Rapamycin inhibits mTOR/p70S6K activation in CA3 region of the hippocampus of the rat and impairs long term memory

The present study was aimed at establishing whether the mTOR pathway and its downstream effector p70S6K in CA3 pyramidal neurons are under the modulation of the cholinergic input to trigger the formation of long term memories, similar to what we demonstrated in CA1 hippocampus. We performed in vivo behavioral experiments using the step down inhibitory avoidance test in adult Wistar rats to evaluate memory formation under different conditions. We examined the effects of rapamycin, an inhibitor of mTORC1 formation, scopolamine, a muscarinic receptor antagonist or mecamylamine, a nicotinic receptor antagonist, on short and long term memory formation and on the functionality of the mTOR pathway. Acquisition was conducted 30 min after i.c.v. injection of rapamycin. Recall testing was performed 1h, 4h or 24h after acquisition. We found that (1) mTOR and p70S6K activation in CA3 pyramidal neurons were involved in long term memory formation; (2) rapamycin significantly inhibited mTOR and of p70S6K activation at 4h, and long term memory impairment 24h after acquisition; (3) scopolamine impaired short but not long term memory, with an early increase of mTOR/p70S6K activation at 1h followed by stabilization at longer times; (4) mecamylamine and scopolamine co-administration impaired short term memory at 1h and 4h and reduced the scopolamine-induced increase of mTOR/p70S6K activation at 1h and 4h; (5) mecamylamine and scopolamine treatment did not impair long term memory formation; (6) unexpectedly, rapamycin increased mTORC2 activation in microglial cells. Our results demonstrate that in CA3 pyramidal neurons the mTOR/p70S6K pathway is under the modulation of the cholinergic system and is involved in long-term memory encoding, and are consistent with the hypothesis that the CA3 region of the hippocampus is involved in memory mechanisms based on rapid, one-trial object–place learning and recall. Furthermore, our results are in accordance with previous reports that selective molecular mechanisms underlie either short term memory, long term memory, or both. Furthermore, our discovery that administration of rapamycin increased the activation of mTORC2 in microglial cells supports a reappraisal of the beneficial/adverse effects of rapamycin administration

The neuron-astrocyte-microglia triad in a rat model of chronic cerebral hypoperfusion: Protective effect of dipyridamole

Chronic cerebral hypoperfusion during aging may cause progressive neurodegeneration as ischemic conditions persist. Proper functioning of the interplay between neurons and glia is fundamental for the functional organization of the brain. The aim of our research was to study the pathophysiological mechanisms, and particularly the derangement of the interplay between neurons and astrocytes-microglia with the formation of triads, in a model of chronic cerebral hypoperfusion induced by the 2-vessel occlusion (2VO) in adult Wistar rats (n=15). The protective effect of dipyridamole given during the early phases after 2VO (4 mg/kg/day i.v., the first 7 days after 2VO) was verified (n=15). Sham-operated rats (n=15) were used as controls. Immunofluorescent triple staining of neurons (NeuN), astrocytes (GFAP) and microglia (IBA1) was performed 90 days after 2VO. We found significantly higher amount of ectopic neurons, neuronal debris and apoptotic neurons in CA1 Str. Radiatum and Str. Pyramidale of 2VO rats. In CA1 Str. Radiatum of 2VO rats the amount of astrocytes (cells/mm2) did not increase. In some instances several astrocytes surrounded ectopic neurons and formed a micro scar around them. Astrocyte branches could infiltrate the cell body of ectopic neurons, and, together with activated microglia cells formed the triads. In the triad, significantly more numerous in CA1 Str. Radiatum of 2VO than in sham rats, astrocytes and microglia cooperated in the phagocytosis of ectopic neurons. These events might be common mechanisms underlying many neurodegenerative processes. The frequency to which they appear might depend upon, or might be the cause of, the burden and severity of neurodegeneration. Dypiridamole significantly reverted all the above described events. The protective effect of chronic administration of dipyridamole might be a consequence of its vasodilatory, antioxidant and anti-inflammatory role during the early phases after 2VO

Microglial distribution, branching, and clearance activity in aged rat hippocampus are affected by astrocyte meshwork integrity: evidence of a novel cell‐cell interglial interaction

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Astrocyte clasmatodendrosis affects clearance mechanisms of Aß-fibrils in the hippocampus of aged rats

