Arrays of MicroLEDs and Astrocytes: Biological Amplifiers to Optogenetically Modulate Neuronal Networks Reducing Light Requirement

In the modern view of synaptic transmission, astrocytes are no longer confined to the role of merely supportive cells. Although they do not generate action potentials, they nonetheless exhibit electrical activity and can influence surrounding neurons through gliotransmitter release. In this work, we explored whether optogenetic activation of glial cells could act as an amplification mechanism to optical neural stimulation via gliotransmission to the neural network. We studied the modulation of gliotransmission by selective photo-activation of channelrhodopsin-2 (ChR2) and by means of a matrix of individually addressable super-bright microLEDs (μLEDs) with an excitation peak at 470 nm. We combined Ca²⁺ imaging techniques and concurrent patch-clamp electrophysiology to obtain subsequent glia/neural activity. First, we tested the μLEDs efficacy in stimulating ChR2-transfected astrocyte. ChR2-induced astrocytic current did not desensitize overtime, and was linearly increased and prolonged by increasing μLED irradiance in terms of intensity and surface illumination. Subsequently, ChR2 astrocytic stimulation by broad-field LED illumination with the same spectral profile, increased both glial cells and neuronal calcium transient frequency and sEPSCs suggesting that few ChR2-transfected astrocytes were able to excite surrounding not-ChR2-transfected astrocytes and neurons. Finally, by using the μLEDs array to selectively light stimulate ChR2 positive astrocytes we were able to increase the synaptic activity of single neurons surrounding it. In conclusion, ChR2-transfected astrocytes and μLEDs system were shown to be an amplifier of synaptic activity in mixed corticalneuronal and glial cells culture.     

TNFα controls glutamatergic gliotransmission in the hippocampal dentate gyrus

Glutamatergic gliotransmission provides a stimulatory input to excitatory synapses in the hippocampal dentate gyrus. Here, we show that tumor necrosis factor-alpha (TNFα) critically controls this process. With constitutive TNFα present, activation of astrocyte P2Y1 receptors induces localized [Ca(2+)](i) elevations followed by glutamate release and presynaptic NMDA receptor-dependent synaptic potentiation. In preparations lacking TNFα, astrocytes respond with identical [Ca(2+)](i) elevations but fail to induce neuromodulation. We find that TNFα specifically controls the glutamate release step of gliotransmission. In cultured astrocytes lacking TNFα glutamate exocytosis is dramatically slowed down due to altered vesicle docking. Addition of low picomolar TNFα promptly reconstitutes both normal exocytosis in culture and gliotransmission in situ. Alternatively, gliotransmission can be re-established without adding TNFα, by limiting glutamate uptake, which compensates slower release. These findings demonstrate that gliotransmission and its synaptic effects are controlled not only by astrocyte [Ca(2+)](i) elevations but also by permissive/homeostatic factors like TNFα. VIDEO ABSTRACT:     

The influence of the astrocyte field on neuronal dynamics and synchronization

Astrocytes can sense local synaptic release of glutamate by metabotropic glutamate receptors. Receptor activation in turn can mediate transient increases of astrocytic intracellular calcium concentration through inositol 1,4,5-trisphosphate production. Notably, the perturbation of calcium concentration can propagate to other adjacent astrocytes. Astrocytic calcium signaling can therefore be linked to synaptic information transfer between neurons. On the other hand, astrocytes can also modulate neuronal activity by feeding back onto synaptic terminals in a fashion that depends on their intracellular calcium concentration. Thus, astrocytes can also be active partners in neuronal network activity. The aim of our study is to provide a computationally simple network model of mutual neuron–astrocyte interactions, in order to investigate the possible roles of astrocytes in neuronal network dynamics. In particular, we focus on the information entropy of neuronal firing of the whole network, considering how it could be affected by neuron–glial interactions.     

