Astrocytes are highly complex cells with many emerging putative roles in brain function. Of these, gliotransmission (active information transfer from glia to neurons) has probably the widest ...implications on our understanding of how the brain works: do astrocytes really contribute to information processing within the neural circuitry? "Positive evidence" for this stems from work of multiple laboratories reporting many examples of modulatory chemical signaling from astrocytes to neurons in the timeframe of hundreds of milliseconds to several minutes. This signaling involves, but is not limited to, Ca
-dependent vesicular transmitter release, and results in a variety of regulatory effects at synapses in many circuits that are abolished by preventing Ca
elevations or blocking exocytosis selectively in astrocytes. In striking contradiction, methodologically advanced studies by a few laboratories produced "negative evidence," triggering a heated debate on the actual existence and properties of gliotransmission. In this context, a skeptics' camp arose, eager to dismiss the whole positive evidence based on a number of assumptions behind the negative data, such as the following: (1) deleting a single Ca
release pathway (IP3R2) removes all the sources for Ca
-dependent gliotransmission; (2) stimulating a transgenically expressed Gq-GPCR (MrgA1) mimics the physiological Ca
signaling underlying gliotransmitter release; (3) age-dependent downregulation of an endogenous GPCR (mGluR5) questions gliotransmitter release in adulthood; and (4) failure by transcriptome analysis to detect vGluts or canonical synaptic SNAREs in astrocytes proves inexistence/functional irrelevance of vesicular gliotransmitter release. We here discuss how the above assumptions are likely wrong and oversimplistic. In light of the most recent literature, we argue that gliotransmission is a more complex phenomenon than originally thought, possibly consisting of multiple forms and signaling processes, whose correct study and understanding require more sophisticated tools and finer scientific experiments than done until today. Under this perspective, the opposing camps can be reconciled and the field moved forward. Along the path, a more cautious mindset and an attitude to open discussion and mutual respect between opponent laboratories will be good companions.
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Astrocytes use calcium signals to process information received from neighboring brain cells and thus generate modulatory responses at the local or network level. Previous studies have relied on ...calcium imaging in line scans or in a single focal plane mostly focusing on the cell bodies of astrocytes. Bindocci et al. used more powerful scanners that can rapidly scan many focal planes. They combined this technique with advanced genetic tools for monitoring calcium gradients with high sensitivity, which allowed three-dimensional calcium imaging of a whole astrocyte. Most of the basal calcium activity occurred in the astrocyte processes, some in the endfeet, and only a small fraction actually in the cell bodies of astrocytes. Science, this issue p. eaai8185 Astrocytes translate incoming information and generate functional outputs via Ca2+ signaling. Thereby, they respond to neuronal activity, producing downstream modulation of synaptic functions, and may participate in hemodynamics regulation. Deciphering the "Ca2+ language" of astrocytes is therefore essential for defining their roles in brain physiology and pathology. However, the specifics of astrocytic Ca2+ signaling are still poorly understood, and recent studies producing inconsistent or contradictory results have fostered debate on the actual role of astrocytes in synaptic and vascular functions. A neglected potential source of inconsistencies lies in the way astrocytic Ca2+ signaling has been studied to date, mostly by conventional two-dimensional (2D) imaging, which assumes that sampling a single (~1 μm) focal plane is representative of the entire astrocytic cell. This is, however, dubious given that astrocytes are highly 3D cells, entertain heterogeneous 3D relations with neighboring structures, and display Ca2+ signals on a local scale. Therefore, we developed a new method to three-dimensionally scan entire astrocytes and observe full-cell Ca2+ dynamics. With our 3D approach, we sampled astrocytes at a sufficient rate to detect events with durations of >1.5 s throughout the cell, and faster ones in selected substructures. We found that Ca2+ activity in an individual astrocyte is heterogeneously scattered throughout the cell, largely compartmented within each region, and preponderantly local. The majority resides in the "gliapil," the peripheral region composed of fine (optically subresolved) structures occupying ∼75% of the astrocyte volume. Within the central (resolvable) "core," the soma is mostly inactive, whereas processes are frequently active yet show widely different activity between them. Even in individual processes, activity distributes heterogeneously, with alternating "hot" and "cold" spots. We performed 3D imaging in awake mice and in adult brain slices. Activity in vivo was faster and more frequent, particularly in endfeet, yet similar in properties and cellular distribution to slices, except for the presence of cell-wide "global" Ca2+ events mainly associated with mouse movement. Contrary to current beliefs, global events were not sweeping waves, but rather consisted of multifocal Ca2+ elevations that started at multiple gliapil loci and then spread to the core. At the vascular interface, astrocytic Ca2+ activity was mostly restricted to individual endfeet, even to their fractions, and only occasionally coordinated with the endfoot process or the rest of the astrocyte. Two or more endfeet were mainly asynchronous, even when enwrapping the same vessel. Astrocytic structures and axons intersected three-dimensionally, and minimal axonal activity (individual action potentials) produced time-correlated astrocytic Ca2+ elevations in small spots (<1% of the volume), which demonstrates that astrocytes can sense even the lowest levels of neuronal activity. We provide the first comprehensive 3D map of Ca2+ activity in an individual astrocyte. Its widespread, heterogeneous, local, and mostly 3D nature confirms the appropriateness of our whole-cell imaging approach. Past 2D studies, often focusing on somatic Ca2+ dynamics, inadequately described the emerging richness and complexity of the astrocyte activity, notably at astrocyte-synapse and astrocyte-vascular interfaces, where activity is small, fast, and frequent. In this context, we can foresee future challenges in extending studies to the gliapil, whose structures fall below current optical resolution, and in reporting the complete gamut of astrocyte Ca2+ signals at the whole-cell scale, both requiring technical advances. Nonetheless, the technique demonstrated here promises to make 3D Ca2+ imaging the state-of-the-art approach for Ca2+ studies addressing the role of astrocytes in brain function. (1) Representation of the 3D structural interactions among astrocyte, axons, and blood vessels. The astrocyte contains optically resolved (c, core) and unresolved (g, gliapil) regions. (2) 3D Ca2+ imaging reveals different activity (intensity color scale) in different astrocytic regions (P, processes; S, soma; EF, endfoot); (3) in different processes; and (4) in different loci of individual processes. (5) It also reveals asynchronous activity in endfeet, (6) often restricted to endfoot subdomains, and (7) local astrocyte responses to minimal axonal firing, or (8) global responses to in vivo locomotion (time color scale). Astrocyte communication is typically studied by two-dimensional calcium ion (Ca2+) imaging, but this method has not yielded conclusive data on the role of astrocytes in synaptic and vascular function. We developed a three-dimensional two-photon imaging approach and studied Ca2+ dynamics in entire astrocyte volumes, including during axon-astrocyte interactions. In both awake mice and brain slices, we found that Ca2+ activity in an individual astrocyte is scattered throughout the cell, largely compartmented between regions, preponderantly local within regions, and heterogeneously distributed regionally and locally. Processes and endfeet displayed frequent fast activity, whereas the soma was infrequently active. In awake mice, activity was higher than in brain slices, particularly in endfeet and processes, and displayed occasional multifocal cellwide events. Astrocytes responded locally to minimal axonal firing with time-correlated Ca2+ spots.
What do we know about gliotransmitter release from astrocytes? Sahlender, Daniela A.; Savtchouk, Iaroslav; Volterra, Andrea
Philosophical transactions of the Royal Society of London. Series B. Biological sciences,
10/2014, Volume:
369, Issue:
1654
Journal Article
Peer reviewed
Open access
Astrocytes participate in information processing by actively modulating synaptic properties via gliotransmitter release. Various mechanisms of astrocytic release have been reported, including release ...from storage organelles via exocytosis and release from the cytosol via plasma membrane ion channels and pumps. It is still not fully clear which mechanisms operate under which conditions, but some of them, being Ca2+-regulated, may be physiologically relevant. The properties of Ca2+-dependent transmitter release via exocytosis or via ion channels are different and expected to produce different extracellular transmitter concentrations over time and to have distinct functional consequences. The molecular aspects of these two release pathways are still under active investigation. Here, we discuss the existing morphological and functional evidence in support of either of them. Transgenic mouse models, specific antagonists and localization studies have provided insight into regulated exocytosis, albeit not in a systematic fashion. Even more remains to be uncovered about the details of channel-mediated release. Better functional tools and improved ultrastructural approaches are needed in order fully to define specific modalities and effects of astrocytic gliotransmitter release pathways.
Changes in the subunit composition of postsynaptic AMPA-type glutamate receptors can be induced at CNS synapses by neural activity and under certain pathological conditions. Fear-induced ...incorporation of GluR2-containing receptors at cerebellar synapses selectively prolongs the decay time of synaptic currents, whereas a switch from GluR2-lacking to GluR2-containing receptors induced by parallel fiber stimulation reduces the amplitude in addition to lengthening the duration of EPSCs. Although it is often assumed that these two forms of synaptic plasticity will alter action potential (AP) firing in the postsynaptic neuron, this has not been directly tested. Using a dynamic current-clamp approach, we now show that the fear-induced increase in EPSC duration increases the size of EPSPs and thereby markedly enhances the AP firing probability. In contrast, the parallel fiber stimulation-triggered switch in GluR2 expression reduces the EPSP-AP coupling because of the decrease in the synaptic current amplitude. The switch also abolished the paired-pulse facilitation that arose from an activity and spermine-dependent unblock of GluR2-lacking receptors and hence reduced the ability of paired stimuli to evoke two consecutive APs. Therefore, fear-induced incorporation of GluR2 receptors enhances the EPSP-AP coupling, but the parallel fiber stimulation-triggered switch reduces both the EPSP-AP coupling and evoked AP doublets. In contrast to long-term potentiation and depression, which modify the amplitude of synaptic currents, this activity-induced change in AMPA receptor phenotype alters synaptic conductance waveform and postsynaptic short-term plasticity. These changes modulate both the probability and pattern of evoked AP firing via a fundamentally different mechanism from long-term potentiation and long-term depression.
