ROLE OF GLUCOCORTIOIDS IN EMOTIONAL MEMORY

Glucocorticoids (GCs; cortisol in humans, corticosterone in rodents) are secreted from

the adrenal cortex following the activation of the hypothalamus-pituitary-adrenal
(HPA) axis. GCs reach peak concentrations after an exposure to a stressful event.
Following stress exposure, they promote an adaptive response to environmental
challenges by modulating physiological and behavioral processes, such as learning
and memory (Joels, Pu, Wiegert, Oitzl, & Krugers, 2006). While GCs enhance the
consolidation of memory, especially for emotional stimuli or in arousing contexts
(Maroun & Akirav, 2008; Roozendaal, 2000), they impair the retrieval of information
that was previously acquired (Buchanan, Tranel, & Adolphs, 2006; Wolf, 2009). In
other words, Gluocorticoids promote ‘memory consolidation mode’ on the expense of
retrieval (Joels et al., 2006; Roozendaal, 2000).

The timing of stress exposure/GCs secretion is, therefore, a major factor determining
the direction of the effect (i.e. enhancement or impairment) on the emotional memory
process. Yet additional learning- and stress-related factors interact in affecting the
memory process (Sandi & Pinelo-Nava, 2007). Among them are the memory type,
stress intensity, source and duration. For instance, fear memories are more strongly
consolidated than neutral memories. This results from GCs and noradrenaline
interaction in the basolateral amygdala (BLA) following an exposure to a stressful
event (Roozendaal, Portillo-Marquez, & Mcgaugh, 1996; Roozendaal, 2000). Stress
intensity has different effects on various memory types. For fear memories
consolidation, a linear or linear-asymptotic dose-response curve was suggested (i.e.
stronger memories following higher GCs levels) (Sandi & Pinelo-Nava, 2007). For
spatial memory, in contrast, an inverted U-shaped curved was demonstrated, with
moderately elevated GCs levels acting as memory facilitators while too high or too
low levels impair it (Joels, 2006).The source of stress also play a role.Whereas
intrinsic stressor (i.e. related to the cognitive task) may enhance memor, the effects of
extrinsic stress (i.e. unrelated to the task) are more heterogeneous (Sandi & Pinelo-
Nava, 2007). Moreover, while acute stress may have various effects on memory,
chronic stress is likely to impair it (McEwen, 2004; Sapolsky, 1999).

In the traditional memory research, memory consolidation was thought to be a one-

time event. According to this view, after an initial fragile period the memory trace
becomes stable (McGaugh, 1966). Yet already in the late 1960s, Misanin, Miller, &
Lewis (1968) challenged this idea, suggesting that retrieval can reactivate the
memory, rendering it fragile again.The postretrieval lability period, later demonstrated
to last up to 6 hours after retrieval (Kindt, Soeter, & Vervliet, 2009; Schiller et al.,
2010), was suggested to serve as an adaptive update mechanism (Alberini, 2011; Sara,
2000). In the last fifteen years, various pharmacological agents and behavioral
manipulations have been found to affect reactivated memories, thereby revealing the
mechanisms mediating the reconsolidation of memory. For instance, (Nader et
al.2000) demonstrated that memory reconsolidation is a protein-synthesis dependent
process, while (Kindt et al.2009) revealed that emotional memory reconsolidation
depends on noradrenergic activity. These findings suggest a similarity between
reconsolidation following retrieval and initial consolidation, a proteinsynthesis
process (Kandel, 2001) that benefits from noradrenergic activity (Roozendaal, et al.,
2006).
Even though timing plays a critical role in determining the effect of stress
and GCs on emotional memory processes.GCs modulation of emotional
memory reconsolidation has been investigated only recently. (Akirav,
2013), the results are often conflicting, as animal studies reported an
impairing effect of stress (Wang, Zhao, 2008), GCs administration but
also GCs antagonists (Pitman et al., 2011) on reactivated memories.
Human studies, usually involving stress induction as opposed to a
pharmacological intervention, demonstrate either an impairing or
enhancing (Bos, & Kindt 2014; Coccoz et al., 2013) effect of stress on
memory reconsolidation, with conflicting results with regard to the
susceptibility of strong emotional memories. Others (Wood et al., 2015)
reported no effect. This is not surprising, as GCs modulation of learning
and memory processes depends on additional factors other than timing of
intervention (e.g. post-retrieval) alone (Sandi & Pinelo-Nava, 2007). Yet
while the literature offers a clearer understanding of the way some factors
interact in modulating memory consolidation (Joels, 2006) and retrieval
(Buchanan & Lovallo, 2001), this is not the case for the emerging field of
reconsolidation. As opposed to stress induction, which leads to the
secretion of GCs and additional stress modulators, such as noradrenaline
(Joels et al., 2006), pharmacological manipulation allows to focus on the
GCs system and to determine the role of its specific receptors.