Astroglial excitability and gliotransmission: an appraisal of Ca2+ as a signalling route

Astroglial cells, due to their passive electrical properties, were long considered subservient to neurons and to merely provide the framework and metabolic support of the brain. Although astrocytes do play such structural and housekeeping roles in the brain, these glial cells also contribute to the brain''s computational power and behavioural output. These more active functions are endowed by the Ca²⁺-based excitability displayed by astrocytes. An increase in cytosolic Ca²⁺ levels in astrocytes can lead to the release of signalling molecules, a process termed gliotransmission, via the process of regulated exocytosis. Dynamic components of astrocytic exocytosis include the vesicular-plasma membrane secretory machinery, as well as the vesicular traffic, which is governed not only by general cytoskeletal elements but also by astrocyte-specific IFs (intermediate filaments). Gliotransmitters released into the ECS (extracellular space) can exert their actions on neighbouring neurons, to modulate synaptic transmission and plasticity, and to affect behaviour by modulating the sleep homoeostat. Besides these novel physiological roles, astrocytic Ca²⁺ dynamics, Ca²⁺-dependent gliotransmission and astrocyte–neuron signalling have been also implicated in brain disorders, such as epilepsy. The aim of this review is to highlight the newer findings concerning Ca²⁺ signalling in astrocytes and exocytotic gliotransmission. For this we report on Ca²⁺ sources and sinks that are necessary and sufficient for regulating the exocytotic release of gliotransmitters and discuss secretory machinery, secretory vesicles and vesicle mobility regulation. Finally, we consider the exocytotic gliotransmission in the modulation of synaptic transmission and plasticity, as well as the astrocytic contribution to sleep behaviour and epilepsy.     

Ammonia induces amyloidogenesis in astrocytes by promoting amyloid precursor protein translocation into the endoplasmic reticulum

Hyperammonemia is known to cause various neurological dysfunctions such as seizures and cognitive impairment. Several studies have suggested that hyperammonemia may also be linked to the development of Alzheimer’s disease (AD). However, the direct evidence for a role of ammonia in the pathophysiology of AD remains to be discovered. Herein, we report that hyperammonemia increases the amount of mature amyloid precursor protein (mAPP) in astrocytes, the largest and most prevalent type of glial cells in the central nervous system that are capable of metabolizing glutamate and ammonia, and promotes amyloid beta (Aβ) production. We demonstrate the accumulation of mAPP in astrocytes was primarily due to enhanced endocytosis of mAPP from the plasma membrane. A large proportion of internalized mAPP was targeted not to the lysosome, but to the endoplasmic reticulum, where processing enzymes β-secretase BACE1 (beta-site APP cleaving enzyme 1) and γ-secretase presenilin-1 are expressed, and mAPP is cleaved to produce Aβ. Finally, we show the ammonia-induced production of Aβ in astrocytic endoplasmic reticulum was specific to Aβ42, a principal component of senile plaques in AD patients. Our studies uncover a novel mechanism of Aβ42 production in astrocytes and also provide the first evidence that ammonia induces the pathogenesis of AD by regulating astrocyte function.     

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca²⁺ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca²⁺ events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.     

Regulation of cell-to-cell communication mediated by astrocytic ATP in the CNS

It has become apparent that glial cells, especially astrocytes, not merely supportive but are integrative, being able to receive inputs, assimilate information and send instructive chemical signals to other neighboring cells including neurons. At first, the excitatory neurotransmitter glutamate was found to be a major extracellular messenger that mediates these communications because it can be released from astrocytes in a Ca(2+)-dependent manner, diffused, and can stimulate extra-synaptic glutamate receptors in adjacent neurons, leading to a dynamic modification of synaptic transmission. However, recently extracellular ATP has come into the limelight as an important extracellular messenger for these communications. Astrocytes express various neurotransmitter receptors including P2 receptors, release ATP in response to various stimuli and respond to extracellular ATP to cause various physiological responses. The intercellular communication "Ca(2+) wave" in astrocytes was found to be mainly mediated by the release of ATP and the activation of P2 receptors, suggesting that ATP is a dominant "gliotransmitter" between astrocytes. Because neurons also express various P2 receptors and synapses are surrounded by astrocytes, astrocytic ATP could affect neuronal activities and even dynamically regulate synaptic transmission in adjacent neurons as if forming a "tripartite synapse". In this review, we summarize the role of astrocytic ATP, as compared with glutamate, in gliotransmission and synaptic transmission in neighboring cells, mainly focusing on the hippocampus. Dynamic communication between astrocytes and neurons mediated by ATP would be a key event in the processing or integration of information in the CNS.     

