A study on cholinergic signal transduction pathways involved in short term and long term memory formation in the rat hippocampus

Neurodegenerative processes alter neuronal and glial physiology and cause cognitive and mnemonic impairments. Aim of this PhD thesis is to investigate the involvement of the cholinergic system and the role of mTOR pathway in the mechanisms of memory encoding in the hippocampus and to study the pathophysiological processes at the base of the cognitive impairments in different experimental models of neurodegeneration: in particular normal brain aging, neuroinfiammation and chronic cerebral hypoperfusion. These mechanisms are studied focusing on the morpho-functional alterations in the neuron-astrocytemicroglia triad

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

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

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

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

Astrocyte clasmatodendrosis in a transgenic mouse model of Alzheimer’s Disease

Aging is frequently accompanied by a low-grade inflammation (inflammaging); on the other hand, inflammation is considered a prodrome of Alzheimer Disease (AD). Indeed, a distinctive event of both aging and AD is the deposition of beta amyloid (Aß) fibrils within the central nervous system, a condition that has been associated to cognitive decline. In a previous research we demonstrated that, in the hippocampus of aged rats, the fragmentation of astrocyte processes (clasmatodendrosis) is associated with a decrease of their activity in terms of Aß-fibril clearance, thus promoting neuron to neuron propagation of Aß-fibrils and therefore their prion like spread [1]. In this study we show preliminary data on the role of clasmatodendrosis in a double transgenic TgCRND8 mouse model, which overexpresses both Swedish and Indiana mutations in the human amyloid precursor protein, and displays early cognitive decline also in young animals [2]. We performed a 3D confocal analysis on optical volumes acquired in the CA1 hippocampal region of young (3m.)- and middle aged (7m)- TgCRND8 mice. We found that young TgCRND8 mice show Aß-amyloid deposition, astrocyte clasmatodendrosis and a decrease of the astrocyte cytoskeletal marker GFAP. In middle aged animals significantly higher levels of GFAP expression, indicating astrogliosis, were in concomitance with both Aß-amyloid deposition. These data appear to link the onset of early cognitive decline in TgCRND8 mice with astrocyte clasmatodendrosis and provide new perspectives on the role of astrocytes in Aß-amyloid deposition and spreading

Morphological analysis of the Hippocampal region of aged rats, role of Clasmatodendrosis

Clasmatodendrosis is a phenomenon first described by Alzheimer in 1910, which was observed in aged nervous system and in the course of neurodegenerative diseases. It consists in the loss of astrocytic distal processes. The occurrence of clasmatodendrosis is frequently associated with an increase of autofluorescent aggregates in different cell types of nervous tissue. In this study we designed a calibrated excitation/emission method of spectral unmixing aimed to discriminate the fluorescence emitted by commercial fluorochrome-conjugated antibodies from the autofluorescent signal, by using confocal microscopy and multiphoton fluorescence lifetime imaging techniques. By this method, the immunolabeled GFAP localization in the CA1 Hippocampal region of aged rats was analyzed. Autofluorescent debris showed a strong positivity to GFAP labeling, suggesting that the detached fragments of clasmatodendrotic astrocytes might take part in the generation of these structures. By 3D confocal analysis we found that these aggregates, were located on neuronal cell surfaces, as well as inside the soma and that, in addition, the presence of autofluorescent aggregates seemed to be related with increased adhesion phenomena among neurons. These data were compared with those obtained in control adult rats and in rats infused with lipopolysaccharide (LPS) in the 4th ventricle to induce a chronic inflammatory state. The presence of autofluorescent aggregates was detected in LPS rats and also in control rats, even if they appeared smaller and with a lesser intensity as compared with the aged rats. These findings suggest that clasmatodendrosis is a process involving the interaction of neurons and astrocytes in a prolonged timespan of life. Its severity increases with aging or under inflammatory and/or neurodegenerative diseases. In conclusion, our results seem to suggest that clasmatodendrosis can affect neuron functionality not only due to a decreased astrocyte activity, but also by direct interaction of the detached astrocytic fragments with neuron somata