et al., 1994). In sheep, betamethasone exposure in utero is associated with delayed
myelination in areas of the brain undergoing active myelination at the time of expo-
sure, such as the optic nerve (Dunlop et al., 1997), with unknown consequences.
Intriguing recent data suggest that deleterious developmental effects of excess glu-
cocorticoids upon the CNS are even more widespread. Prenatal exposure to dexa-
methasone increases the susceptibility of the cochlea to acoustic noise trauma in
adulthood. Interestingly, the mechanism involves increased susceptibility to oxida-
tive stress, and can be treated effectively with antioxidants (Canlon et al., 2003).

CNS programming mechanisms
The brain is clearly important as a target for glucocorticoid programming. Its
mechanisms have been examined at a variety of levels, from structural to gene
expression, for instance recently exploiting emerging microarray technology
(Kinnunen et al., 2003). However, a caution is required since the mechanisms of
programming appear to differ somewhat depending on the timing of the exposure
and the species involved. Nevertheless, some headway has been made.
Indications of the molecular mechanisms by which early life environmental fac-
tors may programme offspring physiology come from the studies of the processes
underpinning postnatal environmental programming of the HPA axis in the
˜neonatal handling™ paradigm (Levine, 1957; 1962; Meaney et al., 1988; 1996). In this
model, 15 min of daily handling of rat pups during the ¬rst 2 weeks of life (Meaney
et al., 1988) permanently increases GR density in the hippocampus and prefrontal
cortex, but not in other brain regions. This increase in receptor density potentiates
the HPA axis sensitivity to glucocorticoid negative feedback and results in lower
plasma glucocorticoid levels throughout life, a state compatible with a good adjust-
ment to environmental stress (Meaney et al., 1989; 1992). Neonatal glucocorticoid
exposure may have similar effects (Catalani et al., 1993). The neonatal handling
model appears to be of physiological relevance, since handling enhances maternal
care-related behaviours and natural variation in such maternal behaviour correlates
similarly with the offspring HPA physiology and hippocampal GR expression
(Liu et al., 1997). The long-term manifestations of some prenatal programming can
be substantially modi¬ed by the immediate postnatal environment (Maccari et al.,
162 J. R. Seckl et al.


1995), suggesting that distinct ˜windows™ occur and showing that apparently similar
early life events may produce different responses depending upon their degree,
duration, developmental timing or sequence. Again, the implications for human
epidemiology are that distinct offspring pathophysiologies may be determined
merely by the timing and severity of the stimulus/stress involved.
For prenatal glucocorticoid exposure, in the rat, while both long- and short-
term exposure to prenatal dexamethasone result in adults with elevated basal cor-
ticosterone levels, the underlying mechanisms differ depending on the timing of
exposure. Exposure to dexamethasone during the last third of pregnancy reduces
MR and GR levels in the hippocampus and increases CRH mRNA in the hypo-
thalamic PVN (Welberg et al., 2001). In contrast, dexamethasone throughout ges-
tation does not alter hippocampal GR or MR, but increases receptor expression in
the amygdala, a structure which stimulates the HPA axis (Welberg et al., 2001).
Thus in the rat, late gestational dexamethasone exposure may permanently alter
the ˜set point™ of the HPA axis at the level of the hippocampus, reducing feedback
sensitivity, whereas continuous exposure may increase forward drive of the HPA
axis through the amygdala. By implication, distinct neural mechanisms underlie
the common outcome of altered HPA axis activity following prenatal glucocorti-
coid exposure. This rather fundamental idea may underlie the subtle but impor-
tant differences in outcome phenotypes seen in various perinatal programming
models and may involve more than the HPA axis. GR and MR programming by
antenatal glucocorticoid exposure also occurs in sheep though the effects appear
less robust (Matthews, 2002).

CRH programming in the amygdala?
The behavioural changes observed in prenatal glucocorticoid-exposed offspring may
be associated with altered functioning of the amygdala, a structure involved in the
expression of fear and anxiety. Intra-amygdala administration of CRH is anxiogenic
(Dunn and Berridge, 1990). Prenatal dexamethasone or 11 -HSD inhibition
increases CRH mRNA levels speci¬cally in the central nucleus of the amygdala
(Figure 5.7), a key locus for the effects of the neuropeptide on the expression of fear
and anxiety (Welberg et al., 2000; 2001). Prenatal stress similarly programmes
increased anxiety-related behaviours along with elevated CRH expression and release
in the amygdala (Cratty et al., 1995). Indeed, corticosteroids facilitate CRH mRNA
expression in this nucleus (Makino et al., 1994; Hsu et al., 1998) and increase GR
and/or MR in the amygdala (Welberg et al., 2000; 2001). The amygdala stimulates the
HPA axis via a CRH signal (Feldman and Weidenfeld, 1998), thus an elevated
corticosteroid signal in the amygdala consequent on the hypercorticosteronaemia
in the adult offspring of dexamethasone-treated dams, may produce the increased
CRH levels in adulthood. CRH, arising from the forebrain, is also important in the
163 Prenatal glucocorticoids and the programming of adult disease


160

140
——
CRH mRNA expression (O.D.)

