Unique patterns of FOS, phospho-CREB and BrdU immunoreactivity in the female rat brain following chronic stress and citalopram treatment.

Affective disorders are common psychiatric illnesses characterized by marked gender-related prevalence. Recent evidence links chronic stress and dysregulation of neurotrophin signaling with the development of depression, while novel theories suggest that antidepressants may act by promoting intracellular adaptations linked to neuroplasticity. Although selective serotonin reuptake inhibitors (SSRIs) efficaciously improve a variety of dysfunctions in males, their neuroendocrine effects and intracellular signaling patterns in females are not well determined. Here we show that chronic footshock stress (21 days) promotes HPA axis hyperactivity (as seen by the increased FOS-ir in the paraventricular hypothalamic nucleus (PVN), plasma corticosterone and adrenal hypertrophy), reduces hippocampal BrdU immunoreactivity and suppresses cortical-limbic CREB phosphorylation in female rats. Long-term citalopram treatment, in contrast, attenuates stress-induced elevation of corticosterone levels and adrenal hypertrophy, although it does not reverse footshock-mediated induction of FOS-ir in the PVN, inhibition of CREB phosphorylation and reduction of hippocampal BrdU-labeling. Moreover, citalopram administration was also associated with significant hypophagic effects and inhibition of CREB phosphorylation. These data suggest that, in female rats, normalization of chronic stress-induced HPA axis abnormalities may represent an initial phase of citalopram-mediated therapeutic actions and despite this SSRI's apparent lack of effects on neuroplasticity, we cannot exclude the possibility that some neurochemical adaptations occur in a later stage which may require more than 3 weeks of treatment to manifest.

Visualization of transcription sites at the electron microscope.

In order to localize at EM level the sites of transcription of both pre-mRNA and pre-rRNA, we have detected the DNA/RNA hybrid molecules and m3Gcapped structures by means of specific antibodies after short bromo-uridine (BrU) incorporation. In addition, the sections have been stained by a selective RNA stain, terbium citrate. Our data indicate that perichromatin fibrils incorporate BrU and are labeled by the anti-hybrid probe; this supports the idea that they are the pre-mRNA transcription sites. On the contrary, interchromatin granules do not incorporate BrU after short pulses and are not labeled by the anti-hybrid probe. Concerning the nucleolus, anti-hybrid and anti-BrdU antibodies colocalize only on the dense fibrillar component, suggesting that this is the site of rRNA transcription. Interestingly, the dense fibrillar component and the granular component, after specific RNA staining, show remarkable structural similarities, both containing fibrogranular RNA structures.

Chronic but not acute foot-shock stress leads to temporary suppression of cell proliferation in rat hippocampus.

Stressful experiences, especially when prolonged and severe are associated with psychopathology and impaired neuronal plasticity. Among other effects on the brain, stress has been shown to negatively regulate hippocampal neurogenesis, and this effect is considered to be exerted via glucocorticoids. Here, we sought to determine the temporal dynamics of changes in hippocampal neurogenesis after acute and chronic exposure to foot-shock stress. Rats subjected to a foot-shock procedure showed strong activation of the hypothalamic-pituitary-adrenal (HPA) axis, even after exposure to daily stress for 3 weeks. Despite a robust release of corticosterone, acute foot-shock stress did not affect the rate of hippocampal cell proliferation. In contrast, exposure to foot-shock stress daily for 3 weeks led to reduced cell proliferation 2 hours after the stress procedure. Interestingly, this stress-induced effect did not persist and was no longer detected 24 hours later. Also, while chronic foot-shock stress had no impact on survival of hippocampal cells that were born before the stress procedure, it led to a decreased number of doublecortin-positive granule neurons that were born during the chronic stress period. Thus, whereas a strong activation of the HPA axis during acute foot-shock stress is not sufficient to reduce hippocampal cell proliferation, repeated exposure to stressful stimuli for prolonged period of time ultimately results in dysregulated neurogenesis. In sum, this study supports the notion that chronic stress may lead to cumulative changes in the brain that are not seen after acute stress. Such changes may indicate compromised brain plasticity and increased vulnerability to neuropathology.

Terminal differentiation of erythroblasts leads to RNP segregation and formation of heterogeneous ectopic RNP-derived structures.

