Functional Features and Physiological Role of Calcium Signalling in Astrocytes

7481 words (30 pages) Dissertation

10th Dec 2019 Dissertation Reference this

Tags: Physiology

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Abstract

  1. Introduction -3000words

The principle of information storage has been at the forefront of scientific research dating back as far as the 19th century. Santiago Ramon y Cajal was the pioneer of astroglial research through developing the first specific stain for astrocytes, which fundamentally demonstrated that astrocytes can divide in the adult brain (Parpura & Verkhratsky, 2012). This paved the way for further discoveries of the stem properties of astroglial such as the role which Calcium ions (Ca2+) play in modulating many astroglial processes such as the release of gliotransmitters.

  1. Classification of glial cells

Healthy brain function is heavily reliant on the interaction between two key cell types, neurons which allows for the transmission of information via electrical impulses and glial cells which have an array of specialised functions ranging from myelination & host defence to modulating synaptic transmissions. Glial cells were traditionally thought to be non-neuronal cells with structural purposes, providing inert scaffolding crucial for neuronal distributions and interactions (Volterra & Meldolesi, 2005). There are a variety of glial cells each differing in function the key ones being oligodendrocytes, microglia, ependymal cells, astrocytes, satellite glial cells and lastly Schwann cells. Oligodendrocytes provide support to the axons of neurons of the central nervous system through the production of the fatty substance myelin which wraps around the axons acting as an electrical insulator. Furthermore, it allows for faster transmission by enabling saltatory conduction. Microglia on the other hand are involved in host defence helping to ensure the clearance of toxic agents or dead cells present in the local environment. Ependymal cells are support cells which form the epithelial lining of the ventricles within the brain and in the central canals of the spinal cord, they are covered in a plethora of cilia which beat in a co-ordinated fashion helping to direct the movement of cerebrospinal fluid hence controlling the movement of nutrients for neurons. Satellite cells and Schwann cells are cells of the Peripheral nervous system. Satellite glial cells surround neurons which not only acts as a protective cushion but also provides nutrients to the neurons, whereas Schwann cells play the same role as oligodendrocytes of the CNS by producing myelin.

The last glia cell of the CNS is astrocytes, these are of particular importance and play a part in numerous functions of the development and physiology of the central nervous system (CNS), some of the most crucial roles include homeostasis of the extracellular ionic environment surrounding neurons, modulation of neuronal communication and the uptake of excess neurotransmitter to name a few (Araque & Navarrete, 2010). In the past few decades, the definition of astrocytes has drastically changed from just being regarded as purely support cells to being involved in a whole host of neuronal modulatory processes. These theories have been strongly supported by experiments demonstrating the ability for astrocytes to respond to neuronal activity through increases in intracellular Ca2+ levels (Figure 1) (Haydon & Carmignoto, 2006; Porter & McCarthy, 1997).

Figure 1: Basic overview of astrocytic modulation of neuron

This diagram very simply shows that changes in calcium concentrations can be caused by the pre-synaptic neuron which in turn stimulates the release of gliotransmitter by astrocytes, hence modulating post synaptic activity.

  1. Neuronal calcium signalling

Calcium signalling is fundamental intracellular signal to controlling a whole host of cell dependent processes. each different cell type is equipped with a specific cellular machinery from the Ca2+ signalling toolkit in order to create environments which differ in their spatial and temporal properties in regards to Ca2+  signalling systems (Berridge et al., 2003). In general, Ca2+ signal derives either from internal or external sources with the former being generally activated via metabotropic G-coupled receptors which can then cause the slow release of internal stores of Ca2+ through the use of messengers such as inositol-1,4,5-trisphosphate (IP3). Whereas the latter generally facilitate the rapid entry of Ca2+ via ligand-gated channels such as N-methyl-D-aspartate receptors (NMDA). In regards to communication at neuronal synapses the arrival of an action potential at the axon terminal of the presynaptic neuron causes the opening of Ca2+ sensitive voltage gated channels causing a large influx of Ca2+. This causes the exocytosis of neurotransmitters from synaptic vesicles by encouraging vesicle fusion events due to the Ca2+ dependent protein synaptotagmin which is located in the membrane of synaptic vesicles (Lodish et al., 2012). The fusion events of the docked vesicles are prevented by the interaction between complexin proteins and the v-SNARE/t-SNARE proteins of the vesicle and the plasma membrane, however upon Ca2+ binding to synaptotagmin there is a conformational change and these interactions are inhibited allowing the release of neurotransmitter (Lodish et al., 2012).

