Glial Cells In Alzheimers Disease Biology Essay

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Alzheimers disease is the most common form of senile dementia, which is characterised by memory loss and confusion. Although currently, specific reasons of AD remain blurred, it is caused by neuropathology changes.

Beta amyloid (Aβ) is formed through Amyloid precursor protein (APP) consequence cleavage via secretase peptides and it is considered as the histopathological hallmark of AD.

There are a significant number of facts supporting the theory that Aβ influences cholinergic signalling in the brain. Some evidence indicates that brain nicotinic acetylcholine receptors (nAChRs) are affected by Aβ in addition to their ability to initiate signalling pathways that preserve from Aβ toxicity. The major neurons that are at risk in AD are cholinergic neurons that express nAChRs that consists of α7 subunits; they are believed to have high affinity interaction with Aβ. This interaction may functionally serve in the synaptic plasticity and homeostasis in non AD individuals, while in AD individuals this interaction could be a contributing factor in elevating Aβ toxicity effect during the disease development. Interestingly, activation of nAChRs leads to membrane depolarization that increases the intracellular Calcium concentration. Could they be the main contributing factor of increasing excess Calcium influx that causes cell death or even increasing the Calcium signalling in astrocytes since nAChRs are expressed on astrocytes?

At present Astrocytes are implied to be excited cells, they are unable to generate action potential instead they communicate through Calcium intracellular concentration transients and oscillations, either by neuron-dependent excitation or spontaneous excitation. Upon Calcium oscillation, excitatory amino acids like glutamate are released by astrocytes and modulate synaptic activity. Glutamate transporters are found on glial and neuronal cells; however, during brain injury, eliminating glutamate overload is inhibited. Glutamate will activate NMDARs (N-Metheyl D-Aspartate) causing increase in Calcium influx that ultimately causes cell death. This process is called excitotoxicity. So could the physiology and pathology of astrocytes be the central cause in aggravating AD by excess glutamate effect?

This study will focus on addressing the following hypotheses: Investigate if there is a link between nAChRs and Aβ deposition that cause disruption in Calcium homeostasis. Will Aβ deposition affect astrocytes Calcium homeostasis or dynamics through nAChRs. The consequence effect of glutamate release and its uptake inhibition by astrocytes on synaptic transmission. Finally, the outcome of excitotoxicity and neurodegeneration caused by Aβ.

Alterations and oscillations in intracellular Ca concentrations are the central methods by which spontaneous movement controls neuronal development, synaptic transmission and plasticity, and regulates the metabolism and apoptosis of the mitochondria. Dysfunction in Ca signalling has been considered as an infective agent of AD, which results in most important neuronal loss and cognitive impairment. Thus, these experiments will involve the use of the Calcium imaging techniques from brain slices preparation, employed from Tg2756 transgenic mice, to analyse and evaluate neuronal and glial Calcium signalling pathways. However, until the hypotheses of AD causes are defined, its treatment will remain unknown. The time therefore is ripe for urgent further investigations in this particular disease.

2. Back Ground:

2.1. Alzheimer's Disease:

Alzheimer's disease (AD) is recognized to be the most common form of dementia and it is often dangerous in onset and progresses gradually over a period of several years. AD affects predominantly the elderly at a rate and level which is very variable. According to NICE guidance, evidence highlights the fact that AD development is dependent on age, and the time from diagnosis to death is about 5-20 years-on average 5 years in patients aged 75−80 years. In individuals with AD, about 50−64% are estimated to suffer from a mild to a moderately severe form of the disease, and around 50% suffer from moderately severe to severe AD.

Although, currently, specific grounds for the presence of AD remain blurred, it is caused by neuropathological changes comprising neuronal loss, intracellular neurofibrillary tangles (NFT) and extracellular amyloid plaques; the best link is due to age. This disease progresses and selectively destroys brain sections and neural circuits important for cognition and memory, these neurons are located predominantly in the hippocampus (Dawbarn and Allen, 2001).

