Fotini Stylianopoulou

Effects of neonatal handling on basal and stress-induced monoamine levels in the male and female rat brain

Neonatal handling has pervasive effects on the rat brain leading to increased ability to cope with and adapt to stressful stimuli. We determined the effects of neonatal handling on the dopaminergic and serotonergic system, in the male and female rat brain, under basal conditions before and after puberty and after short- and long-term forced swimming stress. Exposure of animals to neonatal handling resulted in sex-dependent changes in the concentration and turnover of monoamines in the different brain areas. In the prepubertal brain, the effect of neonatal handling was manifested as an increase in dopamine turnover in the females, particularly in the hypothalamus, an increase in serotonin levels and a decrease in its turnover in all three brain regions examined of both males and females. Certain of the handling-induced effects observed in the prepubertal brain were reversed in the postpubertal animals. Thus, in the postpubertal brain, the handling-induced changes in serotonin levels and its turnover observed in both sexes before puberty were abolished. On the other hand, the handling-induced increase in hypothalamic dopamine turnover was maintained. After exposure to short-term stress, the effect of handling was manifested on one hand as decreased striatal dopamine levels in the females, and decreased dopamine turnover in the hypothalamus of both males and females, and on the other, as increased serotonin levels in the hypothalamus. After exposure to long-term stress, handled females had decreased dopamine turnover in the hypothalamus and the striatum, but there was no effect of handling on the serotonergic system. Our results provide some neurobiological evidence supporting the determinant role of the mother-infant relationship in the development of psychopathology. Neonatal handling, which modifies normal mother-pup interactions, results in alterations in brain dopaminergic and serotonergic systems, both of which are involved in the etiopathogenesis of major psychoses. Exposure to either short- or long-term stress in adult life results in sex-dependent changes in brain monoamines, which are affected by handling thus making coping more efficient and rendering the stressful stimulus less noxious.

Effects of hyperactivity of the maternal hypothalamic-pituitary-adrenal (HPA) axis during pregnancy on the development of the HPA axis and brain monoamines of the offspring

Offspring of mothers with adrenal hyperactivity during pregnancy have been reported to have changes in brain monoamines and altered emotional, reactive, sexual and maternal behavior. Since the hypothalamic‐pituitary‐adrenal (HPA) axis is known to be involved in the expression of such behaviors and is itself under monoaminergic control, we examined the development of the HPA axis and brain monoamines in pups whose mothers had adrenal hyperactivity, reflecting administration of ACTH during the last third of their pregnancy. The adrenals of the experimental animals weighed less and had aberrant morphology. The abnormal histology was more pronounced in the adrenals of the experimental females than of the males, suggesting that females were more vulnerable to the prenatal treatment. In both experimental males and females, basal plasma corticosterone levels were higher compared to the controls, while after exposure to stress, experimental animals attained lower plasma corticosterone levels than the controls. In the brain of the experimental animals, dopaminergic activity appeared to be decreased, while serotonergic activity increased. Our results indicate that the prenatal treatment affected brain development in the offspring and as a consequence programmed the developing HPA axis in such a way as to hyperfunction under basal conditions, leading to its exhaustion and its inability to react properly to stress.

Loss of forebrain cholinergic neurons and impairment in spatial learning and memory in LHX7‐deficient mice

The identification of the genetic determinants specifying neuronal networks in the mammalian brain is crucial for the understanding of the molecular and cellular mechanisms that ultimately control cognitive functions. Here we have generated a targeted allele of the LIM‐homeodomain‐encoding gene Lhx7 by replacing exons 3–5 with a LacZ reporter. In heterozygous animals, which are healthy, fertile and have no apparent cellular deficit in the forebrain, b‐galactosidase activity reproduces the pattern of expression of the wild‐type Lhx7 locus. However, homozygous mutant mice show severe deficits in forebrain cholinergic neurons (FCNs), while other classes of forebrain neurons appear unaffected. Using the LacZ reporter as a marker, we show that in LHX7‐deficient mice FCN progenitors survive but fail to generate cholinergic interneurons in the striatum and cholinergic projection neurons in the basal forebrain. Analysis of behaviour in a series of spatial and non‐spatial learning and memory tasks revealed that FCN ablation in Lhx7 mutants is associated with severe deficits in spatial but only mild impairment of non‐spatial learning and memory. In addition, we found no deficit in long‐term potentiation in mutant animals, suggesting that FCNs modulate hippocampal function independently of its capacity to store information. Overall our experiments demonstrate that Lhx7 expression is required for the specification or differentiation of cholinergic forebrain neurons involved in the processing of spatial information.