ROLE OF GLUCORTICOIDS ON MEMORY CONSOLIDATION

GCs are lipophilic and therefore makes it easy to enter the brain(Mcwen,
1968)when their activation is mediated by two reception types:
Mineralocorticoids (MR) and Glucocorticoid(GR) receptors.The two
receptors differ in affinity and distribution. The Mineralocorticoids
receptors are of higher affinity and are saturated under basal
conditions.They are mainly present in the limbic areas and mediate the
initial response to stress.The glucocorticoids are lowerr affinity,thus
becoming occupied during the circadian peak and exposure to stress.They
contribute to the secession of the HPA negative feedback loop and
mediate the beneficial effect of stress on emotional memory
consolidation. They also tend to exert their effect through gene
expression.

Glucocorticoid receptors

In contrast to the lack of effect of MR antagonism reported above, several

animal studies used the GR antagonist mifepristone and suggested a key
role of GR in the reconsolidation of emotional memories.
Post-retrieval systemic injection of mifepristone (20-30 mg/kg) was
found to impair fear memory reconsolidation in the inhibitory avoidance
(Nikzad, Vafaei, Rashidy-Pour, & Haghighi, 2011) and the cued fear
conditioning paradigms (Pitman et al., 2011). For instance, Pitman et al.
(2011) trained male and female rats to associate a cue with a footshock.
After the reactivation of the conditioned memory (i.e. by an unreinforced
re-exposure to the conditioned cue), mifepristone was systemically
administered to the animals. The result was a reduction in the conditioned
fear, as evident by a shorter duration of freezing to the cue. This effect
was relatively long-lasting (lasting 10 days after treatment) and was
dependent on memory reactivation (i.e. was absent following
mifepristone administration alone), suggesting it resulted from a
disruption to the reconsolidation process. Comparable results were
demonstrated by Achterberg et al. (2014), who showed the impairing
effects of mifepristone on the reconsolidation of appetitive (i.e. social
reward) memories. In this study, however, only preretrieval (as opposed
to post-retrieval) administration led to an effect, probably due to the
longer duration of a reactivation session in this paradigm relative to the
fear conditioning based paradigms (Achterberg et al., 2014). Other
studies have demonstrated the critical role of GR in the BLA and
hippocampus in the reconsolidation of emotional memories. Two studies
reported an impairment of fear memory reconsolidation following post-
retrieval mifepristone administration to the BLA (Jin, Lu, Yang, Ma, &
Li, 2007; Tronel & Alberini, 2007). The effect was dose-dependent, seen
only following the administration of the higher mifepristone dose while
the lower concentrations produced no effect. A dose-dependent effect was
also demonstrated by Nikzad et al. (2011), where post-retrieval
mifepristone administration to the hippocampus led to a more pronounced
reduction of fear using the higher (3 ng/µl) compared with the lower (0.3
ng/µl) dose.

Mifepristone, a GR antagonist, was demonstrated to be an efficient

pharmacological agent for the disruption of emotional memory
reconsolidation. Although the literature provides only scarce data on MR
antagonists, the results suggest they do not play a critical role in this
process. The involvement of GR activation in the consolidation of newly
acquired emotional memories is well established (Roozendaal &
Mcgaugh, 1997; Roozendaal, 2000). The above studies suggest that GR
activation, in the BLA and hippocampus in particular, is also involved in
the reconsolidation of emotional memories after retrieval.