  1. Astrocyte- Neurons interaction

Astrocytes make up more than a third of the brain’s mass and half of the brains cells. They surround a plethora of synapses and dendrites with their multitude of ion channels helping to influence and manipulate the ionic concentration of free ions in the extracellular space and therefore affect the membrane potential of neurons and of the astrocytes themselves (Figure 2).

Figure 2: Visual image of astrocyte and neuron

This micrograph with a scale bar of 10 m shows a protoplasmic astrocyte depicted in green stain surrounding the cell body and processes of a neuron depicted in red stain. The branching nature of the astrocyte can be seen, suggesting its role in controlling many bodily processes.

Source: (Allen & Barres, 2009)

Similarly, to neurons glial cells also possess SNARE Ca2+ dependent proteins which facilitate the release of gliotransmitters such as ATP, glutamate and D-serine, the close proximity of neurons allows these gliotransmitters to help modulate synaptic strength (Lalo et al., 2009). This bidirectional communication is facilitated through the formation of close spatial interactions between neuronal synapses and astrocytic projections, often this is referred to as the tripartite synapse (Figure 3).  The tripartite synapses that occur under specific physiological conditions help to influence the synaptic efficacy mainly through two processes. Firstly, the varying re-uptake of glutamate by astrocytes and secondly the extracellular regulation of the gliotransmitter D-serine which regulates N-methyl-d-aspartate (NMDA) receptor-mediated synaptic transmission (Araque & Navarrete, 2010). The large scale impact this can have on numerous neurons is unprecedented, due to astrocytes being in contact with large numbers of synapses at one time. Experiments by (Angulo et al., 2004) used hippocampal pyramidal cells to truly demonstrate the effects that astrocyte calcium elevations, and the subsequent gliotransmitters released in particular glutamate can have in terms of synchronous excitation of multiple neurons.

Figure 3: Depiction of the tripartite synapse

Astrocytes have many of the same receptors as neurons meaning neurotransmitter release from the presynaptic neuron can bind to astrocytic receptors and evoke an increase in calcium levels. In turn causing the release of active substances which can act back upon the neuron to modulate it.

Source from: (Allen & Barres, 2009)

 

In regards to neurotransmitter uptake astrocytes are not limited to only glutamate as they express transporters for adenosine, dopamine and GABA (Porter & McCarthy, 1997). Although the transport of these neurotransmitters is not as well understood as glutamate experiments are starting to uncover the metabolic roles that they may be contributing to. Dopamine in particular has been demonstrated in cell cultures to evoke Ca2+ signals in astrocytes through the use of reactive oxygen species which inturn activates the PLC pathway (Vaarmann, A. 2010). These findings suggest that dopamine induced calcium release is receptor-independent and hence may have an important physiological role in terms of neurological disorders.

  1. Astrocytic calcium signalling

Perhaps the most crucial and defining features of astrocytes is their inability to generate action potentials unlike neurons. Despite this however it has been found that astrocytes possess a wide range of G protein coupled receptors (GPCRs) linked to Ca2+ mobilisation from internal stores both in culture and in vivo, which in turn could be responsible for a whole host of signalling cascades (Agulhon et al., 2008). Perhaps the most understood GPCR cascade in regards to astrocytic intracellular Ca2+ increases would be the canonical phospholipase C (PLC)/IP3 pathway. The mechanism as described in a review by (Agulhon et al., 2008)  and briefly depicted in (Figure 4) is initiated by the binding of neurotransmitter of the pre-synaptic neuron onto GPCRs, after which PLC hydrolyses the membrane lipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol (DAG) and IP3. This eventually leads to the activation of the IP3 receptor and subsequent release of Ca2+ stores from the endoplasmic reticulum.