Aging-related changes in IGF-II and c-fos gene expression in the rat brain

The protein products of growth factor genes such as IGF‐II and cellular oncogenes such as c‐fos are believed to be necessary for the support of normal neuronal function. Steady‐state levels of c‐fos and IGF‐II mRNA were determined in the brain of young and old rats, using Northern analysis. Both RNAs were found to be decreased in the brain of aged rats. Age‐related decrease was detected in the hippocampus, hypothalamus, striatum, cerebral cortex and cerebellum, for IGF‐II mRNA, and in the cerebral cortex and cerebellum for c‐fos mRNA. Furthermore, changes in the degree and pattern of DNA methylation were noted at both gene loci, in the aged rat brain. Our results could reflect changes at the genomic level possibly related to the process of aging and the accompanying decline in brain function.

Scavenger receptor class B type I (SR-BI) regulates perivascular macrophages and modifies amyloid pathology in an Alzheimer mouse model

Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein receptor that regulates cholesterol efflux from the peripheral tissues to the liver. SR-BI has been identified on astrocytes and vascular smooth muscle cells in Alzheimers disease brain and has been shown to mediate adhesion of microglia to fibrillar amyloid-β (Aβ). Here we report that SR-BI mediates perivascular macrophage response and regulates Aβ-related pathology and cerebral amyloid angiopathy in an Alzheimers mouse model. Reduction or deletion of SR-BI gene in heterozygous or homozygous deficient mice (SR-BI+/−, −/−) resulted in a significant increase in perivascular macrophages in the brain. SR-BI deletion had no effect on apolipoprotein E or apolipoprotein AI levels in the mouse brain. Our analysis revealed increased levels of SR-BI expression in the brains of human amyloid precursor protein (Swedish, Indiana) transgenic mice (J20 line). To evaluate the role of SR-BI in Alzheimers disease pathogenesis, we inactivated one SR-BI allele in J20 transgenic mice. SR-BI reduction in J20/SR-BI+/− mice enhanced fibrillar amyloid deposition and cerebral amyloid angiopathy and also exacerbated learning and memory deficits compared with J20 littermates. Immunohistochemical analysis revealed localization of SR-BI on perivascular macrophages in tight association with Aβ deposits. Our data suggest that SR-BI reduction impairs the response of perivascular macrophages to Aβ and enhances the Aβ-related phenotype and cerebral amyloid angiopathy in J20 mice. These results reveal that SR-BI, a scavenger receptor primarily involved in high-density lipoprotein cholesterol transport, plays an essential role in Alzheimers disease and cerebral amyloid angiopathy.

Effect of neonatal handling and sex on basal and chronic stress-induced corticosterone and leptin secretion.

Neonatal handling is an experimental paradigm for early experiences. It affects the programming of hypothalamo-pituitary-adrenal (HPA) axis function, known to be sexually dimorphic. Recently leptin, a hormone related to energy balance and secreted mainly by adipocytes, has been implicated in the stress response. We thus determined the effect of neonatal handling on plasma concentrations of corticosterone and leptin of male and female rats under basal conditions and after two consecutive chronic stressors: chronic forced swimming stress and chronic restraint. Handling resulted in lower basal corticosterone levels in both males and females and in a more efficient HPA response, with a large corticosterone surge following the first chronic stressor and a return to basal levels following the second. Handling also resulted in decreased plasma leptin concentrations in males, thus abolishing the sex difference in leptin levels. Furthermore, handling increased body weight while it decreased food intake under basal conditions. Food intake and body weight gain during chronic forced swimming was lower in handled than in non-handled males, while in females these parameters were not influenced by handling. In both males and females, handling resulted in decreased food intake and increased body weight loss during chronic restraint stress. Body weight loss during chronic restraint stress, which is considered an index of maladaptation and ‘depression’, was particularly pronounced in the handled females. Our results also showed that non-handled females had higher corticosterone and lower leptin levels than males under basal conditions and following each of the two chronic stressors. The present work suggests that early experiences, such as the mother–infant relationship, interact with endogenous factors, such as gonadal hormones, to determine the organism’s response to stressful stimuli during adulthood.