The main brain areas that underlie the effects of stress and GCs on initial
memory consolidation are the BLA, hippocampus and prefrontal cortex
(Roozendaal, McEwen, & Chattarji, 2009). The role of GR in that process is
well documented (Roozendaal, 2000). GR antagonism (e.g. by mifepristone)
(Yang, Chao, & Lu, 2006) or inhibition of corticosterone synthesis (e.g. by
metyrapone) (Blundell, Blaiss, Lagace, Eisch, & Powell, 2011) were found to
disrupt memory consolidation. In contrast, systemic corticosterone (Blundell et
al., 2011) or GR agonists (e.g. dexamethasone) administration (Ninomiya et al.,
2010; Yang et al., 2006) facilitate memory consolidation (for a review of
consolidation enhancers, also used as extinction-learning facilitators, see:
Singewald, Schmuckermair, Whittle, Holmes, & Ressler, 2015).

IMPORTANT CHARACTERISTICS OF GLUCOCORTICOIDS

Under basal conditions, glucocorticoid secretion exhibits a 24-h circadian

profifile in which glucocorticoid concentrations present a morning

maximum in humans (the circadian peak), and slowly declining levels in

the late afternoon, evening and nocturnal period (the circadian trough),

and an abrupt elevation after the fifirst few hours of sleep. Circulating
glucocorticoids bind with high affifinity to two receptor subtypes; the
mineralocorticoid (MR or Type I) and glucocorticoid (GR or Type II)
receptors.

Although both receptor types have been implicated in mediating corticosteroid

feedback effects (see Reul and deKloet, 1985), there are two major

differences between MR and GR receptors. First, MRs bind glucocorticoids

with an affifinity that is about 6 to 10 times higher than that of GRs. This
differential affifinity results in a striking difference in occupation of the two
receptor types under different conditions and time of day. Thus, during the
circadian trough (the PM phase in humans and the AM phase in rats), the
endogenous hormone occupies more than 90% of MRs, but only 10% ofGRs.
However, during stress and/or the circadian peak of corticosteroid secretion
(the AM phase in humans and the PM phase in rats), MRs are saturated, and
there is occupation of approximately 67–74% of GRs (Reul and deKloet,
1985).

The second major difference between these two receptor types is related to
their distribution in the brain. The MR is exclusively present in the
limbic system, with a preferential distribution in the hippocampus,

parahippocampal gyrus, entorhinal, and insular cortices. On the contrary,

the GR is present in both subcortical (paraventricular nucleus and other

hypothalamic nuclei, the hippocampus and parahippocampal gyrus) and

cortical structures, with a preferential distribution in the prefrontal cortex

(McEwen et al., 1968, 1986; Meaney and Aitken, 1985; Diorio et al., 1993).

As we will see in the following sections, the impact of glucocorticoids on

cognitive function can be best understood in terms of the differential effects of

MR and GR activation (for a complete review, see deKloet et

al.,1999) in both the hippocampus and frontal lobes, two brain structures

critically involved in cognitive function.

MECHANISMS INVOLVED IN GLUCOCORTICOID

ACTIONS ON MEMORY

Among the different cellular and molecular mechanisms that have

been proposed to mediate the changes in synaptic connectivity that are
involved in long-term memory formation, two main events explains the
possible influences of glucocorticoids:

(i)glutamatergic transmission and

(ii) cell adhesion molecules.