Recent advances in fast volumetric imaging have enabled rapid generation of large amounts of multi-dimensional functional data. While many computer frameworks exist for data storage and analysis of ...the multi-gigabyte Ca2+ imaging experiments in neurons, they are less useful for analyzing Ca2+ dynamics in astrocytes, where transients do not follow a predictable spatio-temporal distribution pattern. In this manuscript, we provide a detailed protocol and commentary for recording and analyzing three-dimensional (3D) Ca2+ transients through time in GCaMP6f-expressing astrocytes of adult brain slices in response to axonal stimulation, using our recently developed tools to perform interactive exploration, filtering, and time-correlation analysis of the transients. In addition to the protocol, we release our in-house software tools and discuss parameters pertinent to conducting axonal stimulation/response experiments across various brain regions and conditions. Our software tools are available from the Volterra Lab webpage at https://wwwfbm.unil.ch/dnf/group/glia-an-active-synaptic-partner/member/volterra-andrea-volterra (NB: placeholder link, code to be publicly released pending peer review) in the form of software plugins for Image J (NIH) – a de facto standard in scientific image analysis. Three programs are available: MultiROI_TZ_profiler for interactive graphing of several movable ROIs simultaneously, Gaussian_Filter5D for Gaussian filtering in several dimensions, and Correlation_Calculator for computing various cross-correlation parameters on voxel collections through time.
The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their ...importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.
Changes in emotional state are known to alter neuronal excitability and can modify learning and memory formation. Such experience-dependent neuronal plasticity can be long-lasting and is thought to ...involve the regulation of gene transcription. We found that a single fear-inducing stimulus increased GluR2 (also known as Gria2) mRNA abundance and promoted synaptic incorporation of GluR2-containing AMPA receptors (AMPARs) in mouse cerebellar stellate cells. The switch in synaptic AMPAR phenotype was mediated by noradrenaline and action potential prolongation. The subsequent rise in intracellular Ca(2+) and activation of Ca(2+)-sensitive ERK/MAPK signaling triggered new GluR2 gene transcription and a switch in the synaptic AMPAR phenotype from GluR2-lacking, Ca(2+)-permeable receptors to GluR2-containing, Ca(2+)-impermeable receptors on the order of hours. The change in glutamate receptor phenotype altered synaptic efficacy in cerebellar stellate cells. Thus, a single fear-inducing stimulus can induce a long-term change in synaptic receptor phenotype and may alter the activity of an inhibitory neural network.
Astrocyte Ca(2+) signalling has been proposed to link neuronal information in different spatial-temporal dimensions to achieve a higher level of brain integration. However, some discrepancies in the ...results of recent studies challenge this view and highlight key insufficiencies in our current understanding. In parallel, new experimental approaches that enable the study of astrocyte physiology at higher spatial-temporal resolution in intact brain preparations are beginning to reveal an unexpected level of compartmentalization and sophistication in astrocytic Ca(2+) dynamics. This newly revealed complexity needs to be attentively considered in order to understand how astrocytes may contribute to brain information processing.
Synaptic receptors gate the neuronal response to incoming signals, but they are not homogeneously distributed on dendrites. A spatially defined receptor distribution can preferentially amplify ...certain synaptic inputs, resize receptive fields of neurons, and optimize information processing within a neuronal circuit. Thus, a longstanding question is how the spatial organization of synaptic receptors is achieved. Here, we find that action potentials provide local signals that influence the distribution of synaptic AMPA receptors along dendrites in mouse cerebellar stellate cells. Graded dendritic depolarizations elevate CPEB3 protein at proximal dendrites, where we suggest that CPEB3 binds to GluA2 mRNA, suppressing GluA2 protein synthesis leading to a distance-dependent increase in synaptic GluA2 AMPARs. The activity-induced expression of CPEB3 requires increased Ca2+ and PKC activation. Our results suggest a cell-autonomous mechanism where sustained postsynaptic firing drives graded local protein synthesis, thus directing the spatial organization of synaptic AMPARs.
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•Synaptic GluA2-containing AMPA receptors increase along dendrites of stellate cells•Postsynaptic action potentials and graded Ca2+ entry drive the GluA2 gradient•Action potential firing activates PKC and elevates CPEB3 expression•Elevated CPEB3 levels at proximal dendrites suppress GluA2 protein synthesis
Savtchouk et al. show there is a dendritic gradient in the expression of the GluA2 subunit in synaptic AMPA receptors. The gradient is maintained by tonic postsynaptic firing, which controls the expression of CPEB3, a translational regulator. The postsynaptic AMPA receptor gradient optimizes information processing within a cerebellar circuit.
The subunit composition of synaptic AMPA receptors can undergo dynamic changes during physiological functioning and under pathological conditions. This switch in AMPA receptor phenotype involves ...changes in the level of GluA2 subunits that are mediated via regulated AMPA receptor trafficking, modification of local protein synthesis and altered gene transcription of GluA2 subunits. Incorporation of the GluA2 subunits into an AMPA receptor alters a number of key biophysical properties, including Ca2+ permeability and the waveform of the synaptic current. These changes alter the ability of synaptic currents to evoke an action potential and therefore have a profound effect on the computational capability of individual neurons and thus the output of neuronal circuits.
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