Alterations in IGF-I, BDNF and NT-3 levels following experimental brain trauma and the effect of IGF-I administration

The effects of a unilateral, penetrating brain trauma on IGF-I, BDNF and NT-3 were studied immunocytochemically in the rat. BDNF and NT-3 were decreased in the peritraumatic area, but increased in the adjacent region, 4 and 12 h post-injury. One week following the trauma, BDNF remained low in the peritraumatic area, but was restored to normal levels in the adjacent, while no effect of injury on NT-3 levels was detected in either area. Injury resulted in an increase in IGF-I levels in the peritraumatic area, which was most pronounced 1 week following the trauma, indicating that IGF-I could participate in endogenous repair processes. We thus administered IGF-I immediately following the trauma and investigated its effects on injury-induced changes in neurotrophin levels. Administration of IGF-I partially reversed the injury-induced decrease in BDNF and NT-3 in the peritraumatic area observed 4 and 12 h post-injury, while at the same time-points, it completely cancelled the effects of injury in the adjacent region. One week after the trauma, BDNF levels were dramatically increased in both the peritraumatic and adjacent area, reaching levels even higher than those of the sham-operated animals, following IGF-I administration. Our results showing that IGF-I not only counteracts injury-induced changes in neurotrophins, but can also further increase their levels, indicate that this growth factor could mediate repair and/or protective processes, following brain trauma.

Effect of neonatal handling on adult rat spatial learning and memory following acute stress

Brief neonatal handling permanently alters hypothalamic-pituitary-adrenal axis function resulting in increased ability to cope with stress. Since stress is known to affect cognitive abilities, in the present study we investigated the effect of brief (15 min) handling on learning and memory in the Morris water maze, following exposure to an acute restraint stress either before training or recall. Exposure of non-handled rats to the acute stress prior to training resulted in quicker learning of the task, than in the absence of the stressor. When acute stress preceded acquisition, male handled rats showed an overall better learning performance, and both sexes of handled animals were less impaired in the subsequent memory trial, compared to the respective non-handled. In addition, the number of neurons immunoreactive for GR was higher in all areas of Ammons horn of the handled rats during the recall. In contrast, the number of neurons immunoreactive for MR was higher in the CA1 and CA2 areas of the non-handled males. When the acute restraint stress was applied prior to the memory test, neonatal handling was not effective in preventing mnemonic impairment, as all animal groups showed a similar deficit in recall. In this case, no difference between handled and non-handled rats was observed in the number of GR positive neurons in the CA2 and CA3 hippocampal areas during the memory test. These results indicate that early experience interacts with sex and acute stress exposure in adulthood to affect performance in the water maze. Hippocampal corticosterone receptors may play a role in determining the final outcome.

Effects of an Early Experience Involving Training in a T-Maze Under either Denial or Receipt of Expected Reward through Maternal Contact

The mother is the most salient stimulus for the developing pups and a number of early experience models employ manipulation of the mother-infant interaction. We have developed a new model which in addition to changes in maternal behavior includes a learning component on the part of the pups. More specifically, pups were trained in a T-maze and either received (RER rats) or were denied (DER) the reward of maternal contact, during postnatal days 10–13. Pups of both experimental groups learn the T-maze, but the RER do so more efficiently utilizing a procedural-type of learning and memory with activation of the dorsal basal ganglia. On the other hand, the DER experience leads to activation of the hippocampus, prefrontal cortex, and amygdala in the pups. In adulthood, male DER animals exhibit better mnemonic abilities in the Morris water maze and higher activation of the hippocampus, while they have decreased brain serotonergic activity, exhibit a depressive-like phenotype and proactive aggressive behavior in the resident-intruder test. While male RER animals assume a reactive coping style in this test, and showed increased freezing during both contextual and cued memory recall following fear conditioning.

Enhanced neuronal plasticity and elevated endogenous sAPPα levels in mice over-expressing MMP9

J. Neurochem. (2012) 121, 239–251.