Figure 4: Release of intracellular Ca2+ stores

A brief overview of the basic intracellular Ca2+ release mechanism from the endoplasmic reticulum.         

Although the PLC/IP3 pathway is considered the gold standard in terms of astrocytic calcium increase, there are numerous other ways for calcium entry into astrocytes however little is understood about these mechanisms. Astrocytes in culture have been found to express multiple voltage-gated Ca2+ channels (VGCCs), however experiments which tracked Ca2+ concentrations following neuronal afferent stimulation found that the increase in Ca2+ was attributed to activation of GPCRs not VGCCs (Agulhon et al., 2008). Though not relevant in terms of initiating increased astrocytic Ca2+ levels VGCCs may hold an important role in initiating spontaneous Ca2+ oscillations, which generally occur independently of neuronal input (Aguado et al., 2002; Parri & Crunelli, 2003; Parri et al., 2001).  Radial glia cells communicate with neurons which are developing in order to help guide their migration, with calcium signalling thought be the main method facilitating this communication (Weissman et al.). The study of these specific glia cell types has revealed another major class of ER Ca2+ channels the ryanodine receptors (RyRs) which may help to explain some of the specific fine processes in which astrocytes help to regulate (Zalk et al., 2007). RyRs channels were found to be approximately 10 times larger than VGCCs, and in vitro experiments demonstrated that their activation was dependent upon a gating ring mechanism during times of increasing calcium concentrations (Zalk et al., 2007). Overall the paper found that these channels directly influenced intracellular Ca2+ release within skeletal and cardiac muscles. Lastly, there is increasing evidence for the presence of Ca2+ permeable AMPA receptors on astrocytes, however as of yet there has only been supporting evidence of the presence in specific glial populations. An example of which includes Bergmann glial cells of the cerebellum which possess ionotropic glutamate receptors of the AMPA type, these lack the normally present GluA2 subunit thus allowing them to exhibit high levels of Ca2+ permeability (Droste et al., 2017). These cells resemble hippocampal astrocytes in their capacity to modulate neighbouring neurons via glutamatergic receptor activation hence allowing the data to be extrapolated and applied to astroglial interactions (Auld & Robitaille). This paves the way into understanding how the release of neurotransmitter such as glutamate from neurons can elicit both entry of Ca2+ via extracellular and intracellular sources, both of which may result in different metabolic processes.

Calcium signalling can directly influence and control cerebrovascular responses in the brain as astrocytic end feet ensheath large areas of the brain vasculature (Mathiisen et al., 2010). This allows them to release compounds with can lead to either vasodilation/contraction of the nearby blood vessels, thus allowing for rapid adaptations in response to changing metabolic demands. In terms of metabolic support during increased neuronal activity more energy in the form of lactate will need to be provided to sustain a consistent synaptic transmission. This is facilitated via the lactate shuttle (Figure 5) within astrocytes which in essence is the uptake of blood glucose or the breakdown of intracellular glycogen into lactate which is then released (Brown & Ransom, 2007). Alongside being a key glycogen store in the brain astrocytes are also involved in the synthesis and release of glutamine from the uptake of glutamate. The expression of the enzyme glutamine synthetase allows this reaction and once the glutamine is released into the extracellular environment it can be taken up by neurons in order to be converted back to the neurotransmitter glutamate (Hertz, 2006). Furthermore, a study by (Liang et al., 2006) found that the glutamine which is released can also be taken up by inhibitory neurons to be converted into GABA, thus allowing for another pathway in which astrocytes can control synaptic activity within the CNS. These are just a few of the physiological and functional features that are dependent on calcium signalling, however there is much more to be uncovered about the role which this ion can play within astrocytes which I will explore.  

Figure 5: Lactate shuttle

Proposed mechanism for the synthesis of lactate during times of increased neuronal metabolic demand.

 

  1. Calcium tracking

Through the use of Ca2+ sensitive fluorescent dyes early experiments by (Cornell-Bell et al., 1990) were able to track intracellular Ca2+ levels within living cells, through which astrocytes were found to exhibit cellular excitability which is dependent upon the Ca2+ levels of the cytosol rather than the electrical changes of the outer membrane.