Glutamatergic transmission

Among the early synaptic transients implicated in learning and

memory, glutamatergic transmission proved to be critical in a wide variety of
learning models (Izquierdo & Medina, 1995; Maren &Baudry, 1995). Evidence
that glucocorticoids can increase glutamate concentrations in the hippocampus, as
well as in other brain regions, including the striatum and the frontal cortex, has been
found in mammals (Lowy et al., 1993; Moghaddam et l/.,1994; Abraham et al., 1996;
Venero & Borrell,1998). In this way, glutamatergic activation mightbe a
mechanism by which corticosterone influencesmemory storage. This possibility was
evaluated inthe passive avoidance learning task in the chick because this learning
model is one in which memory has been shown to require the activation of the
glutamate receptor types z-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid
(AMPA) and N-methyl-D-aspartate (NMDA) (Stewart &Rusakov, 1995;
Richard et al., 1994). Using the corticosterone-facilitative model of the weak
training version of this task (see above), long-term retention was evaluated in chicks
injected with either NMDA or AMPA receptor antagonists(Venero & Sandi,
1997). When administered before training, both antagonists prevented the
facilitating effect of corticosterone, but when injected before administration of the
steroid in the post-training period, the antagonists failed to interfere with the steroid
effect. The relatively early effectiveness of the antagonists suggests that the outcome
was related not to corticosterone-induced actions but rather to training-triggered
mechanisms.Nevertheless, pre-training administration of the antagonists might
influence the steroid effects through the delayed expression of their actions. In
addition, the AMPA receptor antagonist, when injected 5.5 hr after training, was also
found to be effective in impairing the long-term memory potentiating effect of corti-
costerone. This finding agre;es with the involvement of this receptor type inmemory
formation for the standard passive avoidance learning at this late time point
(Steele &Stewart, 1995), and implies corticosterone actions on the late-phase
molecular events that are implicated in long-term memory formation.Cell adhesion
molecules

Among the main mechanisms mediating cellular

responses to glucocorticoids are receptor-mediated changes in gene expression
(Burnstein &Cidlowsky, 1989; Jo6ls & de Kloet, 1994). Longterm memory
formation depends on proteinsynthesis mechanisms in a wide number of animal
models (David & Squire, 1984); hence one mechanism by which
glucocorticoids influence consolidation might be the genomic modulation of the
synthesis of certain proteins that are implicated in the synaptic restructuring and
stabilization subserving
memory storage. An increasing number of studies
emphasize a role for certain cell-surface glycoproteins, the cell adhesion molecules
(CAMs)that display recognition and adhesion properties,particularly the neural cell
adhesion molecule(NCAM) and L1 (Scholey et al., 1993; Cremer et

al., 1994; LOthi et al., 1994; Fox et al., 1995;Muller et al., 1996). Interestingly, the
involvementof CAMs in the neural mechanisms of memory appears to occur
several hours after the training experience (from 6-24 hr afterwards, depending
on the animal and the learning task. The hypothesis that corticosterone facilitation
of long-term memory formation might be dependent upon a late-phase
modulation of CAMs was first investigated in the chick passive avoidance learning

task (Sandi et al., 1995). The functional state of

CAMs is modulated through post-translational

glycosylation; glycoprotein fucosylation is involved in the transition mechanisms from
short-term to long-term
memory (Rose, 1995). This mechanism was also
implicated in the facilitative effect of corticosterone on consolidation. Thus,
intracerebral corticosterone injection enhances protein fucosylation in the IMHV of
untrained chicks at 5.5 to 8.5 hr post injection, a result that is analogous to that

elicited by training chicks on the strong passive avoidance task (Sandi et al., 1995). Further
studies involving pharmacological and biochemical experiments, including the
fucosylation inhibitor 2-deoxygalactose, the protein synthesis inhibitor
anisomycin, and radiolabeled fucose, indicated that the late phase of glycoprotein
synthesis involved in the memory-facilitating effect of corticosterone occurs on newly
synthesized proteins (Sandi &Rose, 1997b). NCAM, which shows enhanced
fucosylation as a consequence of training in the standard task (Scholey et al., 1993), is
also implicated in the steroid effect because antibodies against this molecule
(administered 5.5 hr posttraining) prevent the memory-facilitating effect that is induced
by corticosterone in the weak task (Sandi et al., 1995).

