Discussion
In the present study, we hypothesized that the regulation of GRs and MRs
in the PVN may influence depressive-like behaviors by modulating CRF in
the PVN. After exogenous CORT exposure via drinking water for 21 days,
plasma corticosterone rhythms were clearly altered in the present rat
model (Supplementary Fig. S2), which resulted in the
overproduction
of CRF and reductions of GR and MR expression in the PVN. Intra-PVN
administration of only the combination of the GR antagonist RU486 and MR
antagonist spironolactone inhibited depressive- or anxiety-like
behaviors and restored MR, GR, and CRF levels and the MR/GR ratio in the
PVN to normal. However, intra-PVN administration of either RU486 or
spironolactone alone did not inhibit depressive-like behaviors.
Intra-PVN administration of spironolactone alone inhibit anxiety-like
behaviors.
Glucocorticoid
receptors are richly expressed in the medial parvocellular PVN and
co-localize with CRF, thus placing the PVN in a prime position to
control the output of neurons that activate the HPA axis (Liposits et
al., 1987; Uht et al., 1988). Although the
PVN
is well known to be the major site of CRF synthesis in the brain (De
Souza, 1995),
with
projections to many extrahypothalamic areas that are involved in various
behaviors, its role in stress-related behaviors has not been
studied
extensively. Chronic variable
stress reduces the expression of GRs in the medial parvocellular PVN.
CRF expression is negatively regulated by GCs, and GRs may play a role
in the observed upregulation of CRF during chronic stress (Herman et
al., 1995). This negative correlation between GR and CRF levels (i.e.,
downregulation of GRs and upregulation of CRF) was also observed in the
PVN in the present rat model. However, the intra-PVN infusion of RU486
returned GRs level in the PVN to normal, had no effect on MR, decreased
the MR/GR ratio, and increased CRF levels, and depressive- and
anxiety-like behaviors did not improve. These results showed that simply
increasing the expression of GRs in the PVN without reversing CRF to
normal levels could not improve either depressive- or anxiety-like
behavior. Therefore, one unresolved issue is why the recovery of GRs did
not return CRF levels to normal but rather further increased the
abnormally high levels of CRF. One possibility is the recruitment of
other mechanisms beyond the negative regulation of CRF by GRs. Lamberts
et al. reported that RU486 activated the HPA axis, resulting in a
resetting of this system at a higher level at which the diurnal rhythm
and responsiveness to CRF stimulation were maintained, whereas the
sensitivity to dexamethasone diminished, and these changes were caused
by the induction of partial cortisol receptor resistance during RU486
therapy (Lamberts et al., 1991). Based on its mechanism of action and
evidence that the restoration of normal hypothalamic CRF activity by
RU486 appears to be the rate-limiting step in HPA axis recovery (Muglia
et al., 2000), central GR blockade may deprive the hypothalamus from GR
activity, thereby stimulating CRF-producing neurons in the hypothalamus
and resulting in the further enhancement of CRF.
The HPA axis has long been
recognized for its involvement in depression, with a focus on
cortisol/CORT and less of an emphasis on aldosterone as a stress
hormone. A recent study found that
aldosterone acted selectively in mood-regulating brain areas, without
competing with cortisol/CORT (Murck et al., 2014). Although receptors
for aldosterone (i.e., MRs) are known to be expressed in the PVN, its
role in the local regulation of HPA axis function has been less
explored.
The MR antagonist spironolactone inhibits the effects of
mineralocorticoids by displacing them from MRs in the PVN. Wu et al.