After which greater advances in technological allowed for techniques such as patch-clamping, caged compounds and confocal microscopy all of which allowed for the manipulation and imaging of intracellular Ca2+ levels (Haydon, 2001). One particular difficulty in studying astrocytic signalling in situ was that other neural cells also exhibit similar GPCRs which are linked to Ca2+ mobilisation, making it difficult to see the individual effect of just the astrocytic Ca2+ signalling cascades on physiological processes (Agulhon et al., 2008). This was overcome through the use of glass electrodes which allowed non-physiological depolarisation of astrocytes and also by the insertion of caged IP3 or Ca2+ which was loaded via path clamp pipettes allowing for specific targeting of astrocytes (Agulhon et al., 2008). These new techniques allowed for research into oscillations in calcium concentrations and propagating calcium waves, which uncovered possible mechanisms to facilitate these propagations.

In terms of imaging Ca2+ signals in vivo the use of uncharged dyes such as Acetoxymethyl allow for the dye to cross the cell membrane, and once within the cytoplasm the endogenous esterase’s of the cell de-esterify the dye into a charged species (Russell, 2011). This not only allows it to have a high affinity for Ca2+ but also has the added benefit of being highly fluorescent.  Lastly, two-photon microscopy has been a major tool for calcium imaging within the nervous system. This technique of fluorescence imaging typically uses near-infrared excitation light which can excite fluorescent dyes, whilst at the same time minimising light scattering within tissues leading to less background noise within the samples.

  1. Baseline calcium

Baseline levels of calcium can have some physiological function through the utilisation of Ca2+-activated Clchannels present on astrocytes (Agnel et al., 1999). Studies carried out in situ have shown that these channels allow the release of small gliotransmitters such as GABA & glutamate without the need to reach the normal threshold for Ca2+ activation as in sigmoidal activation (Kimelberg et al., 2006).  Ultimately baseline levels of calcium depend on the efficiency of calcium extrusion and calcium uptake into mitochondria and ER.

In the case of neuronal diseases these baseline levels of calcium can often be set too high which has an impact on many neural functions in particular memory formation and consolidation such as in Alzheimer’s disease (AD) (Berridge, 2014). The accumulation of amyloid β-peptide (Aβ) fragments and the continuous disruption of intracellular calcium concentration levels are the main two hypotheses regarding the pathogenesis of AD. Studies by (Abramov et al., 2004a; Abramov et al., 2004b) have found key links between intracellular calcium transients associated with Aβ exposure in astrocytes can often result in the formation in reactive oxygen species within astrocytes, and this alongside with calcium dependent glutathione depletion within neurons often leads to neuronal death. This raises the possibility that both intracellular calcium transients and spontaneous intercellular waves within neuroglia could have a much more profound effect in both ageing and disease than initially thought.

  1. Potential aims and objectives

The research carried out in this project forms part of a larger study into investigating the role of astroglial calcium ion signalling, and the mechanisms and factors which could have a major impact on astroglial signalling.  I aim to review appropriate literature to help uncover the many physiological and functional roles which both astroglial intracellular and spontaneous calcium oscillations play. Furthermore, through the use visual imaging software I will attempt to compare differences in fluorescent intensities between wild type mice and mice with Alzheimer’s disease to try and conclude whether there could be differences in calcium concentrations between the two.

2)Methods – add in pictures of using Image J

The vast majority of this project the methods used mainly include literature searches using online databases to obtain relevant papers and studies on the topic (Figure 6). The main search engines used include PubMed and Google Scholar, with Endnote being used to appropriately store and input the references into the project. Upon searching the relevant topic into the search engine, it was often necessary to then refine the results, leading to me often having to select additional search categories such as publication date topic, publisher, author, topic, title, DOI & address. Due to the topic at hand (Calcium signalling in astrocytes)having a great deal of uncertainty surrounding it, by tuning these categories it allowed me to quickly and efficiently find papers with new hypothesises and data which have built on the knowledge of previous studies.