Role of the Intracellular Nucleoside Transporter ENT3 in Transmitter and High K+ Stimulation of Astrocytic ATP Release Investigated Using siRNA Against ENT3

This study investigates the role of the intracellular adenosine transporter equilibrative nucleoside transporter 3 (ENT3) in stimulated release of the gliotransmitter adenosine triphosphate (ATP) from astrocytes. Within the past 20 years, our understanding of the importance of astrocytic handling of adenosine, its phosphorylation to ATP, and release of astrocytic ATP as an important transmitter has become greatly expanded. A recent demonstration that the mainly intracellular nucleoside transporter ENT3 shows much higher expression in freshly isolated astrocytes than in a corresponding neuronal preparation leads to the suggestion that it was important for the synthesis of gliotransmitter ATP from adenosine. This would be consistent with a previously noted delay in transmitter release of ATP in astrocytes but not in neurons. The present study has confirmed and quantitated stimulated ATP release in response to glutamate, adenosine, or an elevated K⁺ concentration from well-differentiated astrocyte cultures, measured by a luciferin–luciferase reaction. It showed that the stimulated ATP release was abolished by downregulation of ENT3 with small interfering RNA (siRNA), regardless of the stimulus. The concept that transmitter ATP in mature astrocytes is synthesized directly from adenosine prior to release is supported by the postnatal development of the expression of the vesicular transporter SLC17A9 in astrocytes. In neurons, this transporter carries ATP into synaptic vesicles, but in astrocytes, its expression is pronounced only in immature cells and shows a rapid decline during the first 3 postnatal weeks so that it has almost disappeared at the end of the third week in well-differentiated astrocytes, where its role has probably been taken over by ENT3.     

Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles

The astrocyte is a major glial cell type of the brain, and plays key roles in the formation, maturation, stabilization and elimination of synapses. Thus, changes in astrocyte condition and age can influence information processing at synapses. However, whether and how aging astrocytes affect synaptic function and maturation have not yet been thoroughly investigated. Here, we show the effects of prolonged culture on the ability of astrocytes to induce synapse formation and to modify synaptic transmission, using cultured autaptic neurons. By 9 weeks in culture, astrocytes derived from the mouse cerebral cortex demonstrated increases in β-galactosidase activity and glial fibrillary acidic protein (GFAP) expression, both of which are characteristic of aging and glial activation in vitro. Autaptic hippocampal neurons plated on these aging astrocytes showed a smaller amount of evoked release of the excitatory neurotransmitter glutamate, and a lower frequency of miniature release of glutamate, both of which were attributable to a reduction in the pool of readily releasable synaptic vesicles. Other features of synaptogenesis and synaptic transmission were retained, for example the ability to induce structural synapses, the presynaptic release probability, the fraction of functional presynaptic nerve terminals, and the ability to recruit functional AMPA and NMDA glutamate receptors to synapses. Thus the presence of aging astrocytes affects the efficiency of synaptic transmission. Given that the pool of readily releasable vesicles is also small at immature synapses, our results are consistent with astrocytic aging leading to retarded synapse maturation.     

Astrocytic connectivity in the hippocampus

Little is known about the functional connectivity between astrocytes in the CNS. To explore this issue we photo-released glutamate onto a single astrocyte in murine hippocampal slices and imaged calcium responses. Photo-release of glutamate causes a metabotropic glutamate receptor (mGluR)-dependent increase in internal calcium in the stimulated astrocyte and delayed calcium elevations in neighboring cells. The delayed elevation in calcium was not caused by either neuronal activity following synaptic transmission or by glutamate released from astrocytes. However, it was reduced by flufenamic acid (FFA), which is consistent with a role for adenosine triphosphate (ATP) release from astrocytes as an intercellular messenger. Exogenous ligands such as ATP (1 mircoM) increased the number of astrocytes that were recruited into coupled astrocytic networks, indicating that extracellular accumulation of neurotransmitters modulates neuronal excitability, synaptic transmission and functional coupling between astrocytes.     

