Two decades of astrocytes in neurovascular coupling.

The brain is a highly energy demanding organ, which accounts in humans for the 20% of total energy consumption at resting state although comprising only 2% of the body mass. The necessary delivery of nutrients to brain parenchyma is ensured by the cerebral circulatory system, through the exchange of glucose and oxygen (O2) at the capillary level. Notably, a tight spatial and temporal correlation exists between local increases in neuronal activity and the subsequent changes in regional cerebral blood flow. The recognized concept of neurovascular coupling (NVC), also named functional hyperemia, expresses this close relationship and stands at the basis of the modern functional brain imaging techniques. Different cellular and molecular mechanisms have been proposed to mediate this tight coupling. In this context, astrocytes are ideally positioned to act as relay elements that sense neuronal activity through their perisynaptic processes and release vasodilator agents at their endfeet in contact with brain parenchymal vessels. Two decades after the astrocyte involvement in neurovascular coupling has been proposed, we here review the experimental evidence that contributed to unraveling the molecular and cellular mechanisms underlying cerebral blood flow regulation. While traveling through the different controversies that moved the research in this field, we keep a peculiar focus on those exploring the role of astrocytes in neurovascular coupling and conclude with two sections related to methodological aspects in neurovascular research and to some pathological conditions resulting in altered neurovascular coupling.

Rescue of astrocyte activity by the calcium sensor STIM1 restores long-term synaptic plasticity in female mice modelling Alzheimer's disease.

Calcium dynamics in astrocytes represent a fundamental signal that through gliotransmitter release regulates synaptic plasticity and behaviour. Here we present a longitudinal study in the PS2APP mouse model of Alzheimer's disease (AD) linking astrocyte Ca2+ hypoactivity to memory loss. At the onset of plaque deposition, somatosensory cortical astrocytes of AD female mice exhibit a drastic reduction of Ca2+ signaling, closely associated with decreased endoplasmic reticulum Ca2+ concentration and reduced expression of the Ca2+ sensor STIM1. In parallel, astrocyte-dependent long-term synaptic plasticity declines in the somatosensory circuitry, anticipating specific tactile memory loss. Notably, we show that both astrocyte Ca2+ signaling and long-term synaptic plasticity are fully recovered by selective STIM1 overexpression in astrocytes. Our data unveil astrocyte Ca2+ hypoactivity in neocortical astrocytes as a functional hallmark of early AD stages and indicate astrocytic STIM1 as a target to rescue memory deficits.

Astrocytes mediate long-lasting synaptic regulation of ventral tegmental area dopamine neurons.

The plasticity of glutamatergic transmission in the ventral tegmental area (VTA) represents a fundamental mechanism in the modulation of dopamine neuron burst firing and phasic dopamine release at target regions. These processes encode basic behavioral responses, including locomotor activity, learning and motivated behaviors. Here we describe a hitherto unidentified mechanism of long-term synaptic plasticity in mouse VTA. We found that the burst firing in individual dopamine neurons induces a long-lasting potentiation of excitatory synapses on adjacent dopamine neurons that crucially depends on Ca2+ elevations in astrocytes, mediated by endocannabinoid CB1 and dopamine D2 receptors co-localized at the same astrocytic process, and activation of pre-synaptic metabotropic glutamate receptors. Consistent with these findings, selective in vivo activation of astrocytes increases the burst firing of dopamine neurons in the VTA and induces locomotor hyperactivity. Astrocytes play, therefore, a key role in the modulation of VTA dopamine neuron functional activity.

Calcium Signals in Astrocyte Microdomains, a Decade of Great Advances.

The glial cells astrocytes have long been recognized as important neuron-supporting elements in brain development, homeostasis, and metabolism. After the discovery that the reciprocal communication between astrocytes and neurons is a fundamental mechanism in the modulation of neuronal synaptic communication, over the last two decades astrocytes became a hot topic in neuroscience research. Crucial to their functional interactions with neurons are the cytosolic Ca2+ elevations that mediate gliotransmission. Large attention has been posed to the so-called Ca2+microdomains, dynamic Ca2+ changes spatially restricted to fine astrocytic processes including perisynaptic astrocytic processes (PAPs). With presynaptic terminals and postsynaptic neuronal membranes, PAPs compose the tripartite synapse. The distinct spatial-temporal features and functional roles of astrocyte microdomain Ca2+ activity remain poorly defined. However, thanks to the development of genetically encoded Ca2+ indicators (GECIs), advanced microscopy techniques, and innovative analytical approaches, Ca2+ transients in astrocyte microdomains were recently studied in unprecedented detail. These events have been observed to occur much more frequently (∼50-100-fold) and dynamically than somatic Ca2+ elevations with mechanisms that likely involve both IP3-dependent and -independent pathways. Further progress aimed to clarify the complex, dynamic machinery responsible for astrocytic Ca2+ activity at microdomains is a crucial step in our understanding of the astrocyte role in brain function and may also reveal astrocytes as novel therapeutic targets for different brain diseases. Here, we review the most recent studies that improve our mechanistic understanding of the essential features of astrocyte Ca2+ microdomains.