In terms of investigating the effects of AD on astrocytes calcium signalling, the software ImageJ was used to track the astroglial cells present in different samples of mice. The individual cells were traced using the in-app selection tool for both the green and red dyed cells. After which measurements were taken using the multi-measure function which gave me the mean sizes and intensities of the different astrocytes, from this I could then work out if there were any significant differences in calcium signalling between wild type mice and mice with AD. Significant differences were calculated using the Red/Green ratios, after which using the inbuilt excel formulas the averages were taken from each of the different samples as well as the standard deviation. Lastly T tests were carried out between different groups to test whether there was any significance between the Red/Green ratios using a 95% confidence interval.

Furthermore, the clear majority of figures in the project were made using Microsoft PowerPoint and the figures taken from other papers were once again referenced using Endnote.

Figure 6: Overview of research techniques used to find relevant papers

An initial search term related to the topic of interest into a search engine gives a large number of results. The results are then refined by specific categories leading to a more manageable number of papers after which I can read the abstracts of the papers with relevant titles. Once a relevant paper is found and the full paper is read I can then use the references cited in the paper to find other relevant sources of information.

 

3)Results

3.1 maybe put role of calcium in reg synaptic transmission etc here

 

3.2 Propagation of calcium waves

The presence of Ca2+ waves which propagate between cultured astrocytes has been shown to travel over long distances, meaning the activation of astrocytes in one location could modulate a wide range of neurons. In vitro experiments have helped to uncover prominent hypotheses which underline the mechanisms of Ca2+ wave propagations. One theory is the diffusion of IP3 through gap junctions to evoke Ca2+ signals in neighbouring unstimulated astrocytes, whilst another leading hypothesis is that the release of ATP from an astrocyte can in turn activate purinergic G protein-coupled receptors (P2Y) on adjacent astrocytes thus stimulating Ca2+ signals (Haydon & Carmignoto, 2006). These are a family of G protein-coupled receptors which are stimulated b nucleotides such as ATP. It was eventually concluded that both these pathways are not mutually exclusive however despite this they are likely to work in conjunction (Scemes, 2006) in order to provide co-ordinated Ca2+ signals (Figure 7), with the IP3 diffusion pathway facilitating short range calcium oscillation. On the other hand the activation of membrane P2Y receptors by ATP allows for long range calcium oscillations by facilitating the production of IP3 in neighbouring astrocytes.

Figure 7: Radially propagating wave of elevated calcium

Putative mechanisms for astrocytic calcium ion propagation from internal sources (i.e. Endoplasmic Reticulum), in response to increased levels of IP3. IP3 can passively diffuse into neighbouring astrocytes via gap junctions (connexins) causing short-range signals whereas longer range Ca2+ signals are mediated by the release of ATP via connexins which causes the regenerative production of IP3 and therefore acts as a positive feedback loop to cause the release of more ATP onto neighbouring astrocytes.

As mentioned above calcium waves can be propagated via the diffusion of ATP acting on membrane receptors, however this has not been found to be consistent in striatal astrocytes.  Early experiments helped demonstrate that they instead have a prominent Ca2+ response to glutamate (Cornell-Bell et al., 1990) compared to ATP, paving the way to question whether astrocytes from different brain regions vary in mechanisms of Ca2+ propagations.

Another particular area of interest are the neuronal progenitor radial glial cells many of which postnatally transform into astrocytes (Rakic, 2003). Whilst little is definitive in terms of the mechanisms which regulate these cells links have been found which suggest the role of calcium transients in aiding cell division, neuronal differentiation and neuronal migration. Due to astrocytes and radial glia cells being developmentally linked studies were carried out to find whether Ca2+ dynamics could contribute to the functional radial glia signalling mechanisms. Alongside the regular means of Ca2+ wave spread via ATP/IP3 pathways, these glial cells were also found to be able to initiate spontaneous intercellular Ca2+ waves (Weissman, 2004). The underlying mechanism which initiates these waves was proposed to involve the opening of radial glial connexin hemichannels, therefore allowing the release of factors that initiate local calcium waves upon neighbouring cells. The experiments carried out by Weissman, used the ATP receptor antagonist suramin to help show that extracellular ATP may be involved in spontaneous oscillations in addition to mediating electrically or mechanically stimulated calcium waves. This research paves the way to explore the possibility of spontaneous Ca2+ oscillations in astrocytes which are independent of neuronal activity and the physiological implications this can have.