In rats, glucocorticoids have also been found to influence the expression and
modulation of CAMs. Thus, an intraperitoneal corticosterone injection of adose
(5 mg/kg) that facilitates memory for weak versions of the water maze (Sandi et
al., 1997a) and contextual fear conditioning ( Cordero & Sandi,1998) tasks,
results in decreased glycoprotein synthesis in the hippocampus and the striatum at3
hr post-injection (Venero et al., 1996). The same dose was also shown to induce
increased levels of NCAM expression in the frontal, including the prefrontal cortex at 8 hr
and 24 hr post-injection.(Sandi & Loscertales, submitted). Given the key role of
the brain areas showing corticosteroneinduced modulation of CAMs on the
mechanism of learning and memory, these findings suggest that these molecules are
potential mediators of glucocorticoid actions that determine the strength of memory
consolidation. In fact, NCAM levels at the hippocampus appeared to be
increased after training on the contextual fear conditioning paradigm (Sandiet
al., 1998).The possibility that the CAMs were modulated by chronic
glucocorticoid treatments that are known to produce morphological (Wooley et al.,
1990) and cognitive (Luine et al., 1993; Sandi et al., 1997b; Loscertales et al.,
1998) deficits has also been addressed. Exposure of rats to a 21-day
corticosterone treatment results in reduced glycoprotein fucosylation in the
hypothalamus (Venero et al., 1996), as well as reduced NCAM expression in this
brain region and in the frontal/ prefrontal cortex (Sandi & Loscertales, submitted). It
is.interesting to note that NCAM expression in the frontal cortex shows a reversed
regulation by corticosterone treatments that results in opposite cognitive actions (see
above), which might have implications in understanding the cellular and molecular
mechanisms by which glucocorticoid actions at the brain turn from facilitating to
deleterious as the period of exposure increases.Work is currently in progress to
elucidate further the relation between glucocorticoids, CAMs, and cognition.
THE NEGATIVE EFFECTS OF ACUTE GLUCOCORTICOID

INCREASES ON HUMAN EMOTIONAL MEMORY

Memory Sustained by the Hippocampus

Given the presence of MRs and GRs in the human hippocampus, it has

been suggested that acute increases of glucocorticoids should lead to

defificits in memory functions sustained by this brain region. The seminal

work of Scoville and Milner (1957) in amnesic patients having undergone

bilateral hippocampal ablation demonstrated that this structure plays a

critical role in memory formation, particularly in declarative memory

function. Declarative memory refers to the conscious or voluntary recollection

of learned information (such as remembering what one had for

breakfast; Squire, 1982, 1987; Cohen, 1984; Thyompson, 1986), whereas

non-declarative memory refers to the facilitation of recollection of previous

information without a conscious and deliberate intention to retrieve this

information (such as measured in priming; Schacter, 1987). This somewhat

specialized role of the hippocampus served as the basis for specifific

hypotheses regarding the effects of glucocorticoids on human learning and

memory.

In 1996, Kirschbaum and collaborators took advantage of the declarative/non-

declarative memory dissociation within the hippocampus in

order to assess whether glucocorticoids would have a specifific impact on

declarative memory function in humans. They reported that the administration

of a low dose of synthetic glucocorticoids led to a signifificant

decrease in declarative memory performance, while it had no effect on

non-declarative memory performance. These results suggested that
glucocorticoids interact with hippocampal neurons to induce cognitive
defificits in humans.