120

100

80

60

40

20

0
Control DEX1-3 DEX3
Figure 5.7 Administering dexamethasone prenatally increased CRH mRNA levels in the amygdala of
adult rats. Adapted from Welberg et al. (2000)



hippocampus where it facilitates acetylcholine transmission. Intriguingly prenatal
stress potentiates this action of CRH, though the molecular basis is obscure (Day
et al., 1998).
A direct relationship between brain corticosteroid receptor levels and anxiety-
like behaviour is supported by the phenotype of cre-lox transgenic mice with select-
ive loss of GR gene expression in the brain, which show markedly reduced anxiety
(Tronche et al., 1999). Dexamethasone exposure increases GR and MR gene
expression in the amygdala, though the subnuclei involved depend upon timing of
exposure (Welberg et al., 2001). In such details will doubtless lie the understanding
of the links between programming and phenotype.
How might such mechanistically distinct effects come about with glucocorticoid
exposure at different times during development? It seems reasonable to propose
that programming may only happen at critical times during organ development.
Thus, glucocorticoid exposure in the last days of gestation in the rat can target CNS
regions actively developing, such as the hippocampus, but not those yet to develop
or those already in their ¬nal state. The long and complex pre- and postnatal
ontogeny of the brain makes it a prime target for programming. The complex pat-
terns of expression of the key candidate genes GR, MR and the 11 -HSDs in the brain
may underlie this (Diaz et al., 1998; Matthews, 1998). Whilst the details of brain
ontogeny patterns are species speci¬c, the broad impression of tissues protected
from or allowing timed exposure to glucocorticoids appears a tenable interpreta-
tion of these exquisite patterns of gene expression. Clearly exogenous (or endoge-
nous) steroids can only have developmental effects on speci¬c target genes and
systems during their individual ontogenic windows of susceptibility.
164 J. R. Seckl et al.


Neuronal pathways and mechanisms
In recent years, the precise pathways involved in HPA axis programming associated
with neonatal handling and variations in maternal care have been dissected.
Handling acts via ascending serotonergic (5-hydroxytryptamine, 5HT) pathways
from the midbrain raphe nuclei to the hippocampus (Smythe et al., 1994).
Activation of 5HT induces GR gene expression in fetal hippocampal neurones in
vitro (Mitchell et al., 1990) and in neonatal (O™Donnell et al., 1994) and adult hip-
pocampal neurones in vivo (Yau et al., 1997a). The ˜handling™ induction of 5HT
requires thyroid hormones that are elevated by the stimulus. Consistent with this,
administration of dexamethasone to fetal guinea pigs leads to an elevation of fetal
thyroid hormone and an upregulation of hippocampal GR mRNA (Dean and
Matthews, 1999). At the hippocampal neuronal membrane, some recent ¬ndings
implicate the ketanserin-sensitive 5HT7 receptor subtype, which is regulated by gluco-
corticoids (Yau et al., 1997b) and positively coupled to cAMP generation, in the han-
dling effects (Meaney et al., 2000). In vitro, 5HT stimulation of GR expression in
hippocampal neurones is blocked by ketanserin and mimicked by cAMP analogues
(Mitchell et al., 1990; 1992). 5HT7 receptors appear to play a key role in this action
(Laplante et al., 2002). In vivo, handling also stimulates cAMP generation in the
hippocampus (Diorio et al., 1996). The next step appears to involve stimulation of
cAMP associated and other transcription factors, most notably nerve growth fac-
tor-inducible factor A (NGFI-A) and activator protein 2 (AP-2) (Meaney et al.,
2000). NGFI-A and AP-2 may bind to the GR gene promoter (Encio and Detera-
Wadleigh, 1991), though direct evidence for this is lacking. This pathway might also
be involved in some prenatal programming paradigms affecting the HPA axis since
last trimester dexamethasone exposure increases 5HT transporter expression in the
rat brain (Fumagalli et al., 1996; Slotkin et al., 1996b), an effect predicted to reduce
5HT availability in the hippocampus and elsewhere. This may well induce a fall of
GR and MR, the converse of postnatal handling.

The GR gene: a common programming target?
Expression of the GR gene is regulated in a complex tissue-speci¬c manner.
Although GR are expressed in all cells, their density and regulation vary considerably
between tissues, and even within a tissue (Herman et al., 1989). Transgenic mice with
a reduction of 30“50% in tissue levels of GR have major neuroendocrine, metabolic
and immunological abnormalities (Pepin et al., 1992; King et al., 1995). The level of
expression of GR is thus critical for cell function. As discussed, there is much
evidence to suggest that GR gene transcription can be programmed in a tissue-
speci¬c manner by perinatal events. The GR promoter is extremely complex, with
multiple tissue-speci¬c alternate untranslated ¬rst exons in rats (McCormick et al.,

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