The exposition to high levels of stress hormones during development can cause long-term effects 
. Changes in the microenvironment and in cell survival in the hippocampus have been reported in response to maternal stress 
. Here we show that prenatal stress decreases proliferation markers in the hypothalamus of adult male rats, in agreement with our previous report 
, but also that a similar phenomenon occurs in the hippocampus and pituitary. Although previous studies have shown increased cell death in neurons of the hypothalamic paraventricular nucleus in fetal rats 
, studies in adult rats show a reduction in hippocampus cell proliferation in response to prenatal restraint stress and report that it is not accompanied by an increase in pyknosis 
. This is in accordance with the data presented here as we found that prenatal stress not only reduced the rate of cell proliferation, but also inhibited cell death in the adult hippocampus. This reduction in cell death also occurred in the hypothalamus and the pituitary. The long-term effect of prenatal stress on cell death and proliferation reported here could be related to the “glucocorticoid cascade” hypothesis, which proposes that stressful experiences are responsible for alterations in the structure and function of the hippocampal formation through an excessive release of corticosterone 
. However, this effect would most likely be due only to prenatal exposition to corticosterone, as the levels of corticosterone at sacrifice were similar in all rats 
and there was no effect on adrenal gland weight, as reported here. Taken together, these data suggest a slowing of the cell cycle in the HHP axis of prenatally stressed rats. Prenatal stress induces apoptosis in different areas of the fetal or neonatal brain, including neurons of the hypothalamic paraventricular nucleus 
. This suggests that stress may have a greater effect on immature cells, which may be more susceptible to cell death prior to their establishment of firm connections 
To study the intracellular mechanisms involved in the reduction of cell death, apoptotic pathways were analyzed. Fragmentation of caspase-8 was reduced in the HHP axis in prenatally stressed rats. Calpains, a family of Ca2+
-dependent cystein proteases involved in neuronal apoptotic processes after different injuries 
are known to act as regulator of caspases 
and many calpain substrates are similar to, or their functions overlap with, those of caspases 
. Prenatal stress inhibited cleavage of calpain-2 in the HHP axis of adult offspring. Calpain levels are regulated by an endogenous inhibitor, calpastatin that exerts neuroprotective actions 
. We observed an increase of calpastatin levels in the three areas studied in prenatally stressed rats. Thus, this up-regulation of calpastatin could help to explain the decrease in calpain-2 and apoptosis.
An important regulatory step in apoptosis occurs at mitochondrial membranes involving the members of the Bcl-2 family of proteins. The levels of the anti-apoptotic protein Bcl-2 were increased in the HHP axis in the prenatally stressed rats. In contrast, the levels of the pro-apoptotic protein Bax were reduced in the hippocampus and hypothalamus of prenatally stressed rats. Hence, prenatal stress up-regulated Bcl-2 and down-regulated Bax, resulting in an anti-apoptotic balance. This rise in Bcl-2 possibly is involved in the inhibition of caspase-8 activation 
. The increased expression of Bcl-2 also provides a mechanism to inhibit the opening of ionic channels resulting in an accumulation of calcium, as a consequence the cell is more resistant to calcium induced injuries 
. The prolonged exposure to calcium would lead to an increase in calpastatin expression, which could be a cellular mechanism of protection against alterations of the intracellular calcium homeostasis 
The tumor suppressor protein p53 is activated in response to cell stress leading to cell cycle arrest and apoptotic cell death. p53-induced cell death leads to the activation of caspases by release of apoptogenic factors from mitochondria with this process being regulated by the Bcl-2 family of proteins 
. Our data show that phosphorylation of p53 is decreased in the hippocampus and in the pituitary with no effect in the hypothalamus in rats subjected to prenatal stress. The observed inhibition of p53 could be the result of the increase in Bcl-2 in those areas.
The transcription factor CREB is proposed to be involved in protecting the brain after a stressful situation 
. In addition, the calpastatin promoter sequence contains single cAMP-response element 
. Prenatal stress increased CREB phosphorylation in the HHP axis, which could explain the increase in calpastatin levels and the following inhibition of calpain and caspase activation. Moreover, CREB regulates the expression of Bcl-2, Bax, and p53 in different cells 
. As CREB is involved in IGF-I induced neuron survival 
and is involved in metabolic homeostasis and growth during development 
, IGF-I could be involved in the changes observed here. In support of this, we found that prenatal stress increased IGF-I mRNA levels in the three areas studied. Circulating levels of IGF-I in blood were similar in both experimental groups. These data suggest that IGF-I could be acting in an autocrine-paracrine manner regulating the calpastatin-calpain system via CREB to inhibit cell death.
In summary, our data suggest that prenatal stress induces a long-term slowing or deceleration in the cell death and proliferation rate in the HHP axis. The increase in local IGF-I levels could be involved in the increase in calpastatin levels via CREB that would in turn, inhibit calpain-2 resulting in decreased activation of the extrinsic apoptosis pathway (). Exposure of the developing brain to stress is known to increase the individual’s vulnerability to mental disorders 
. In addition, not only can the long-term stress response be affected by this slowing of cell-turnover in the HHP axis, but other endocrine functions could be modulated in the adult rat due to the affection of the hypothalamus and pituitary. This slowing of cell turnover could result in a reduction in their sensitivity to both physiological and pathophysiological changes with, for example, their susceptibility to future metabolic challenges possibly being modified. It is known that a high fat diet induces obesity 
and this is associated with an inhibition of neurogenesis 
, as well as the induction ofapoptosis of hypothalamic neurons 
. Hence, it is possible that the slowing down of cell turnover of the HHP axis could be involved in the vulnerability to metabolic imbalances or other diseases.
Diagram representing the mechanism proposed for prenatal stress inhibition of cell death