More recently, DeQuervain and collaborators (2000) tested the impact of an

acute increase of glucocorticoids as a function of the nature of memory
processing. A medium dose of synthetic glucocorticoids was administered
either before the acquisition of a word list, immediately after, or justbefore the
retrieval of the list. The results revealed signifificant impairmentsin memory
when the drug was administered just before retrieval, thus sug- 30 S. J. Lupien
et al.gesting specifific effects of glucocorticoids on the retrieval of previously
learned information. A specifific effect of acute glucocorticoid elevations on
retrieval process in humans has recently been replicated by Wolf and
collaborators (2001). Young and aged men were given a medium dose of
synthetic glucocorticoids after having learned a list of 10 words. A second
word list was learned and recalled after drug administration. Results showed
that glucocorticoids impaired recall of the word list learned before treatment in
both groups but did not inflfluence recall of the list learned after treatment.
These results go along with previous data obtained by de Quervain et al.,
(2000) showing that acute exogenous administrations of glucocorticoids have
impairing effects on retrieval process. The in vivo demonstration of
glucocorticoid effects on memory retrieval process was recently performed by
the group of de Quervain and collaborators (2003) using positron emission
tomography (PET). Young subjects were administered a medium dose of
synthetic glucocorticoids 24 hours after learning various declarative memory
tasks. Brain activation was measured by PET 1 hour after drug administration.
Results showed that glucocorticoids induced a large decrease in regional
cerebral blood flflow in the right posterior medial temporal lobe coupled with
impaired cued recall of word pairs learned 24 hour earlier. These results were
the fifirst to provide an invivo demonstration that acutely elevated
glucocorticoid levels can impair declarative memory retrieval processes that are
related to a disturbance of medial temporal lobe function. A similar impairment
of retrieval function was recently reported by Buss and collaborators (in press).
These authors administered a small dose of synthetic glucocorticoids to young
adults, and measured retrieval of past events in their life (autobiographical
memory). Results showed that when compared to placebo, glucocorticoids
signifificantly impaired retrieval of past personal events.

EMOTIONAL MEMORY Sustained by the Frontal Lobes

In 2000, Sanchez and collaborators (2000) reported that, in contrast

to its well established distribution in the rat brain, GR mRNA is only

weakly detected in the dentate gyrus and Cornu Ammonis of the macaque
hippocampus. In contrast, GR mRNA is strongly detected in the pituitary,
cerebellum, hypothalamic paraventricular nucleus and prefrontal

cortices. Additionally, using a specifific squirrel monkey antibody Patel

and collaborators (2000) found that GR receptors were well expressed in

the hippocampus, but were more prominently found in the prefrontal

cortex.

The almost exclusive presence of GRs in the primate and human

frontal lobes led scientists to study the impact of glucocorticoids on frontal

lobes functions. Studies in nonhuman primates (Goldman-Rakic, 1987,1995)
and humans (Petrides and Milner, 1982; Owen et al., 1990) showed that lesions
of the dorsolateral prefrontal cortex (DLPFC) give rise to impairments in
emotional memory. Emotional memory is the cognitive mechanism that allows
us to keep a limited amount of information active for a limited period of time
(see Baddeley, 1995). Thus, working memory impairments have been found in
several experiments using a variety of delay task procedures. In these tasks, a
temporal gap is introduced between a stimulus and a response, which creates
the need to maintain the stimulus in temporary memory storage. Data obtained
in monkeys showed that cells in the lateral prefrontal cortex become
particularly active during delayed response tasks, suggesting that these cells are
actively involved in maintaining the information during the delay (Goldman-
Rakic et al., 1990, 1995). Neuropsychological evidence suggests that humans
with prefrontal damage are impaired in emotional memory (Luria, 1966;
Fuster, 1980). These patients are also highly susceptible to cognitive
interference and they perform poorly on neuropsychological tests that require
response inhibition such as the Wisconsin Card Sorting Test (Stuss et al., 1982;
Shimamura, 1995). Moreover, recent neuroimaging data summarized and
reviewed by Smith et al., (1998; see also Dolan and Fletcher, 1997;
Ungerleider et al., 1998) show a signifificant relationship between working
memory processing, and activation observed in the prefrontal cortex (Smith et
al.,1998; Ungerleider et al., 1998).In 1999, we reported data showing
impairments in working memory

function with a high dose of synthetic glucocorticoids in young male subjects

(Lupien et al., 1999).

THE ABSENCE OF EFFECTS OF ACUTE GLUCOCORTICOID

INCREASES ON HUMAN MEMORY

Although many studies reported impairing effects of acute increases in

glucocorticoids on declarative memory performance, other studies reported no
such impairing effects on memory functions sustained by the

hippocampus and frontal lobe region.

Hypothalamic-Pituitary-Adrenal (HPA) axis

The HPA axis is a vital component of both the central and the peripheral limb of the

stress system . As such, HPA axis integrity and precise regulation of its function are

essential characteristics of the successful adaptive response to any stressor. At the

level of the hypothalamic-pituitary unit, CRH is released into the hypophyseal portal

system and acts as the principal regulator of the anterior pituitary ACTH secretion.

