Dorota Dudys

Early-life stress affects the structural and functional plasticity of the medial prefrontal cortex in adolescent rats.

Early life experiences are crucial factors that shape brain development and function due to their ability to induce structural and functional plasticity. Among these experiences, early‐life stress (ELS) is known to interfere with brain development and maturation, increasing the risk of future psychopathologies, including depression, anxiety, and personality disorders. Moreover, ELS may contribute to the emergence of these psychopathologies during adolescence. In this present study, we investigated the effects of ELS, in the form of maternal separation (MS), on the structural and functional plasticity of the medial prefrontal cortex (mPFC) and anxiety‐like behavior in adolescent male rats. We found that the MS procedure resulted in disturbances in mother–pup interactions that lasted until weaning and were most strongly demonstrated by increases in nursing behavior. Moreover, MS caused atrophy of the basal dendritic tree and reduced spine density on both the apical and basal dendrites in layer II/III pyramidal neurons of the mPFC. The structural changes were accompanied by an impairment of long‐term potentiation processes and increased expression of key proteins, specifically glutamate receptor 1, glutamate receptor 2, postsynaptic density protein 95, αCa2+/calmodulin‐dependent protein kinase II and αCa2+/calmodulin‐dependent protein kinase II phosphorylated at residue Thr305, that are engaged in long‐term potentiation induction and maintenance in the mPFC. We also found that the MS animals were more anxious in the light/dark exploration test. The results of this study indicate that ELS has a significant impact on the structural and functional plasticity of the mPFC in adolescents. ELS‐induced adaptive plasticity may underlie the pathomechanisms of some early‐onset psychopathologies observed in adolescents.

ACTIVATION OF CB1 CANNABINOID RECEPTORS IMPAIRS MEMORY CONSOLIDATION AND HIPPOCAMPAL POLYSIALYLATED NEURAL CELL ADHESION MOLECULE EXPRESSION IN CONTEXTUAL FEAR CONDITIONING

We investigated the role of CB1 receptors in hippocampal-dependent memory consolidation mediated by polysialylated neural cell adhesion molecule (PSA-NCAM) during contextual fear conditioning (CFC). The CB1 receptor agonist 3-(1,1-dimethylheptyl)-(-)-11-hydroxy-Delta(8)-tetrahydrocannabinol (HU-210) (0.1 mg/kg) was given immediately after training during the memory consolidation phase, and freezing behavior was measured 24 h after conditioning. Administration of HU-210 attenuated freezing behavior measured in CFC. Western blot analysis showed that CFC induced a decrease in the expression of NCAM-180, but did not change the level of NCAM-140 and increased PSA-NCAM expression measured 24 h after training in the rat hippocampus. HU-210 (0.1 mg/kg) injection did not affect the reduction in NCAM-180 levels induced by CFC, but it blocked the increase in PSA-NCAM expression. Since the dentate gyrus (DG) of the hippocampus is known to be involved in memory consolidation and expresses a high level of PSA-NCAM protein, we measured the effects of CFC and HU-210 administration on PSA-NCAM-immunoreactive (IR) cells in the DG. CFC caused an increase in the number of PSA-NCAM-IR cells in the DG, but not K(i)-67- or doublecortin (DCX)-IR cells. This increase in PSA-NCAM-IR cells was abolished by HU-210 injection. Administration of the CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM-251) (3 mg/kg immediately before HU-210) inhibited the effects of HU-210 on freezing behavior and PSA-NCAM expression in the DG. These results indicate that activation of CB1 receptors disturbs consolidation of fear memory in CFC, likely by affecting PSA-NCAM expression in the DG, which plays an important role in synaptic rearrangement during the formation of memory traces.