2.2. The role of Beta amyloid:

Amyloid precursor protein (APP) is a part of a large family of amyloid precursor- like proteins (APLP) and APP homologues. Beta amyloid (Aβ) is formed through APP cleavage through a series of enzymes. First of all, APP is cleaved by an enzyme called α secretase forming sAPPα, which is believed to be a neuron protector from the excitotoxic action of Aβ and glutamate. Next the APP undergoes further cleavage, amyloidogenic pathway, via β and γ secretase enzymes liberating sAPPβ and Aβ. Senile plaques are microscopic regions of deterioration which enclose deposits of Aβ, these are considered the histopathological hallmarks of AD. The most popular proteins of Aβ that are associated with AD are Aβ1-40 and Aβ1-42 (Dawbarn and Allen, 2001).

Studies examined the Aβ1-42/Aβ1-40 ratio in the human brain, cerebrospinal fluid and plasma, in addition to transgenic animals; all showed that the ratio is constantly increasing in AD (Kumar-Singh et al., 2006).

According to the amyloid hypothesis, build up of (Aβ) in the brain is the chief control motivating AD pathogenesis, though symptoms may not appear for many years. The rest of the disease process, including formation of neurofibrillary tangles containing tau protein, is suggested to result from a disproportion between Aβ generation and Aβ removal (Kumar-Singh et al., 2006). However, very little is implicit about the system through which APP/Aβ cause the principal neurodegeneration.

Furthermore, assessment of the cause and progress of AD examined the physical properties of Aβ and found that Aβ exists in soluble and insoluble forms as well as monomer, dimer or oligomer and fibrils. The study showed that there was a three-fold increase in soluble Aβ in AD. Moreover,it also proved that there is a correspondence between the soluble Aβ level and severity of the disease,whereas the insoluble level of Aβ is found only to differentiate AD from controls, and does not associate with neither the development of the disease nor the amount of amyloid plaques (McLean et al., 1999).

A study completed on cultured cells of AD brain tissue and AD animal models showed that Aβ1-40 and Aβ1-42 are competent to adopt many differently produced aggregates, such as amyloid fibrils in addition to non-fibril aggregates that are also known as oligomers Aβ. It is not yet well-known which Aβ form, nor the specific subcellular location, is the primary cause of AD. Aβ peptide and Aβ plaques naturally accumulate outside the cell, although a number of proved conformations suggest that there is a considerable amount of Aβ that exists in the intracellular compartment (McLean et al., 1999).

Results of the Friedrich study (2009) revealed a mechanism for the development of Aβ plaques. Firstly, soluble and extracellular Aβ peptide enters and distributes into the multivascular bodies (MVBs). In the presence of some factors that are suitable for the formation of fibril, fibril will grow and spread out, ultimately causing cell death which will lead to the discharge of all intercellular Aβ into the extracellular area.

It is vital to highlight, however, that the cell death witnessed in Friedrich cultured cells signifies that cell death cause is associated with plaque biogenesis. This cell death is not necessarily the same as the one accountable for the tremendous neuronal loss in AD patients. Neuronal loss has been shown not to be associated directly with the formation of plaque in AD patients. Moreover, the AD mouse model showed massive plaque aggregates despite very little neuronal damage. Overall, these in vivo experiments clarify that different types of cells are involved in the formation of Aβ.

Nevertheless, some evidence points towards Aβ formation as being essential for neuronal survival. Plant et al. (2003) conducted a study on rodents, which demonstrated that inhibition of β and γ secretase will reduce neuron capability. The concept that Aβ is vital in controlling neurotoxicity is reinforced from the evidence that represents the substitution of endogenous Aβ with concentrations of exogenous Aβ improves neuron viability. Crucially, the recovery of cell death via Aβ depends on the isomers type of peptides used. This effect is most obvious when the physiologically Aβ1-40 is used. The research witnessed a complete rescue of neuron toxicity at a concentration of 10 pM and 1 nM of Aβ1-40, which correlates with that reported from human CSF. However, Aβ1-42, which is circulated in a less concentrated amount in human CSF, plays a minor role in rescuing neuron viability.

A noticeable contrast to the protecting effect of Aβ1-40 was observed with the Aβ25-35 amino acids. When Aβ25-35 was treated with one of the secretase inhibitors they demonstrated no alteration in neuron toxicity.