Knockdown of astrocytic Grin2a aggravates β‐amyloid‐induced memory and cognitive deficits through regulating nerve growth factor

Synapse degeneration correlates strongly with cognitive impairments in Alzheimer''s disease (AD) patients. Soluble Amyloid‐beta (Aβ) oligomers are thought as the major trigger of synaptic malfunctions. Our earlier studies have demonstrated that Aβ oligomers interfere with synaptic function through N‐methyl‐D‐aspartate receptors (NMDARs). Our recent in vitro study found the neuroprotective role of astrocytic GluN2A in the promotion of synapse survival and identified nerve growth factor (NGF) derived from astrocytes, as a likely mediator of astrocytic GluN2A buffering against Aβ synaptotoxicity. Our present in vivo study focused on exploring the precise mechanism of astrocytic GluN2A influencing Aβ synaptotoxicity through regulating NGF. We generated an adeno‐associated virus (AAV) expressing an astrocytic promoter (GfaABC1D) shRNA targeted to Grin2a (the gene encoding GluN2A) to perform astrocyte‐specific Grin2a knockdown in the hippocampal dentate gyrus, after 3 weeks of virus vector expression, Aβ were bilaterally injected into the intracerebral ventricle. Our results showed that astrocyte‐specific knockdown of Grin2a and Aβ application both significantly impaired spatial memory and cognition, which associated with the reduced synaptic proteins PSD95, synaptophysin and compensatory increased NGF. The reduced astrocytic GluN2A can counteract Aβ‐induced compensatory protective increase of NGF through regulating pNF‐κB, Furin and VAMP3, which modulating the synthesis, mature and secretion of NGF respectively. Our present data reveal, for the first time, a novel mechanism of astrocytic GluN2A in exerting protective effects on synapses at the early stage of Aβ exposure, which may contribute to establish new targets for AD prevention and early therapy.     

A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters

Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca²⁺ transients in astrocytes, and these Ca²⁺ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4⁺ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4⁺ astrocytes. After activation, both TRPV4⁺ and TRPV4⁻ astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4⁺ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4⁺ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands.     

Amyloid β-peptide directly induces spontaneous calcium transients,delayed intercellular calcium waves and gliosis in rat cortical astrocytes

The contribution of astrocytes to the pathophysiology of AD (Alzheimer''s disease) and the molecular and signalling mechanisms that potentially underlie them are still very poorly understood. However, there is mounting evidence that calcium dysregulation in astrocytes may be playing a key role. Intercellular calcium waves in astrocyte networks in vitro can be mechanically induced after Aβ (amyloid β-peptide) treatment, and spontaneously forming intercellular calcium waves have recently been shown in vivo in an APP (amyloid precursor protein)/PS1 (presenilin 1) Alzheimer''s transgenic mouse model. However, spontaneous intercellular calcium transients and waves have not been observed in vitro in isolated astrocyte cultures in response to direct Aβ stimulation in the absence of potentially confounding signalling from other cell types. Here, we show that Aβ alone at relatively low concentrations is directly able to induce intracellular calcium transients and spontaneous intercellular calcium waves in isolated astrocytes in purified cultures, raising the possibility of a potential direct effect of Aβ exposure on astrocytes in vivo in the Alzheimer''s brain. Waves did not occur immediately after Aβ treatment, but were delayed by many minutes before spontaneously forming, suggesting that intracellular signalling mechanisms required sufficient time to activate before intercellular effects at the network level become evident. Furthermore, the dynamics of intercellular calcium waves were heterogeneous, with distinct radial or longitudinal propagation orientations. Lastly, we also show that changes in the expression levels of the intermediate filament proteins GFAP (glial fibrillary acidic protein) and S100B are affected by Aβ-induced calcium changes differently, with GFAP being more dependent on calcium levels than S100B.     