CRH binding on CRH-R1 of the corticotrophs is permissive for ACTH secretion,

whilst AVP acts as a potent synergistic factor to CRH with little ACTH secretagogue

activity by itself(Grammatopoulos et al,2012). Under non-stressful conditions, both

CRH and AVP are secreted into the portal system in a circadian and highly

concordant pulsatile fashion(Abou-Samra et al,1987). The HPA axis activity is

characterized not only by a typical circadian rhythm, but also by an ultradian pattern

of discrete pulsatile release of glucocorticoids, with a pulse of production every 1-2

hours (Redekopp et al,1986.). The amplitude of the CRH and AVP pulses increases in

the early morning hours, consequently resulting in increased amplitude and frequency

of ACTH and cortisol secretory bursts in the systemic circulation. Of note, recent data

indicate that various factors including age, body mass index (BMI), and gender, are

individually and in some cases jointly associated with endogenous ACTH-induced

stimulation of overnightpulsatile cortisol secretion(Iranmanesh, 1990).

The circadian release of CRH/AVP/ACTH/cortisol in their characteristic pulsatile

manner appears to be controlled by one or more CNS pace makers, as will be more

precisely described in the following section on the “CLOCK system”(Veldhuis et

al). These diurnal variations are perturbed by changes in lighting, feeding and

physical activity patterns, whilst they are disrupted when a stressor is imposed.

During acute stress, the amplitude and synchronization of both CRH and AVP

secretory pulses increases, with additional recruitment of PVN CRH and AVP

secretion. Furthermore, angiotensin II, various cytokines and lipid mediators of

inflammation are also secreted, depending on the stressor, and act on various levels of

the HPA axis to mainly stimulate its activity.

Interestingly, nicotine can also induce the HPA axis via both CRH-R and AVP V(1b)

receptors; hence, when CRH-R is blocked, nicotine may utilize the AVP V(1b)

receptor to induce its action and increase the secretion of ACTH and

glucocorticoids(Veldhuis et al).

The adrenal cortex constitutes the principal target organ of the pituitary-derived

circulating ACTH. The latter is the key regulator of glucocorticoid and adrenal

androgen secretion by the zona fasciculata and zona reticularis, respectively, whilst it
is also implicated in the regulation of aldosterone secretion by the zona glomerulosa

(Aguilera, 1993). Notably, existing evidence suggests that the adrenal cortisol

secretion is further regulated by other hormones and/or cytokines coming from the

adrenal medulla or the systemic circulation, and by neuronal signals via the

autonomic innervation of the adrenal cortex.

Glucocorticoids are the final hormonal effectors of the HPA axis, exerting their

pleiotropic effects via their ubiquitously distributed intracellular receptors (GRα and

GRβ; both members of the nuclear receptor superfamily)(Munck A,1984). The non-

activated glucocorticoid receptor resides in the cytosol as a hetero-oligomer with heat

shock proteins and immunophilin(Smith DF, 1993). Upon ligand binding,

glucocorticoid receptors dissociate from the rest of this hetero-oligomer, and

subsequently homodimerize and translocate into the nucleus, where they interact with

specific glucocorticoid response elements (GREs) of the DNA to transactivate or

transrepress appropriate hormone-responsive genes(Pratt WB, 1990). Transactivation

has been suggested as mediating most of the adverse effects of glucocorticoids, while

transrepression is considered to mediate mostly anti-inflammatory glucocorticoid

effects by inhibiting several inflammatory mediators/pathways (e.g. AP-1, NF-κB).

Posttranslational modifications of glucocorticoid receptors (e.g. phosphorylation,

acetylation, ubiquitination and sumoylation) regulate the receptor stability and nuclear

localization, as well as its interaction with other proteins. Furthermore, glucocorticoid

receptor activation causes changes in the stability of other mRNAs and, hence, the

translation rates of several glucocorticoid-responsive proteins(Ismaili et al). Notably,

glucocorticoids influence the secretion rates of specific proteins and alter the

electrical potential of neuronal cells, through mechanisms that remain to be

elucidated. Glucocorticoids can further induce rapid nongenomic effects, via

mechanisms which are also not fully clarified yet. Moreover, there are also data

indicating that glucocorticoids have the ability to regulate mitochondrial functions and

energy metabolism. Indeed, the presence of both GRα and GRβ in mitochondria of

animal and human cells has been associated with modulation of mitochondrial

functions indicating that the cross-talk of glucocorticoid receptors with mitochondria

may be involved in cell survival(Koufali, et al 1990).