To conclude, Plant et al. (2003) showed that in neuronal cells of rodents and humans the inhibition of secretase peptides which forms the basis of the production and secretion of Aβ results in neuron toxicity. This might be an attractive area for further investigation - rescuing cell viability through adding physiologically appropriate concentrations of Aβ1-40, the proposition being that this peptide may play an essential role in the normal function of neuronal cells.

2.3. Nicotinic Achetyl Choline Receptors:

The attention of AD progress research has shifted in recent years further than toxicity towards previous events, for instance alteration in synaptic function. Accordingly, some evidence demonstrated that Aβ affects cholinergic signalling independent of its cytotoxic act.

Nicotinic Achetyl Choline Receptors (nAChRs) are ligand gated ion channels composed of five subunits to form a central, cation permeate channel, the permeability of which is controlled by the binding effect of acetylcholine (ACh) neurotransmitter. The most common nAChRs in the CNS are composed of α7, α4β2 and α3β4 (Lindstrom, 2003).

A number of studies demonstrate a relationship between nAChRs in the brain and AD development. Biochemical, electrophysiological, and pharmacological analysis showed that the brain of an AD patient has a scarcity of nAChRs and both the active decrease of cholinergic synthesis and the activation of acetylcholinesterase (AChE) lead to a drop in acetylcholine neurotransmitter (ACh) (Bartus et al., 1982).

The focus of pharmaceutical companies is to target the destroying factors of ACh. Up to now, the AChE inhibitors have been the most broadly prescribed drugs for this disease and have been fairly beneficial in slowing the loss of cognition (Arneic et al., 2007).

The α7 nAChRs are located in specific areas of the brain which are vulnerable to damage by this disease; predominantly in the hippocampus. Dineley (2001) showed the responsibility of nAChRs in ruling synaptic transmission and synaptic plasticity. They are strongly linked with neurons that build up Aβ. Since this breakthrough discovery, investigations of how this high affinity may influence the physiology of functionally serve in the synaptic plasticity and homeostasis in normal physiology as well as being a contributing factor in the AD etiology (Parri and Dineley, 2009).

Generally, activation of nAChRs is the reason for membrane depolarization and this process increases the intracellular Calcium concentration. When nAChRs are present presynaptically, their activation usually raises the possibility of neurotransmitter release: whereas, when they are located postsynaptically, nAChRs originate Calcium signals and depolarization that activate intracellular signalling mechanisms. The effects of nAChRs cause and assist the initiation of long-term changes in synaptic transmission. The direction of hippocampal nAChR-mediated synaptic plasticity, depends on the period of nAChRs activation in relation to concurrent presynaptic and postsynaptic electrical action, and in addition depends on the location of cholinergic stimulation within the local network. Therapeutic activation of nAChRs may show success in the treatment of neurodegeneration where synaptic transmission is endangered, as in AD (McKay et al., 2007).

The Sharma (2009) study showed that astrocytes are sensitive to α7- particular antagonists. Stimulation of these receptors enhances Calcium transients in cells through calcium induced calcium release (CICR) triggered by ion flux through the receptor channels. The results suggest that α7 receptors stimulate a Calcium signalling pathway in astrocytes that is distinctive from that stimulated by these receptors on neurons, proposing that astrocytes might play a crucial role in cholinergic signalling.

Dineley (2002) focused on the connection of learning impairments with the α7 nAChRs in the dentate gyrus. Two main findings were achieved. Firstly, Aβ1-42 stimulates the mitogen-activated protein kinase (MAPK) pathway via α7 nAChRs. Secondly, the study verified that the elevation of Aβ resulted in an up-regulation of α7 nAChRs. This latter discovery, the up-regulation, is associated negatively with the performance of the mice in the Moriss water maze. The study shows a cognitive shortage in the AD animal models that observed impairment in hippocampal function and suggested that the up-regulation of α7 nAChRs could be a biochemical marker in indentifying hippocampal dysfunction, which could be an area for further research. Since nAChRs are highly permeable for Calcium ions and they are present on astrocytes, this might mean that the increase in Calcium influx enhances astrocytes Calcium signalling.