In situ experimentations within astrocytes originally uncovered the presence of spontaneous Ca2+ oscillations within the ventrobasal thalamus, however it has since been found that other brain regions exhibiting these oscillations include the hippocampus & cortex (Parri, 2003). In the cortex a possible pathological role of these oscillations has been linked to epilepsy due to only cortical samples of samples with epilepsy exhibited these oscillations (Tashiro, 2002). The underlying mechanisms hypothesised in the study were that the neocortical circuit is hyper-excitable in epileptic patients meaning that subsequently levels of extracellular potassium or glutamate is significantly elevated. Therefore leading to chronic depolarisation of astrocytes which in turn leads to large influx of Ca2+ via voltage-sensitive calcium channels, this can then constantly replenish internal stores calcium stores triggering astrocytic oscillations with spontaneous kinetics.

3.2 Metabolic role of calcium

Astrocytes are metabolically coupled to neurons by supplying energy (lactate) to neurons in an activity dependant manner. Astrocytes are a major source of localised glycogen within the brain which can be utilised during times of high neuronal activity via the trafficking of glucose and lactate (Brown and Ransom, 2007). In terms of the pathways involved in metabolic role of astrocytes, there is much debate. One view is that glucose is the sonly energy substrate utilised by neurons, however a more widely accepted hypothesis involves astrocyte derived lactate via the lactate shuttle for use as an energy substrate (Pellerin and Magistretti, 2003).  This is known as the astrocyte-neuron lactate shuttle hypothesis (ANLSH). A study in 2004 used two-photon microscopy by fluorescently tagging versions of the electron carrier β-nicotinamide adenine dinucleotide (NADH) in hippocampal astrocyte samples to help track the level of astrocyte glycolysis upon axonal stimulation of nearby neurons (Kasischke et al. (2004)). The ultimate conclusions drawn were that upon increased neuronal stimulation the rate of glycolysis increased proportionally through the ANLSH pathway. The specific cellular mechanisms surrounding the lactate shuttle were investigated by (Magistretti., et al 1990) to begin with at the pre synaptic neuron there is a Ca2+ dependent release of the neurotransmitter glutamate, which then along with Na+ is transported into the astrocyte via a sodium/glutamate co-transporter channel. This leads to an increase in intracellular Na+ levels which in turn activates the Na+ /K+ ATPase pump leading to the initiation of glycolysis by ATP consumption. This increase in glycolysis is accompanied by higher levels of both glucose utilisation and lactate production. The lactate produced inside the astrocyte is released and quickly taken up by the neurons via a monocarboxylate transporter, where it can then be used as a metabolic substrate.

A striking feature of energy metabolism in the brain is the close relationship which exists between both energy demand and supply which is reflected by changes in the vasculature surrounding the brain in order to regulate glucose and oxygen delivery. The increased need for glucose consumption leads to the greater uptake of glucose from surrounding blood vessels via the glucose transporter 1 (GLUT-1), which is possible due to the astrocytic endfeet ensheathing the vascular of the brain. The level of blood flow in the surrounding vasculature determines the sustainability of metabolic processes occurring within the neuronal network, with strong evidence proposing that astrocytes can release vasoactive compounds to cause either vasodilation or vasoconstriction of the surrounding vasculature (Zonta, M. 2003). There are two main hypothesis in regards to how exactly the changes in vasculature are brought about, both of which ultimately rely on astrocytic Ca2+ signals. The first mechanism proposed involves Ca2+ elevations in astrocyte endfeet which leads to the opening of Ca2+ sensitive K+ channels, thus allowing the release of K+ onto smooth muscle cells causing either vasodilation/vasoconstriction (Filosa et al., 2006). However a multitude of studies have demonstrated the ability of astrocytes to produce a wide array of vasoactive substances, hinting at the possibility that microcirculation by astrocytes could not be simply controlled on the spatial buffering of the K+ hypothesis (Haydon, 2006). An alternate mechanism was therefore proposed which involves the increase of internal Ca2+ levels within astrocytes which in turn leads to the formation of arachidonic acid (AA) metabolites (Figure 8). These include namely, epoxyeicosatrienoic acid (EET) which is formed from AA via cytochrome P-450 epoxygenase (CPY2C) and prostaglandins which are also formed from AA however via cyclooxygenase-2 (COX) which both cause vasodilation (Roman, Richard J.2002). On the other hand vasoconstriction is caused by the accumulation of 20-hydroxyeicosatetraenoic acid (20-HETE), which is caused due to derivative of AA is which causes vasoconstriction as the diffusion of AA to the smooth muscle of the vasculature containing high levels of CPY4A which stimulates the accumulation of 20-HETE (Haydon,2006).