Cellular and molecular mechanisms of new onset seizure generation.

New onset epilepsy and seizures are common neurological disorders in aged people, second only to stroke and dementia. They are frequently related to other pathological conditions including stroke, trauma, tumors and neurological diseases whereas in about one-third of cases the origin is unknown. Besides the origin, the cellular and molecular events that suddenly trigger seizures are poorly defined. Using an acute model of seizure generation that better resembles new onset seizures, we studied GABAergic interneurons and astrocytes during seizure generation. We found that seizures are preceded by a GABAergic rhythmic hyperactivity that synchronizes pyramidal neurons by inducing a rebound spiking that favors seizures' onset. Furthermore, the intense activity in GABAergic interneurons evokes Ca2+ elevations in astrocytes that, by releasing glutamate, further excite neuronal network. Elucidating the cellular and molecular events that generate seizures may reveal new targets for treatment of new onset seizures and epilepsy.

Correction to: Optogenetic Interneuron Stimulation and Calcium Imaging in Astrocytes.

Figures 1 and 2 were inadvertently switched during the production and this has been corrected so the figures appear in the proper order.

Optogenetic Interneuron Stimulation and Calcium Imaging in Astrocytes.

In brain networks, neurons are constantly involved in a dynamic interaction with the other cell populations and, particularly, with astrocytes, the most abundant glial cells in the brain. Astrocytes respond to neurotransmitters with Ca2+ elevations which represent a key event in the modulation of local brain circuits played by these glial cells. Due to technical limitations, the study of Ca2+ signal dynamics in astrocytes has focused for decades almost exclusively on somatic and perisomatic regions. Accordingly, Ca2+ signal in astrocytic fine protrusions, which are in close contact with the synapse, has been poorly investigated. Over the last years, the diffusion of novel tools such as the viral vector gene delivery of genetically encoded Ca2+ indicators (GECI), the optogenetics, and multiphoton laser scanning microscopy has boosted significantly our capability to study astrocytic Ca2+ signals in the different subcellular compartments. Here we report a protocol that combines these techniques to study astrocyte Ca2+ signaling in response to somatostatin (SST)-expressing interneurons, one of the main classes of GABAergic inhibitory interneurons.

Dynamic interactions between GABAergic and astrocytic networks.

Brain network activity derives from the concerted action of different cell populations. Together with interneurons, astrocytes play fundamental roles in shaping the inhibition in brain circuitries and modulating neuronal transmission. In this review, we summarize past and recent findings that reveal in neural networks the importance of the interaction between GABAergic signaling and astrocytes and discuss its physiological and pathological relevance.

mGlu1 Receptors Monopolize the Synaptic Control of Cerebellar Purkinje Cells by Epigenetically Down-Regulating mGlu5 Receptors.

In cerebellar Purkinje cells (PCs) type-1 metabotropic glutamate (mGlu1) receptors play a key role in motor learning and drive the refinement of synaptic innervation during postnatal development. The cognate mGlu5 receptor is absent in mature PCs and shows low expression levels in the adult cerebellar cortex. Here we found that mGlu5 receptors were heavily expressed by PCs in the early postnatal life, when mGlu1α receptors were barely detectable. The developmental decline of mGlu5 receptors coincided with the appearance of mGlu1α receptors in PCs, and both processes were associated with specular changes in CpG methylation in the corresponding gene promoters. It was the mGlu1 receptor that drove the elimination of mGlu5 receptors from PCs, as shown by data obtained with conditional mGlu1α receptor knockout mice and with targeted pharmacological treatments during critical developmental time windows. The suppressing activity of mGlu1 receptors on mGlu5 receptor was maintained in mature PCs, suggesting that expression of mGlu1α and mGlu5 receptors is mutually exclusive in PCs. These findings add complexity to the the finely tuned mechanisms that regulate PC biology during development and in the adult life and lay the groundwork for an in-depth analysis of the role played by mGlu5 receptors in PC maturation.