2.4. The role of Calcium:

Calcium is essential for living organisms, particularly in cell physiology, where movement of the calcium ion into and out of the cytoplasm functions as a signal for many cellular processes, for example, neuronal development, synaptic transmission and plasticity, and regulates the metabolism and apoptosis of the mitochondria (Tsien, 1990). Alterations and oscillations in intracellular Calcium concentrations are the central system by which spontaneous movement controls neural development. Dysfunction in Ca signalling has been considered as an infective agent of AD, which results in most important neuronal loss and cognitive impairment (Rang et al., 2003).

The neurotoxic effect of Aβ remains controversial. Recent studies show that Aβ is a pore former in intact neuronal membranes; it appeared that Aβ has an effect on Calcium ion-selective channels as well as on voltage-gated Calcium permeable channels (Arispne et al, 1993). A slight alteration in Calcium concentration signalling was indicated after long term exposure of Aβ. This drew attention to the possibility that an interruption in Calcium concentration homeostatic mechanism may cause changes in cellular metabolism (Abramov et al., 2003).

2.5. The role of Astrocytes:

Astrocytes and other cells of the glial family such as oligodendrocytes and microglia, were supposed to be only structural cells. Their purpose was merely to hold neurons collectively because they are unable to generate action potential. However, only lately has this family of glial cells been considered to be involved in organizing the dynamics and having an active role in the neuronal arrangement in the CNS (Volterra and Meldolesi, 2005).

There are opposing reports on Calcium concentration in astrocytes. One side demonstrates that Aβ increases astrocyte Calcium concentration, while the other side suggests that it decreases astrocyte Calcium concentration. This is vital as there is a bidirectional Calcium signalling connection between neuronal and glial cells. Astrocytes play a major role in providing metabolic substrates and the precursors of the antioxidant glutathione (Ambramov et al., 2003). Also they contain glutaminergic transporters that eliminate excitatory amino acids from the extracellular space; these courses of action are important in neuroprotection. However, in AD, the neuroprtection action of astrocytes is disturbed, resulting in a reduction of glutamate clearance. In addition, astrocytes have been revealed to contribute in glutamate production through calcium-mediated vesicular release of glutamate. Neuronal harm is a consequence of glutamate overload (Markesbery, 1997; Hansson, 2003).

Studies that prepared cultured astrocytes showed that free Ca concentration in the cytosol experiences great alterations either in a spontaneous manner or as a result of several pharmacological and physiological effects; for instance, mechanical stimulation, membrane potential depolarization and activation of metabotropic and ionotropic glutamate receptors, mainly NMDARs (Pasti et al., 1997). These actions intervene with intracellular supplies of Calcium release, which can induce a strong effect on neuronal activity. These facts suggest a possible homeostatic position of astrocytes in the regulation of extracellular accumulating neurotransmitters that can lead to the increase of Calcium influx into neurons through NMDARs, due to glutamate activation, results in cell death.

Current research has announced that healthy adult astrocytes have a major defending task, since they specifically connect, internalize and powerfully condense Aβ. In AD, this mechanism is disturbed; in addition to the unusual expression of secretase peptides can change the role of astrocytes to a promoter of Aβ accumulation. The exact method by which astrocytes identify and condense Aβ is not yet acknowledged (Volterra and Meldoles, 2005).

2.6. Objective:

Investigate if the link between nAChRs and Aβ deposition causes disruption in Calcium homeostasis.

Examine if Aβ deposition would affect astrocytes Calcium homeostasis or dynamics through nAChRs.

Investigate the effects of glutamate release and its uptake inhibition by astrocytes on synaptic transmission.

Distinguish astrocytes from neuronal effects that contribute to AD progression.

Study the outcome of excitotoxicity and neurodegeneration caused by Aβ.

In conclusion, AD is the most common of the devastating dementias affecting humans. The amyloid hypothesis of AD states that an abnormal build up of Aβ in the brain leads to the symptoms and neurodegeneration associated with the condition. There is evidence that amyloid causes Calcium increase in cultured strocytes. This project will test the hypothesis that Aβ is an agonist at receptors on astrocytes and neurons in brain slices. Specifically it will focus initially on nAChRs in brain slices.

It is known that cholinergic afferents are one of the first neurodegenerative steps that are seen in AD. Could this be due to an agonist effect of Aβ at AChRs?