Maternal separation affects the number, proliferation and apoptosis of glia cells in the substantia nigra and ventral tegmental area of juvenile rats

Adverse early life experiences can increase the risk of psychiatric and neurological disorders as the result of interference with brain development and maturation. In the present study, we tested whether early life stress, that is, maternal separation (MS), affects cell number, cell proliferation and constitutive apoptotic processes in the substantia nigra (SN) and the ventral tegmental area (VTA) of juvenile male and female rats. It was found that MS decreased the total number of glia but not neuronal cells in the SN pars compacta (SNc) and VTA of males. Moreover, MS reduced the number of S-100β-immunoreactive (IR) glial cells in the SN of females and in the VTA of males. It was also observed that MS decreased the rate of proliferation (as measured by Ki-67-immunoreactivity) in the SN pars reticulata (SNr) and VTA of both males and females. Additionally, MS reduced the number of TUNEL-positive cells in the SNc of both males and females and in the SNr and VTA of males only. Moreover, MS decreased the number of cleaved caspase-9-IR cells in the SN and VTA of male rats. Cleaved caspase-9 was present in microglia cells, which exhibited the morphological hallmarks of apoptosis, but not in neuronal, astrocytic or oligodendrocytic cells. On the contrary, MS increased the number of cleaved caspase-3-IR cells in the SN and VTA of both male and female rats. Cleaved caspase-3-IR cells did not display signs of apoptosis and had an astrocytic phenotype (S-100β-IR). In males exposed to MS, a decrease in caspase-3 enzymatic activity in the SN was also observed. In summary, the results of the present study revealed that early life stress affects the number, proliferation and naturally occurring apoptosis of glia cells in the SN and VTA in a sex-dependent manner and consequently may impair brain functions that are regulated by these structures.

Impact of maternal separation on neural cell adhesion molecules expression in dopaminergic brain regions of juvenile, adolescent and adult rats

Stressful experiences in the early stages of life can influence brain development and maturation, and they can also increase the risk for some psychiatric disorders; however, the specific mechanisms of this effect are still poorly understood. Neural cell adhesion molecules (NCAM 120, 140, 180 kDa) are known to play an important role in normal brain development and synaptic plasticity. Therefore, we decided to investigate whether maternal separation (MS) in rats, a paradigm which models an early life stress, has any impact on the expression of NCAM proteins in the juvenile, adolescent and adult brains of both male and female rats. Specifically, we focused our efforts on the brain regions associated with dopaminergic neurotransmission. In juvenile rats, MS decreased the levels of NCAM-140 in the substantia nigra (SN) of females and NCAM-180 in the ventral tegmental area of males. During adolescence, a reduction in NCAM-180 levels in the SN and medial prefrontal cortex (mPFC) of MS females was revealed. Finally, in adulthood, a decrease in NCAM-180 expression was observed in the mPFC of MS males. The results that we obtained indicate that early life stress can affect maturation and NCAM-driven plasticity in dopaminergic brain areas at different stages of ontogenesis and with a sex-specific manner.

The effects of early-life adversity on fear memories in adolescent rats and their persistence into adulthood.

Adolescence is a developmental period characterized by extensive morphological and functional remodeling of the brain. The processes of brain maturation during this period may unmask malfunctions that originate earlier in life as a consequence of early-life stress (ELS). This is associated with the emergence of many psychopathologies during adolescence, particularly affective spectrum disorders. In the present study, we applied a maternal separation (MS) procedure (3h/day, on postnatal days 1-14) as a model of ELS to examine its effects on the acquisition, expression and extinction of fear memories in adolescent rats. Additionally, we studied the persistence of these memories into adulthood. We found that MS decreased the expression of both contextual (CFC) and auditory (AFC) fear conditioning in adolescent rats. Besides, MS had no impact on the acquisition of extinction learning. During the recall of extinction MS animals both, those previously subjected and not subjected to the extinction session, exhibited equally low levels of freezing. In adulthood, the MS animals (conditioned during adolescence) still displayed impairments in the expression of AFC (only in males) and CFC. Furthermore, the MS procedure had also an impact on the expression of CFC (but not AFC) after retraining in adulthood. Our findings imply that ELS may permanently affect fear learning and memory. The results also support the hypothesis that, depending on individual predispositions and further experiences, ELS may either lead to a resilience or a vulnerability to early- and late-onsets psychopathologies.