Glucocorticoids play a crucial role in the regulation of the basal HPA axis activity and

in the termination of the stress response by acting at multiple levels, including extra-

hypothalamic regulatory centers, the hypothalamus and the pituitary. As such, the

inhibitory glucocorticoid feedback on the ACTH secretory response limits the

duration of the total tissue exposure to glucocorticoids, thus minimizing the catabolic,

anti-reproductive and immunosuppressive effects of these hormones. Interestingly, a

dual glucocorticoid receptor system exists in the CNS, including both type I

glucocorticoid receptors (mineralocorticoid receptor) which respond to low levels of

glucocorticoids and primarily act to induce activation; and the classic glucocorticoid

receptor (type II) which responds to higher levels of glucocorticoids, stress-related or

not, and can either dampen some systems or activate other. The negative feedback

control of the CRH and ACTH secretion is mediated through type II glucocorticoid

receptors(de Kloet, 2000).

Glucocorticoid secretion pulsatility is among the main factors determining the HPA

axis responsiveness to stress and the transcriptional responses of glucocorticoid

responsive genes. Data on the downstream effects of short-term fluctuations in serum

glucocorticoid concentrations indicate that ultradian cortisol pulsatility can impact on

gene expression and phenotype of target cells. Importantly, pulsatile cortisol hasbeen

shown to significantly reduce cell survival due to increased apoptosis compared to

continuous exposure to the same cumulative dose(Lightman SL, 2008).

Amygdala/Hippocampus

The amygdala/hippocampus complex is activated during stress primarily by ascending

catecholaminergic neurons originating in the brain stem or by inner emotional

stressors (e.g. conditioned fear) possibly from cortical association areas. The

amygdala nuclei constitute the principal CNS center for fear-related behaviors and

their activation is important for both retrieval and emotional analysis of all relevant

stored information for any given stressor. In response to emotional stressors, the

amygdala can directly stimulate central stress system components and the

mesocorticolimbic dopaminergic system. Interestingly, there are CRH peptidergic

neurons in the amygdala which respond positively to glucocorticoids and whose

activation leads to stress system stimulation and anxiety. CRH neurons in the central

nucleus of the amygdala send projections to the PVN parvocellular regions and the

parabrachial nucleus of the brain stem which are considered crucial for CRH-induced

neuroendocrine, autonomic and behavioral effects. CRH fibers also interconnect the

amygdala with the bed nucleus of the stria terminalis and the hypothalamus.

Conversely to the stimulatory CRH and norepinephrine effect, the hippocampus

exerts a tonic and stimulated inhibitory effect on the amygdala activity and the PVN

CRH and LC/NE-sympathetic systems. Indeed, the hippocampus plays an important

role in shutting off the HPA stress response. Hippocampal atrophy or damage impairs

this shut off function and can lead to prolonged HPA responses to psychological

stressors. These findings led to the "glucocorticoid cascade hypothesis" of stress and

aging. Accordingly, Lupien et al. have shown that progressively increased salivary

cortisol levels during annual exams over a 5-year period can predict reduced

hippocampal volume and decreased performance on hippocampal-dependent learning

and memory tasks. Moreover, Refojo et al. have demonstrated, through specific CRH-

R1 deletions in glutamatergic, GABAergic, dopaminergic and serotonergic cells, that

CRH-R1 absence in forebrain glutamatergic circuits reduces anxiety and impairs

neurotransmission in the amygdala and hippocampus, whilst elective CRH-R1

deletion in midbrain dopaminergic neurons results in increased anxiety-like behavior,

suggesting a bidirectional model for the CRH-R1 role in anxiety.