We show an as yet unnoticed feature of mammalian erythrocyte maturation, i.e., the formation of heterogeneous ectopic RNP-derived structures in the nucleus of erythroblasts, occurring in parallel with chromatin condensation. Inside these structures, RNPs are always recognized by specific antibodies, which demonstrates that the protein moieties of RNPs still preserve (at least partially) their native organization. This phenomenon shares extensive similarity with the segregation and clustering of nuclear RNPs occurring during spontaneous apoptosis of rat thymocytes and in several other cell models in which transcription is physiologically arrested.

Selective chronic stress-induced in vivo ERK1/2 hyperphosphorylation in medial prefrontocortical dendrites: implications for stress-related cortical pathology?

Stress has been shown to affect brain structural plasticity, promote long-term changes in multiple neurotransmitter systems and cause neuronal atrophy. However, the mechanisms involved in these stress-related neural alterations are still poorly understood. Mitogen-activated protein kinase (MAPK) cascades play a crucial role in the transduction of neurotrophic signal from the cell surface to the nucleus and are implicated in the modulation of synaptic plasticity and neuronal survival. An intriguing possibility is that stress might influence brain plasticity through its effects on selective members of such intracellular signalling cascades responsible for the transduction of neurotrophin signals. Here, we have investigated the effects of stress on the expression of three members of the MAPK/extracellular-regulated kinase (ERK) pathway such as phospho-ERK1, phospho-ERK2 and phospho-cAMP/calcium-responsive element-binding protein (CREB) in the adult rat brain. Male rats were subjected to mild footshocks and the patterns of protein expression were analysed after 21 consecutive days of stress. We found that chronic stress induced a pronounced and persistent ERK1/2 hyperphosphorylation in dendrites of the higher prefrontocortical layers (II and III) and a reduction of phospho-CREB expression in several cortical and subcortical regions. We hypothesized that defects in ERK signalling regulation combined with a reduced phospho-CREB activity may be a crucial mechanism by which sustained stress may induce atrophy of selective subpopulations of vulnerable cortical neurons and/or distal dendrites. Thus, ERK-mediated cortical abnormalities may represent a specific path by which chronic stress affects the functioning of cortical structures and causes selective neural network defects.

Molecular correlates of impaired prefrontal plasticity in response to chronic stress.

Disturbed adaptations at the molecular and cellular levels following stress could represent compromised neural plasticity that contributes to the pathophysiology of stress-induced disorders. Evidence illustrates atrophy and cell death of stress-vulnerable neurones in the prefrontal cortex. Reduced plasticity may be realized through the destabilized function of selective proteins involved in organizing the neuronal skeleton and translating neurotrophic signals. To elucidate the mechanisms underlying these effects, rats were exposed to chronic footshock stress. Patterns of c-fos, phospho-extracellular-regulated protein kinases 1/2 (ERK1/2), calcineurin and phospho-cyclic-AMP response-element binding protein (CREB) expression were subsequently investigated. The results indicate chronic stress-induced impairments in prefrontal and cingulate signal transduction cascades underlying neuronal plasticity. The medial prefrontal cortex, demonstrated functional hyperactivity and dendritic phospho-ERK1/2 hyperphosphorylation, while reduced c-fos and calcineurin immunoreactivity occurred in the cingulate cortex. Significantly reduced phospho-CREB expression in both cortical regions, considering its implication in brain-derived neurotrophic factor (BDNF) transcription, suggests reduced synaptic plasticity. This data confirms the damaging effect of stress on cortical activity, on a molecular level. Due to the association of these markers in the regulation of BDNF signalling, these findings suggest a central role for intracellular neurotrophin transduction members in the pathways underlying cellular actions of stress in the brain.

Immunohistochemical changes induced by repeated footshock stress: revelations of gender-based differences.

As a growing literature has proven, adverse experiences, particularly when severe and persistent, play a pivotal role in the development of neuronal dysfunctions and psychopathology. In the present study, the neurochemical changes induced by acute and repeated footshock exposure were investigated at the molecular and cellular level, using c-fos and phospho-ERK1/2 immunoreactivity and gene expression arrays. Marked gender-related differences were found following both acute and prolonged footshock exposure. Acute aversive conditioning resulted in significant immunohistochemical changes that might be critically involved in the modulation of fear-related responses, especially in males. Prolonged footshock exposure, on the contrary, was associated with sustained hypothalamic-pituitary-adrenal axis hyperactivity, differential gender-related patterns of cortical-limbic activity, and abnormal neuronal plasticity, especially in medial prefrontocortical regions. These data may provide additional insights into the understanding of the neural circuits underlying the effects of acute and repeated footshock exposure as well as clarify some of the mechanisms involved in the development of stress-related neuronal abnormalities.