Glial cells are increasingly recognised as partners in CNS function and recent focus on astrocytes has implicated them in such diverse roles as synchronising neuronal activity to controlling cerebral blood flow. While it is known that astrocytic morphological changes occur in AD and that they are involved in amyloid, nothing is known about changes in astrocytic transmitter release.

This study will therefore focus on changes in glial excitatory and inhibitory neurotransmitters released during the development of amyloid over expression in these mice and determine the role of these changes in relation to known synaptic efficacy changes which parallel the cognitive decline in AD.

This is a motivating project and will broaden the knowledge of glial signalling in human disease. Also, finding early onset markers of AD could prove useful in screening new therapies.

3. Intended design and methods of investigation:

Mice brain tissue will be used in this project because it has several advantages; it is easy to isolate as an intact tract and it can be kept for several hours in a brain slice chamber without dying, allowing multiple tests to be carried out and also because it has been found that learning and memory aspects in mice appear to be similar to those in the human brain. Furthermore, brain will be used in order to optimize the results through using living tissue preparation to create reliable AD conditions and most of the contributory factors. To achieve this, Tg2756 transgenic mice, will be used as a model of AD, that express two mutant human Aβ proteins in the neurons. This mutation leads to Aβ production and deposition.

Mouse brain dissection:

Mice will be humanely killed according to the regulations provided by the Home Office of the United Kingdom under the Animals (Scientific Procedure) Act 1986.

Mice will be anaesthetized with an I.M. injection of Ketamine and also xylazine and then will be decapitated.

Following this, cardiac-perfused with artificial cerebrospinal fluid (aCSF) will be applied in order to flush the blood. The brain should be removed rapidly and immersed in oxygenatied aCSF ice-cold.

Brain Slice preparation:

Sagittal brain slices of the hippocampal will be cut using a vibrating tissue slicer, and should be stored in ice-cold aCSF (contain: 124 mM NaCl, 2.5 mM KCl, 1.3 mM MgCl2, 1 mM Na2HPO4, 26.2 mM NaHCO3 2.5 mM CaCl2, 26.2 and 20 mM D glucose) and bubbled with 95% O2 - 5% CO2. Following slicing, in order to keep the tissue alive, pieces will be preserved in aCSF for 30 minutes at 35°C and then for up to eight hours at 25°C and should always be perfused with oxygenated aCSF throughout the experiments (Kim et al., 2007).

Calcium imaging experiments:

Changes of intracellular Calcium signalling have been observed in a variety of disease states and AD is one of them. In order to study and understand the hypothetical causes of AD, this study will be mainly measuring the alteration of Calcium ions influx within cells. Calcium imaging makes this target a possible one by allowing Calcium concentration to be identified as modifications in fluorescence.

Calcium imaging is an advanced technique carried out by scientific researchers. This technique takes advantage of calcium indicators, molecules that can reflect to the binding of Calcium as well as Calcium excitation or emission through altering their spectral characteristics. Chemical Indicators: are small molecules that can chelate Calcium ions.


In this project, use will be made of 35-mm dishes with glass bottoms, which should be coated before plating the brain slices. Before being placed on the Nikon fn1 microscope, the cells are loaded with Fluo-4 chemical indicator for 30 minutes at room temperature. Excitation lights of 488nm wave length will be applied. This type of light can be obtained by using a standard fluorescein filter set, this procedure will result in a rapid and transient increase in cytosolic calcium as will be indicated by changes in fluo-4 fluorescence.

Images are then collected with Hamamatsu orca cameras. Data will be acquired by taking images every five seconds at various frequencies at various frequencies. Acquirement and analyses will be performed through compix software.

First set of experiments:

Dysfunction of Calcium signalling is one of the AD pathogenesis. Aβ in previous experiments was seen to participate in the alteration of ca intracellular concentration. It is agreed that Aβ has a high affinity for α7 nAChRs, in which these receptors are considered to have high permeability for Calcium ions. To prove this Aβ1-42 will be applied (Aβ agonist) then monitor to see if there is any enhancement in the Calcium oscillations. If there are changes then, methyllycaconatine (MLA)( nAChRs antagonist) will be applied to block the receptors. However, if the oscillations are inhibited, then that should confirm Aβ is an agonist and this should help us to move to the next stage of the project.