(2013) found that chronic
subcutaneous CORT treatment triggered several depressive-like behaviors
and downregulated MR expression in parallel in the hippocampus and
hypothalamus. On the other hand, they also reported that intraperitoneal
spironolactone administration for 7 days in animals that were
subcutaneously treated with CORT reduced immobility time in the FST and
improved performance in a novel object recognition test (Wu et al.,
2013). In the present study, CORT-exposed rats via drinking water also
exhibited lower MR expression in the prefrontal cortex, hippocampus, and
amygdala (data not shown). Bitran et al. (1998) hypothesized that the
anxiolytic effects of an MR antagonist may be more pronounced in animals
whose hippocampal MRs are activated by CORT or the MR agonist
aldosterone. In the present study, the intra-PVN infusion of
spironolactone restored MR levels in the PVN to normal and improved
anxiety-like behaviors in the NSFT and SIT, without altering CRF levels
in the PVN, but it did not improve depressive-like behaviors in the FST
or SPT. However, still needing to be confirmed is whether the anxiolytic
effects were caused by the restoration of MRs in the PVN in the present
rat model. Thus, further studies should seek to determine the
pharmacological specificity of the effects of intra-PVN spironolactone
administration to exclude possible actions of spironolactone at sites
other than MRs.
The balance between MRs and GRs during chronic stress plays an important
role in the etiology of depression (De Kloet et al., 1998).
The
pharmacological modulation of GRs and MRs also influences the endocrine
stress response and depressive-like behaviors in rodents. The ways in
which MRs and GRs interact to influence the endocrine system and related
behaviors are still being clarified. In the present study, RU486 alone
restored the downregulation of GR levels in the PVN to normal but did
not influence the downregulation of MRs, thus making the MR/GR ratio
significantly lower than normal and failing to suppress depressive- and
anxiety-like behaviors (Fig. 3). Spironolactone treatment alone restored
MR levels in the PVN to normal but did not influence the downregulation
of GRs, thus making the MR/GR ratio significantly higher than normal and
inhibiting anxiety-like behaviors in the NSFT and SIT (Fig. 4). When
RU486 and spironolactone were administered together, MR and GR levels
and the MR/GR ratio returned to normal, and depressive- and anxiety-like
behaviors significantly improved (Fig. 5). The present findings suggest
that the normalization of MR, GR, and CRF levels and the MR/GR ratio may
be necessary conditions to inhibit depressive- and anxiety-like
behaviors in the present rat model. Nonetheless, the restoration of MR
levels in the PVN to normal may be a sufficient condition to improve
anxiety-like behaviors.
CRF also acts within other regions of the brain where it directly
contributes to stress-responsive behavior. Indeed, some depressed
patients had high CSF levels of CRF, and these levels are normalized in
patients who respond to antidepressant treatment, suggesting that these
alterations of CRF levels contribute to the symptomology of depression
(Pandey et al., 2019). In the central nervous system, patients had high
levels of CRF in the PVN and in monoaminergic nuclei, including the
locus coeruleus and raphe (Austin et al., 2003; Bissette et al., 2003).
Elevations of plasma CRF levels in depressed individuals are ameliorated
by successful electroconvulsive shock therapy (Nemeroff et al., 1991)
and antidepressant treatment (Heuser et al., 1998). The causal
relationship between depression and CRF hyperactivity and receptor
expression is unclear. The present results suggest that a state of
equilibrium between GRs and MRs in the PVN may improve the function of
the HPA axis, which can improve depressive- and anxiety-like behaviors.
However, the specific ways in which GRs, MRs, and CRF in the PVN are
involved in the pathogenesis of depression and anxiety need to be
elucidated.
The present study has some limitations. We only examined male rats to
eliminate gender influences that are known to modulate experimentally
induced emotion and HPA activity. The significance of sex differences in
PVN function is virtually unexplored. Therefore, we do not know whether
our results are generalizable to female rats. Furthermore, we did not
detect the activity of HPA function. Therefore, unclear was the state of
the HPA axis when antidepressant effects were induced by GR and MR
blockade. One possibility is that the target sites of the antidepressant
effects of RU486 and spironolactone may not be in the PVN.
Overall, the present study found that the combination of pharmacological
GR and MR antagonism in the PVN prevented the pathogenesis of
depressive- and anxiety-like behaviors in rats that were chronically
treated with CORT, confirming that both MRs and GRs in the PVN play an
important role in CRF function. Such a combined pharmacological approach
may be a novel strategy to prevent hypercortisolism- or chronic
stress-induced depression and its metabolic complications. Much work
still needs to be done to achieve a more complete understanding of the
role of GRs, MRs, and CRF in the PVN in depressive- and anxiety-like
behaviors.