Computational modeling of paroxysmal depolarization shifts in neurons induced by the glutamate release from astrocytes

Recent experimental studies have shown that astrocytes respond to external stimuli with a transient increase of the intracellular calcium concentration or can exhibit self-sustained spontaneous activity. Both evoked and spontaneous astrocytic calcium oscillations are accompanied by exocytosis of glutamate caged in astrocytes leading to paroxysmal depolarization shifts (PDS) in neighboring neurons. Here, we present a simple mathematical model of the interaction between astrocytes and neurons that is able to numerically reproduce the experimental results concerning the initiation of the PDS. The timing of glutamate release from the astrocyte is studied by means of a combined modeling of a vesicle cycle and the dynamics of SNARE-proteins. The neuronal slow inward currents (SICs), induced by the astrocytic glutamate and leading to PDS, are modeled via the activation of presynaptic glutamate receptors. The dependence of the bidirectional communication between neurons and astrocytes on the concentration of glutamate transporters is analyzed, as well. Our numerical results are in line with experimental findings showing that astrocyte can induce synchronous PDSs in neighboring neurons, resulting in a transient synchronous spiking activity.     

Astrocytes are important mediators of A��-induced neurotoxicity and tau phosphorylation in primary culture

Alzheimer''s disease (AD) is pathologically characterised by the age-dependent deposition of β-amyloid (Aβ) in senile plaques, intraneuronal accumulation of tau as neurofibrillary tangles, synaptic dysfunction and neuronal death. Neuroinflammation, typified by the accumulation of activated microglia and reactive astrocytes, is believed to modulate the development and/or progression of AD. We have used primary rat neuronal, astrocytic and mixed cortical cultures to investigate the contribution of astrocyte-mediated inflammatory responses during Aβ-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aβ-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aβ-induced tau phosphorylation observed in primary neurons. The release of soluble inflammatory factor(s) from astrocytes accompanies these events, and inhibition of astrocyte activation with the anti-inflammatory agent, minocycline, reduces astrocytic inflammatory responses and the associated neuronal loss. Aβ-induced increases in caspase-3 activation and the production of caspase-3-truncated tau species in neurons were reduced when the astrocytic response was attenuated with minocycline. Taken together, these results show that astrocytes are important mediators of the neurotoxic events downstream of elevated Aβ in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy.     

Astrocyte and Neuron Intone Through Glutamate

The unexpected finding of astrocytes to release glutamate as gliotransmitter challenges the traditional concepts on astrocyte being “passive” in CNS communications. Glutamate is the major excitatory transmitter in transferring information between neurons, but is now also known to activate astrocyte through transporters and receptors. Together with the sensitive swelling response, astrocytes could respond directly to glutamate and neuronal activity. Other new functions of astrocytes include modulation of synaptic plasticity and cerebral blood flow (CBF). The classic glutamate deplenishment through glutamine synthesis and CO₂ production does not account for the total glutamate internalized into astrocytes. This leads us to speculate there are many hidden functions of glutamate in neurons and astrocytes waiting to be discovered. In this review, we attempted to reexamine some of these new and older functions of glutamate and to reevaluate the roles of glutamate intoning these two cell types. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Is astrocyte calcium signaling relevant for synaptic plasticity?

Astrocytes constitute a major group of glial cells which were long regarded as passive elements, fulfilling nutritive and structural functions for neurons. Calcium rise in astrocytes propagating to neurons was the first demonstration of direct interaction between the two cell types. Since then, calcium has been widely used, not only as an indicator of astrocytic activity but also as a stimulator switch to control astrocyte physiology. As a result, astrocytes have been elevated from auxiliaries to neurons, to cells involved in processing synaptic information. Curiously, while there is evidence that astrocytes play an important role in synaptic plasticity, the data relating to calcium's pivotal role are inconsistent. In this review, we will detail the various mechanisms of calcium flux in astrocytes, then briefly present the calcium-dependent mechanisms of gliotransmitter release. Finally, we will discuss the role of calcium in plasticity and present alternative explanations that could reconcile the conflicting results published recently.     

Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes

Astroglial excitability operates through increases in Ca²⁺_cyt (cytosolic Ca²⁺), which can lead to glutamatergic gliotransmission. In parallel fluctuations in astrocytic Na⁺_cyt (cytosolic Na⁺) control metabolic neuronal-glial signalling, most notably through stimulation of lactate production, which on release from astrocytes can be taken up and utilized by nearby neurons, a process referred to as lactate shuttle. Both gliotransmission and lactate shuttle play a role in modulation of synaptic transmission and plasticity. Consequently, we studied the role of the PMCA (plasma membrane Ca²⁺-ATPase), NCX (plasma membrane Na⁺/Ca²⁺ exchanger) and NKA (Na⁺/K⁺-ATPase) in complex and coordinated regulation of Ca²⁺_cyt and Na⁺_cyt in astrocytes at rest and upon mechanical stimulation. Our data support the notion that NKA and PMCA are the major Na⁺ and Ca²⁺ extruders in resting astrocytes. Surprisingly, the blockade of NKA or PMCA appeared less important during times of Ca²⁺ and Na⁺ cytosolic loads caused by mechanical stimulation. Unexpectedly, NCX in reverse mode appeared as a major contributor to overall Ca²⁺ and Na⁺ homoeostasis in astrocytes both at rest and when these glial cells were mechanically stimulated. In addition, NCX facilitated mechanically induced Ca²⁺-dependent exocytotic release of glutamate from astrocytes. These findings help better understanding of astrocyte-neuron bidirectional signalling at the tripartite synapse and/or microvasculature. We propose that NCX operating in reverse mode could be involved in fast and spatially localized Ca²⁺-dependent gliotransmission, that would operate in parallel to a slower and more widely distributed gliotransmission pathway that requires metabotropically controlled Ca²⁺ release from the ER (endoplasmic reticulum).     

Exocytosis in Astrocytes: Transmitter Release and Membrane Signal Regulation

Astrocytes, a type of glial cells in the brain, are eukaryotic cells, and a hallmark of these are subcellular organelles, such as secretory vesicles. In neurons vesicles play a key role in signaling. Upon a stimulus—an increase in cytosolic concentration of free Ca²⁺ ([Ca²⁺]_i)—the membrane of vesicle fuses with the presynaptic plasma membrane, allowing the exit of neurotransmitters into the extracellular space and their diffusion to the postsynaptic receptors. For decades it was thought that such vesicle-based mechanisms of gliotransmitter release were not present in astrocytes. However, in the last 30?years experimental evidence showed that astrocytes are endowed with mechanisms for vesicle- and non-vesicle-based gliotransmitter release mechanisms. The aim of this review is to focus on exocytosis, which may play a role in gliotransmission and also in other forms of cell-to-cell communication, such as the delivery of transporters, ion channels and antigen presenting molecules to the cell surface.     

Astrocytes mediate in vivo cholinergic-induced synaptic plasticity

Long-term potentiation (LTP) of synaptic transmission represents the cellular basis of learning and memory. Astrocytes have been shown to regulate synaptic transmission and plasticity. However, their involvement in specific physiological processes that induce LTP in vivo remains unknown. Here we show that in vivo cholinergic activity evoked by sensory stimulation or electrical stimulation of the septal nucleus increases Ca²⁺ in hippocampal astrocytes and induces LTP of CA3-CA1 synapses, which requires cholinergic muscarinic (mAChR) and metabotropic glutamate receptor (mGluR) activation. Stimulation of cholinergic pathways in hippocampal slices evokes astrocyte Ca²⁺ elevations, postsynaptic depolarizations of CA1 pyramidal neurons, and LTP of transmitter release at single CA3-CA1 synapses. Like in vivo, these effects are mediated by mAChRs, and this cholinergic-induced LTP (c-LTP) also involves mGluR activation. Astrocyte Ca²⁺ elevations and LTP are absent in IP₃R2 knock-out mice. Downregulating astrocyte Ca²⁺ signal by loading astrocytes with BAPTA or GDPβS also prevents LTP, which is restored by simultaneous astrocyte Ca²⁺ uncaging and postsynaptic depolarization. Therefore, cholinergic-induced LTP requires astrocyte Ca²⁺ elevations, which stimulate astrocyte glutamate release that activates mGluRs. The cholinergic-induced LTP results from the temporal coincidence of the postsynaptic activity and the astrocyte Ca²⁺ signal simultaneously evoked by cholinergic activity. Therefore, the astrocyte Ca²⁺ signal is necessary for cholinergic-induced synaptic plasticity, indicating that astrocytes are directly involved in brain storage information.