Interneuron-specific signaling evokes distinctive somatostatin-mediated responses in adult cortical astrocytes.

The signaling diversity of GABAergic interneurons to post-synaptic neurons is crucial to generate the functional heterogeneity that characterizes brain circuits. Whether this diversity applies to other brain cells, such as the glial cells astrocytes, remains unexplored. Using optogenetics and two-photon functional imaging in the adult mouse neocortex, we here reveal that parvalbumin- and somatostatin-expressing interneurons, two key interneuron classes in the brain, differentially signal to astrocytes inducing weak and robust GABAB receptor-mediated Ca2+ elevations, respectively. Furthermore, the astrocyte response depresses upon parvalbumin interneuron repetitive stimulations and potentiates upon somatostatin interneuron repetitive stimulations, revealing a distinguished astrocyte plasticity. Remarkably, the potentiated response crucially depends on the neuropeptide somatostatin, released by somatostatin interneurons, which activates somatostatin receptors at astrocytic processes. Our study unveils, in the living brain, a hitherto unidentified signaling specificity between interneuron subtypes and astrocytes opening a new perspective into the role of astrocytes as non-neuronal components of inhibitory circuits.

Fast spiking interneuron control of seizure propagation in a cortical slice model of focal epilepsy.

In different animal models of focal epilepsy, seizure-like ictal discharge propagation is transiently opposed by feedforward inhibition. The specific cellular source of this signal and the mechanism by which inhibition ultimately becomes ineffective are, however, undefined. We used a brain slice model to study how focal ictal discharges that were repetitively evoked from the same site, and at precise times, propagate across the cortex. We used Ca(2+) imaging and simultaneous single/dual cell recordings from pyramidal neurons (PyNs) and different classes of interneurons in rodents, including G42 and GIN transgenic mice expressing the green fluorescence protein in parvalbumin (Pv)-fast spiking (FS) and somatostatin (Som) interneurons, respectively. We found that these two classes of interneurons fired intensively shortly after ictal discharge generation at the focus. The inhibitory barrages that were recorded in PyNs occurred in coincidence with Pv-FS, but not with Som interneuron burst discharges. Furthermore, the strength of inhibitory barrages increased or decreased in parallel with increased or decreased firing in Pv-FS interneurons but not in Som interneurons. A firing impairment of Pv-FS interneurons caused by a membrane depolarization was found to precede ictal discharge onset in neighbouring pyramidal neurons. This event may account for the collapse of local inhibition that allows spatially defined clusters of PyNs to be recruited into propagating ictal discharges. Our study demonstrates that Pv-FS interneurons are a major source of the inhibitory barrages that oppose ictal discharge propagation and raises the possibility that targeting Pv-FS interneurons represents a new therapeutic strategy to prevent the generalization of human focal seizures.

An excitatory loop with astrocytes contributes to drive neurons to seizure threshold.

Seizures in focal epilepsies are sustained by a highly synchronous neuronal discharge that arises at restricted brain sites and subsequently spreads to large portions of the brain. Despite intense experimental research in this field, the earlier cellular events that initiate and sustain a focal seizure are still not well defined. Their identification is central to understand the pathophysiology of focal epilepsies and to develop new pharmacological therapies for drug-resistant forms of epilepsy. The prominent involvement of astrocytes in ictogenesis was recently proposed. We test here whether a cooperation between astrocytes and neurons is a prerequisite to support ictal (seizure-like) and interictal epileptiform events. Simultaneous patch-clamp recording and Ca2+ imaging techniques were performed in a new in vitro model of focal seizures induced by local applications of N-methyl-D-aspartic acid (NMDA) in rat entorhinal cortex slices. We found that a Ca2+ elevation in astrocytes correlates with both the initial development and the maintenance of a focal, seizure-like discharge. A delayed astrocyte activation during ictal discharges was also observed in other models (including the whole in vitro isolated guinea pig brain) in which the site of generation of seizure activity cannot be precisely monitored. In contrast, interictal discharges were not associated with Ca2+ changes in astrocytes. Selective inhibition or stimulation of astrocyte Ca2+ signalling blocked or enhanced, respectively, ictal discharges, but did not affect interictal discharge generation. Our data reveal that neurons engage astrocytes in a recurrent excitatory loop (possibly involving gliotransmission) that promotes seizure ignition and sustains the ictal discharge. This neuron-astrocyte interaction may represent a novel target to develop effective therapeutic strategies to control seizures.

Glutamate-mediated astrocyte-to-neuron signalling in the rat dorsal horn.