The impact of maternal separation on the number of tyrosine hydroxylase-expressing midbrain neurons during different stages of ontogenesis

Early life stressors have life-long functional and anatomical consequences. Though many neurotransmitters are involved in the functional impact of early life stress, dopamine seems to be important because of its roles in motor control, adaptation to stressful conditions, mood, cognition, attention and reward. Thus, in the present study, we investigated the way that early life stress, in the form of maternal separation (MS), affects the populations of tyrosine hydroxylase-immunoreactive (TH-IR) dopaminergic neurons in rat midbrain structures during ontogenesis. We included in the study the sub-regions of the substantia nigra (SN) and the ventral tegmental area (VTA). In both the control and MS rats, we found that the estimated total number of TH-expressing neurons fluctuated during ontogenesis. Moreover, MS influenced the number of TH-IR cells, especially in the SN pars reticulata (SNr) and VTA. Shortly after the termination of MS, on postnatal day (PND) 15, a decrease in the estimated total number of TH-IR neurons was observed in the SNr and VTA (in both males and females). On PND 35, MS caused a transient increase in the number of TH-IR cells only in the SNr of female rats. On PND 70, MS affected the number of TH-IR neurons in the VTA of females; specifically, an increase in the number of these cells was observed. Additionally, MS did not alter TH-IR cell sizes or the total levels of TH (measured by Western blot analysis) in the SN and VTA for all stages of ontogenesis in both males and females. The results from the study herein indicate that early life stress has enduring effects on the populations of midbrain TH-expressing dopaminergic neurons (especially in female rats), which are critically important for dopamine-regulated brain function throughout ontogenesis.

Repeated risperidone treatment increases the expression of NCAM and PSA-NCAM protein in the rat medial prefrontal cortex.

The present study investigates whether the anti-schizophrenic drug risperidone may evoke changes in the expression of NCAM/PSA-NCAM proteins, an indispensable element in the remodeling of synaptic arrangements, in the medial prefrontal cortex (mPFC). Rats were treated with risperidone (0.2 mg/kg, i.p.) either once or repeatedly (once a day, for 21 days). The expression of NCAM and PSA-NCAM proteins was analyzed via western blot and immunohistochemistry at intervals of 3 h and 3, 6, and 9 days after the single or the last risperidone dose. Repeated (but not acute) administration of risperidone was found to increase the expression of NCAM-180, NCAM-140 and PSA-NCAM proteins at 3 or 6 days after treatment. PSA-NCAM immunoreactivity was found in cell bodies, perisomatic-like sites, and in the neuropil of the mPFC. Neither single nor repeated risperidone administration changed the number of PSA-NCAM neurons in the mPFC. In contrast, the repeated risperidone treatment increased the number of PSA-NCAM perisomatic-like sites and the length density of PSA-NCAM positive neuropil at 3 days after the last injection. The data obtained indicate that risperidone, given repeatedly, may promote the remodeling of the structure of presumably GABA-ergic interneurons and that it may evoke the rearrangement of the synaptic contact in the mPFC.

Impact of early-life stress on the medial prefrontal cortex functions – a search for the pathomechanisms of anxiety and mood disorders

Although anxiety and mood disorders (MDs) are the most common mental diseases, the etiologies and mechanisms of these psychopathologies are still a matter of debate. The medial prefrontal cortex (mPFC) is a brain structure that is strongly implicated in the pathophysiology of these disorders. A growing number of epidemiological and clinical studies show that early-life stress (ELS) during the critical period of brain development may increase the risk for anxiety and MDs. Neuroimaging analyses in humans and numerous reports from animal models clearly demonstrate that ELS affects behaviors that are dependent on the mPFC, as well as neuronal activity and synaptic plasticity within the mPFC. The mechanisms engaged in ELS-induced changes in mPFC function involve alterations in the developmental trajectory of the mPFC and may be responsible for the emergence of both early-onset (during childhood and adolescence) and adulthood-onset anxiety and MDs. ELS-evoked changes in mPFC synaptic plasticity may constitute an example of metaplasticity. ELS may program brain functions by affecting glucocorticoid levels. On the molecular level, ELS-induced programming is registered by epigenetic mechanisms, such as changes in DNA methylation pattern, histone acetylation and microRNA expression. Vulnerability and resilience to ELS-related anxiety and MDs depend on the interaction between individual genetic predispositions, early-life experiences and later-life environment. In conclusion, ELS may constitute a significant etiological factor for anxiety and MDs, whereas animal models of ELS are helpful tools for understanding the pathomechanisms of these disorders.