Figure 8: Mechanism which underlies vasoconstriction/vasodilation of vasculature surrounding astrocytes

High synaptic activity leads to glutamate release from axon terminal, upon binding of glutamate to the astrocyte membrane metabotropic glutamate receptors (mGluRs) internal calcium oscillations are triggered. These oscillations spread to the astrocytic endfeet and regulate the release of vasoactive agents.

The transport of neurotransmitters by astrocytes has also proposed to pose a possible role in helping to facilitate cellular metabolism. Adenosine in particular is transported through ENT1 & ENT2 channels expressed on astrocytes, with in vitro experiments showing that this increase of intracellular adenosine was in turn used for cellular metabolism by facilitating adenine synthesis (Nagai et al., 2005; Peng et al., 2005).

All these mechanisms seem to fundamentally rely upon sarcoplasmic reticulum (SR)/ER Ca2+ ATPase (SERCA) release channels to facilitate the rapid, localised release of intracellular Ca2+. There are two main families of release channels on the SR/ER which are widely recognised, these include the IP3 receptors and also ryanodine receptors (Zalk, 2007). The RyRs are less understood in terms of how widespread they are within different regions of the brain, for instance RyR-mediated Ca2+ signals were observed in the thalamus following caffeine stimulation (Parri and Crunelli, 2003) but the presence of RyRs were not found in hippocampal astrocytes (Beck et al., 2004;). What is known is the role in which RyR channels directly play in regards to controlling intracellular Ca2+ release within not onky skeletal and cardiac muscles in order to stimulate muscle contractions, but also within neurons to help modulate action potential and neurotransmitter release (zalk).

  1. Glia modulate synaptic transmission – GABA inhib, glutamate excite (Although it is certain that astrocytes are responsible for transmitter uptake from the synaptic cleft, it is also possible that they release glutamate to modulate synaptic transmission) – Haydon review paper

Glutamate recycling (check intro to see what is already written)

  1. Long term plasticity – cannabinoid receptors http://rstb.royalsocietypublishing.org/content/369/1654/20140077
  2. Elimination of Ca2+ permeable AMPA receptors from Bergmann glial leads to retraction of processes, which affects neuronal activity at Purkinje cells synapses (Iino et al., 2001). (page 14 example dissi)
  3. Why does Aβ cause spontaneous calcium waves, page 11 AD example dissi LINK IMAGE J DATA

Or role of astrocyte in ischaemic heart disease https://mospace.umsystem.edu/xmlui/handle/10355/9656

http://journals.sagepub.com/doi/full/10.1042/AN20090035

http://www.biochemj.org/content/474/3/333.full

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063224/

Discussion

  1. New fields reasearching into NG2 glia.  Classic studies recognize two functionally segregated macroglial cell types in the central nervous system (CNS), namely astrocytes and oligodendrocytes. A third macroglial cell type has now been identified by its specific expression of the NG2 chondroitin sulphate proteoglycan (NG2-glia). These NG2-glia exist abundantly in both grey and white matter of the mature CNS and are almost as numerous as astrocytes. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1571571/ and on page 7 (example dissi)
  2. Talk about the areas of confusion so we don’t know exactly how calcium progrates to neighbouring astrocytes etc
  3. Drawbacks of current models/experiments used to study calcium signals in astrocytes