Second set of experiments:

It is proved that nAChRs are present on astrosytes and as previously mentioned; nAChRs have a high Calcium permeability. This consequently results in an elevated Calcium concentration in astrocytes. Testing this idea will be done through measuring Calcium concentration in astrocytes. If high Calcium concentration is witnessed in astrocytes then the next step will be to apply α-bungarotoxin (α-BTX) (nAChRs antagonist) and monitor whether Calcium concentration will be reduced in astrocytes. Consequently, if the results follow this plan, two main conclusions can be drawn. First, this proves that nAChRs (which should be enhanced by Aβ future suggestion from the outcome of experiment one) elevate Calcium influx in astrocytes. Secondly, increasing Calcium concentration in astrocytes means enhancing their Calcium signalling, which is the main signalling connection with neurons. This shows the effect of astrocytes on neurons.

Third set of experiments:

It is implicated in the pathophysiology of AD that neurotoxicity is due to an increase in NMADRs stimulation. In AD there is an increase in astrocytes activity. Might this result in glutamate elevation? As cited previously, there is an alteration in astrocytes function in glutamate uptake. This glutamate overload activates NMDARs that enhance excess Calcium influx in cells and cause their death. To examine this theory, first observe if there is a rise in Calcium oscillation in astrocytes then Ketamine (non-competitive NAMDRs blocker) will be applied. After assessing the effect of Ketamine, the results should reveal a decline in spontaneous Calcium oscillation.

Fourth set of experiments:

Depending on how the experiments will proceed and upon the results collected, it would be interesting to apply at some stage tetrodotoxin (TTX) to block the activity of neurons since nAChRs are present on neuron membranes as well as on astrocytes. Here it focuses on excluding any neuron contribution in Calcium oscillation.

These experiments aim to prove that astrocytes are the main contributing agents in neuronal damage through the effect of Aβ agonist.

4. Bibliography Reference:

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Arispe, N., Rojas, E. and Pollard, H.B. (1993). Alzheimer disease amyloid _ protein forms calcium channels in bilayer membranes: blockade by tromethamine and aluminum. Proc Natl Acad Sci USA[online], 90, p.567-571. Available from: [accessed 13 Feb 2010]. (Proved that Aβ could be an ion channel former).

Arneric, S. P., Holladay, M. and Williams M. (2007). Neuronal nicotinic receptors: a

perspective on two decades of drug discovery research. Biochem Pharmacol [online], 74, p.1092-1101. Available from: [Accessed on 22 Feb 2010]. (Understanding the molecular structure and kinetic properties of nAChRs).

Bartus, R.T., Dean, R.L., Beer, B. and Lippa, A.S. (1982). The cholinergic hypothesis of geriatric memory dysfunction. Science [online], 217, p.408-414.

Available from: [Accessed on 20 Feb 2010]. (Considers the pharmacological effects of nAChRs ).

Dawbarn, D. And Allen, S.J. (2001). Neurobiology of Alzheimer's disease: Neuropathology of Alzeihimer's disease . 2nd ed. United States: Oxford University Press. (Back ground knowledge on AD).

Dineley, K.T., Xia, X., Bui, D., Sweatt, J.D. and Zheng, H. (2002). Accelerate Plaque Accumulation, Associateve Learning Deficits, and Up-regulating of α7 Nicotinic Receptor Protein in Transgenic Mice Co-expressing Mutant Human Presenilin 1 and Amyloid Precursor Proteins. The Journal of Biological Chemistry, 21, p.22768-22780. (Suggesting a biochemical marker for AD).

Dineley, K.T., Westrman, M., Bui, D., Sweatt, J.D,Bell, K. And Ashe, K.H. (2001). Β-Amyloid Activates the Mitogen-Activated Protein Kinase Cascade via Hippocampal α7 Nicotinic Achetylcholine Receptors: in Vitro and in Vivo Mechanism Related to Alzheimer's Disease. The Journal of Neuroscience, 21(12), p.4125-4133.

(Up regulation consequences of α7 nAChRs through MAKP pathway activation).