Cocaine enhances ST8SiaII mRNA expression and neural cell adhesion molecule polysialylation in the rat medial prefrontal cortex.

The present study investigated whether cocaine (COC) administration evokes changes in the mRNA and protein levels of neural cell adhesion molecule (NCAM) and polysialylated neural cell adhesion molecule (PSA-NCAM) in the medial prefrontal cortex (mPFC) of rats. NCAM/PSA-NCAM is required for neuronal structural plasticity and is constitutively expressed in the mPFC. Rats were treated with a single dose of COC (15 mg/kg, i.p.), and mRNA levels of NCAM and the polysialyltransferases ST8SiaII and ST8SiaIV, enzymes involved in polysialylation of NCAM, were measured at 3, 6 and 24 h after COC treatment. At the same time points, the protein levels of NCAM and PSA-NCAM were measured via western blotting. Acute COC injection did not affect mRNA levels of NCAM and ST8SiaIV, but it increased the mRNA level of ST8SiaII 3 h after injection. At the same time point, an increase in PSA-NCAM, but not in NCAM, protein was observed. Morphological studies of PSA-NCAM protein expression patterns (immunocytochemistry/stereology) performed 3 h after COC administration revealed an enhancement of PSA-NCAM immunostaining in perisomatic-like sites and in the length density of PSA-NCAM-positive neuropil. Double immunofluorescence staining showed that PSA-NCAM perisomatic-like sites surround excitatory neurons. We also observed that a single injection of raclopride (0.4 mg/kg) or SCH 23390 (0.5 mg/kg), D2/D3 and D1 dopamine receptors antagonists, respectively, which were ineffective when given alone, abolished the effects of COC administration on mRNA and protein expression. The data in the present study indicate that COC administration may modify constitutive synaptic plasticity in the mPFC by increasing the NCAM polysialylation in perisomatic innervations of pyramidal neurons via activation of dopamine D1 and D2/D3 receptors.

MK-801, a NMDA receptor antagonist, increases phosphorylation of histone H3 in the rat medial prefrontal cortex.

BACKGROUND The present study investigated whether MK-801, when given in doses that cause psychomimetic effects in rats, could alter the phosphorylation of histone 3 (H3) at serine 10 (H3S10p) and the acetylation of H3 at lysine 14 (H3K14ac) in the medial prefrontal cortex (mPFC). These posttranslational modifications of H3 promote chromatin relaxation and increase the probability of gene expression. METHODS Stereological counting, immunoblot analysis and confocal laser scanning microscopy. RESULTS Treatment with MK-801 (0.4 mg/kg) evoked a time-dependent increase in the number of H3S10p positive nuclei in both the II/III and V/VI layers of the mPFC, reaching the peak of activation 30 min after injection. MK-801 treatment (0.4 mg/kg) failed to alter H3K14ac. These effects were confirmed by immunoblot analysis on tissue samples from the mPFC. Analysis of cortical cells expressing H3S10p positive nuclei revealed that constitutive and MK-801-induced expression of H3S10p was observed only in neurons and not in glia cells (H3S10p colocalized with NeuN but not with S-100β). Moreover, it has been found that H3S10p is exclusively present in pyramidal (glutamate-positive) but not in cortical GABA-ergic interneurons (GABA-positive). The effects of MK-801 can be attenuated or blocked by the neuroleptic drug risperidone. In the cortical layer II/III, risperidone was effective at doses of 0.2 and 1 mg/kg, while it was only active at a dose of 1 mg/kg in the V/VI layer. Again, these stereological data were confirmed by immunoblot analysis. CONCLUSIONS Our results indicate that MK-801 may increase the transcriptional activity of mPFC via the activation of the epigenetic program associated with H3S10p phosphorylation during the course of experimental psychosis.