E.g. Astroglial Ca2+signaling can easily be assessed in vitro using purified cultured astroglia. However, most investigators in the field acknowledge that cultured astroglia present a very poor model for studying the functions of astrocytic Gq GPCRs in situ or in vivo. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623689/

Page 42 example dissi, current models ie calcium uncaging bypasses secondary messenger creation

  1. Evidence for Gliotransmission Is Not Monolithic page 139 example dissi
  1. Look at results lit review and see physiological/pathological to link to different diseases and how can these have implications for future healthcare e.g. RYR DYSFUNCTION AND DISEASES http://www.annualreviews.org/doi/full/10.1146/annurev.biochem.76.053105.094237

Astrocytes role in ischaemic heart disease https://mospace.umsystem.edu/xmlui/handle/10355/9656

Talk about NMDA receptors and disease

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3166701/

Talk about statistical errors i.e. false negs

Although under particular circumstances, such as brain hypoxia or ischemia, this process may indeed affect blood vessels, the time course of the neurovascular coupling argues against this hypothesis http://physrev.physiology.org/content/86/3/1009.full

So like in heart disease talk about maybe we cud administer caged calcium or IP3 etc in order to cause vasodilation etc, we learnt this by studying lactate shuttle.

References

Abramov, A. Y., Canevari, L. & Duchen, M. R. (2004a) Calcium signals induced by amyloid β peptide and their consequences in neurons and astrocytes in culture. Biochimica et Biophysica Acta (BBA) – Molecular Cell Research, 1742 (1): 81-87.

Abramov, A. Y., Canevari, L. & Duchen, M. R. (2004b) Beta-amyloid peptides induce mitochondrial dysfunction and oxidative stress in astrocytes and death of neurons through activation of NADPH oxidase. J Neurosci, 24 (2): 565-575.

Agnel, M., Vermat, T. & Culouscou, J.-M. (1999) Identification of three novel members of the calcium-dependent chloride channel (CaCC) family predominantly expressed in the digestive tract and trachea 1. FEBS Letters, 455 (3): 295-301.

Aguado, F., Espinosa-Parrilla, J. F., Carmona, M. a. A. & Soriano, E. (2002) Neuronal Activity Regulates Correlated Network Properties of Spontaneous Calcium Transients in Astrocytes <em>In Situ</em&gt. The Journal of Neuroscience, 22 (21): 9430.

Agulhon, C., Petravicz, J., McMullen, A. B., Sweger, E. J., Minton, S. K., Taves, S. R., Casper, K. B., Fiacco, T. A. & McCarthy, K. D. (2008) What Is the Role of Astrocyte Calcium in Neurophysiology? Neuron, 59 (6): 932-946.

Allen, N. J. & Barres, B. A. (2009) Neuroscience: Glia [mdash] more than just brain glue. Nature, 457 (7230): 675-677.

Angulo, M. C., Kozlov, A. S., Charpak, S. & Audinat, E. (2004) Glutamate Released from Glial Cells Synchronizes Neuronal Activity in the Hippocampus. The Journal of Neuroscience, 24 (31): 6920.

Araque, A. & Navarrete, M. (2010) Glial cells in neuronal network function. Philosophical Transactions of the Royal Society B: Biological Sciences, 365 (1551): 2375-2381.

Auld, D. S. & Robitaille, R. Glial Cells and Neurotransmission. Neuron, 40 (2): 389-400.

Berridge, M. J. (2014) Calcium regulation of neural rhythms, memory and Alzheimer’s disease. The Journal of Physiology, 592 (Pt 2): 281-293.

Berridge, M. J., Bootman, M. D. & Roderick, H. L. (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol, 4 (7): 517-529.

Brown, A. M. & Ransom, B. R. (2007) Astrocyte glycogen and brain energy metabolism. Glia, 55 (12): 1263-1271.

Cornell-Bell, A. H., Finkbeiner, S. M., Cooper, M. S. & Smith, S. J. (1990) Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. Science, 247 (4941): 470.

Droste, D., Seifert, G., Seddar, L., Jädtke, O., Steinhäuser, C. & Lohr, C. (2017) Ca2+-permeable AMPA receptors in mouse olfactory bulb astrocytes. Scientific Reports, 7 44817.

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