Aging is frequently accompanied by a low-grade pro-inflammatory condition which is considered a prodrome of Alzheimer Disease (AD). Indeed a common event of aging and AD is the deposition of beta amyloid (Aß) fibrils in the central nervous system, that has been associated to cognitive decline also in normal aging. Identifying traits of amyloid aggregates are the irreversibility of their molecular interactions, the ability to propagate from one cell to another and resist to clearance mechanisms [1]. We previously reported that, in aged rats, astrocyte clasmatodendrosis promote neuron to neuron propagation of Aß-fibrils and, therefore, their prion like spread. Clasmatodendrosis is a phenomenon described in astrocytes that consists in the loss of their distal processes. It’s known that astrocytes constitutively remove Aß-fibrils from neurolemma with their cytoplasmic processes and, in pathological conditions, this activity may cause their necrosis. Moreover, they regulate microglial activity in the central nervous system. Although microglial cells are involved in Aß-fibril clearance, they have also been associated to neuronal cell death in Alzheimer Disease. In this work we found that clasmatodendrosis is associated with a decrease of astrocyte activity of Aß fibril clearance in the pyramidal layer and affects microglial phagocytic activation in the hippocampus of aged rats. To discriminate immunofluorescence signals from autofluorescence typical of amyloid aggregates on confocal acquisitions, we designed a specific method of linear unmixing. Moreover, multiphoton microscopy analyses were implemented with an innovative method of fluorescence lifetime analysis (FLIM/Phasor), suitable to discriminate multiple fluorescences. On the whole our data suggest that clasmatodendrosis consistently affects clearance mechanisms of Aß fibril in the central nervous system and foreshadow new strategies in the development of therapeutical protocols against AD

Use, Attitudes and Knowledge of Complementary and Alternative Drugs (CADs) Among Pregnant Women: a Preliminary Survey in Tuscany

To explore pregnant women's use, attitudes, knowledge and beliefs of complementary and alternative drugs (CADs) defined as products manufactured from herbs or with a natural origin. A preliminary survey was conducted among 172 pregnant women in their third trimester of pregnancy, consecutively recruited in two obstetrical settings; 15 women were randomly selected to compute a test-to-retest analysis. Response rate was 87.2%. Test-to-retest analysis showed a questionnaire's reproducibility exceeding a K-value of 0.7 for all items. Mean age was 32.4 ± 0.4 years; most women were nulliparae (62.7%). The majority of subjects (68%) declared to have used one or more CADs during their lifetime; 48% of pregnant women reported taking at least one CAD previously and during the current pregnancy. Women's habitual use of CADs meant they were at higher risk of taking CADs also during pregnancy (adjusted odds ratio = 10.8; 95% confidence interval: 4.7–25.0). Moreover, 59.1% of the subjects were unable to correctly identify the type of CADs they were using. The majority of women resorted to gynecologists as the primary information source for CADs during pregnancy, while they mainly referred to herbalists when not pregnant. Habitual use of CADs seems to be a strong predictor for their ingestion also during pregnancy; in addition most subjects were unable to correctly identify the products they were taking. In the light of the scanty data concerning the safety of CADs during pregnancy, these preliminary results confirm the need to investigate thoroughly the situation of pregnant women and CADs consumption

Interactions between astrocytes and microglial cells in the hippocampus

A great amount of data is currently available on the role played by astrocytes and microglial cells in normal and pathological conditions, due to their relevance in the progression of neurodegenerative diseases. It is known that astrocytes provide homeostasis for neuronal networks, regulate neuronal maturation and synapse formation, modulate neurotransmission, act as progenitor cells in the neurogenic zone, and may influence microglial phagocytosis. On the other hand, they may enhance an inflammatory condition by producing and releasing inflammatory cytokines and amyloid fibrils. Microglial cells are the immunocompetent cells of the central nervous system, they remove damaged neurons and dysfunctional synapses in normal and pathological brain. They constantly act as sensor cells in normal brain, their ramified processes constantly scanning brain environment and eventually extending toward their targets. The same molecular pathways characterizing these activities are also utilized by microglia to influence nervous system development and connectivity in the normal and developing brain. However, inflammation may lead to deregulation of microglial cells, resulting in aggravation of disease progression. In this scenario, the comprehension of the interactions occurring between astrocytes and microglial cells, could be essential to get an inclusive synthesis of the evidence on their functions which are constantly accumulating. This study is aimed to verify whether the contacts occurring between astrocyte and microglia processes may undergo significant changes in number and spatial distribution according to different functional states of glial cells. To this aim we performed 3D particle analysis on confocal optical volumes acquired in the CA1 hippocampal region of control- and chronically inflamed- young and old rats