Friedrich, R.P.,Tepper, K., Rönicke, R., Soom, M., Westermann, M., Reymann, K., Kaether, C. and Fändrich, M. (2009). Mechanism of amyloid plaque formation suggests an intracellular basis of Aβ pathogenicity. The Journal of Neuroscience, 107(5), p.1942-1947. Available from: [Accessed on 28 Feb 2010]. (Explaines the mechanism of Aβ development).

Hansson, E. and Ronnback, L. (2003). Glial neuronal signaling in the central nervous system. The Faseb Journal [online], 17, p. 341-348.

Available from: [Accessed on 11 Mar 2010]. (Broad knowledge on glial cells ).

Kim, S.J., Jin, Y., Kim, J., Worley, P.F. and Linden, D.J. (2007). Long-term depression of mGluR1 signaling. Neuron[online], 55(2), p.277-287. Available from: [Accessed on 1 Mar 2010]. (Demonstrating brain slicing procedure).

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Free Rad Biol Med[online], 23, p.134-147. Available from: [Accessed 1 Mar 2010]. (The role of oxidative stress induced by Aβ1-42 in AD).

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Beyreuther, K., Bush, A.I. and Masters, C.L. (1999). Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease. Ann Neurol[online], 46, p.860-866. Available from: [Accessed on 18 Feb 2010]. (Outlines locations and forms of Aβ and its sequential relation to the neurodegenerative process).

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and plasticity by neuronal nicotinic acetylcholine receptors.

Biochem Pharmacol.[online], 74(8), p.1120-1133.

Available from: [Accessed 28 Feb 2010]. (Role of nAChRs in synaptic transmission in the hippocampus).

Kumar-Singh, S., Theuns, J., Broeck, B.V., Pirici, D., Vennekens, K., Corsmit, E., Cruts, M., Dermaut, B., Wang, R. and Broeckhoven, C.V. (2006). Mean Age-of-Onset of Familial Alzheimer Disease Caused by Presenilin Mutations Correlates

With Both Increased Aβ42 and Decreased Aβ40. Human Mutation [online], 27(7), p.686-695. Available from: [Accessed on 18 Feb 2010]. (A review showing the correlation between Aβ1-42 and Aβ1-40).

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Parri, H.R., Dineley, K.T. (2009). Nicotinic Acetylcholone Receptor Interaction with β amyloid: Molecular Cellular, and Physiological Consequences. Current Alzheimer Research, 7(1). (Emphasis link between Aβ and nAChRs in AD etiology).

Parri, H.R., Gould, T.M. and Crunelli, V. (2001). Spontaneous astrocytic Ca2+ oscillations in situ drive NMDA-mediated neuronal excitation. Nat Neurosci [online], 4, p.803-812. Available from: [Accessed 2 Mar 2010]. (Evidence presents that astrocytes can act as prime cause in neurodegenerating).

Pasti, L., Volterra, A., Pozzan, T. and Carmignoto, G. (1997). Intracellular calcium oscillations in astrocytes: A highly plastic, bidirectional form of communication between neurons and astrocytes in situ. The Journal of Neuroscience [online], 17, p.7817-7830. Available from: [Accesssed on 23 Feb 2010]. (The investigation of Ca oscillations in astrocytes).

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Sharma, G. and Vijayaraghavan, S. (2001). Nicotinic cholinergic signaling in hippocampal astrocytes involves calcium-induced calcium release from intracellular stores. Proceeding of the National Academy of Sciences of the United States of America, 98(7), p. 4148-4153. (Proves nAChRs function as Ca influx pathway on astrocytes).

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Volterra, A and Meldolesi, J. (2005). Astrocytes from brain glue to communication elements: the revolution continues. Nature, 6, p. 626-640. (Recent and general information about astrocytes ).

5. Project management plan:

Apply Aβ1-42 to test if Aβ is an agonist on nAChRs, measure Ca oscillation.

Apply MLA to block nAChRs, measure Ca oscillations, analyse results.

Apply αBTX block nAChRs on astrocytes, analyse results.

Apply Kitamine to block NMDARs, analyse results.

Might repeat an experiment and apply TTX to block neuron activity, to eliminate neuron action ,analyse results.

Spear week for unexpected experiments or to compensate for any delays if not for analysis and reviw.

Review and write the project.