By releasing neuroactive agents, including proinflammatory cytokines, prostaglandins and neurotrophins, microglia and astrocytes are proposed to be involved in nociceptive transmission, especially in conditions of persistent, pathological pain. The specific action on dorsal horn neurons of agents released from astrocytes, such as glutamate, has been, however, poorly investigated. By using patch-clamp and confocal microscope calcium imaging techniques in rat spinal cord slices, we monitored the activity of dorsal horn lamina II neurons following astrocyte activation. Results obtained revealed that stimuli that triggered Ca(2+) elevations in astrocytes, such as the purinergic receptor agonist BzATP and low extracellular Ca(2+), induce in lamina II neurons slow inward currents (SICs). Similarly to SICs triggered by astrocytic glutamate in neurons from other central nervous system regions, these currents (i) are insensitive to tetrodotoxin (TTX), (ii) are blocked by the NMDA receptor (NMDAR) antagonist d-AP5, (iii) lack an AMPA component, and (iv) have slow rise and decay times. Ca(2+) imaging also revealed that astrocytic glutamate evokes NMDAR-mediated episodes of synchronous activity in groups of substantia gelatinosa neurons. Importantly, in a model of peripheral inflammation, the development of thermal hyperalgesia and mechanical allodynia was accompanied by a significant increase of spontaneous SICs in dorsal horn neurons. The NMDAR-mediated astrocyte-to-neuron signalling thus represents a novel pathway that may contribute to the control of central sensitization in pathological pain.

Presynaptic functional trkB receptors mediate the release of excitatory neurotransmitters from primary afferent terminals in lamina II (substantia gelatinosa) of postnatal rat spinal cord.

A subset of primary sensory neurons produces BDNF, which is implicated in control of nociceptive neurotransmission. We previously localized full-length trkB receptors on their terminals within lamina II. To functionally study these receptors, we here employed patch-clamp recordings, calcium imaging and immunocytochemistry on slices from 8-12 days post-natal rats. In this preparation, BDNF (100-500 ng/mL) enhances the release of sensory neurotransmitters (glutamate, substance P, CGRP) in lamina II by acting on trkB receptors expressed by primary afferent fibers of the peptidergic nociceptive type (PN-PAFs). Effect was blocked by trk antagonist K252a or anti-trkB antibody clone 47. A pre-synaptic mechanism was demonstrated after (i) patch-clamp recordings where the neurotrophin induced a significant increase in frequency, but not amplitude, of AMPA-mediated mEPSCs, (ii) real time calcium imaging, where sustained application of BDNF evoked an intense response in up to 57% lamina II neurons with a significant frequency rise. Antagonists of ionotropic glutamate receptors and NK(1) receptors completely inhibited the calcium response to BDNF. Reduction of CGRP (a specific marker of PN-PAFs) and substance P content in dorsal horn following BDNF preincubation, and analysis of the calcium response after depletion with capsaicin, confirmed that the neurotrophin presynaptically enhanced neurotransmitter release from PN-PAFs. This is the first demonstration that trkB receptors expressed by PN-PAF terminals in lamina II are functional during postnatal development. Implications of this finding are discussed considering that BDNF can be released by these same terminals and microglia, a fraction of which (as shown here) contains BDNF also in unactivated state.

Synaptobrevin2-expressing vesicles in rat astrocytes: insights into molecular characterization, dynamics and exocytosis.

The SNARE-dependent exocytosis of glutamate-containing vesicles in astrocytes is increasingly viewed as an important signal at the basis of the astrocyte-to-neurone communication system in the brain. Here we provide further insights into the molecular features and dynamics of vesicles in cultured astrocytes. We found that immunoisolated synaptobrevin2 vesicles are clear vesicles quite heterogenous in size and contain the vesicular glutamate transporter v-Glut-2. Moreover, they are immunopositive for synaptotagmin IV, for AMPA receptor subunits GluR2,3 and, to a lesser extent, for GluR1. We also provide direct evidence for the functional expression of v-Glut-2 in astrocytes and demonstrate that synaptobrevin2-positive vesicles can specifically take up (3H)L-glutamate via a bafilomycin-sensitive mechanism. Finally, by time lapse confocal microscopy, we show that a subpopulation of vesicles (tagged with a synaptobrevin2-EGFP chimera) is highly mobile and can fuse with the plasma membrane, preferentially at the level of the astrocyte processes, in a Ca2+-dependent manner. These latter observations, together with the evidence reported here for the expression of functional v-Glut-2 in synaptobrevin2-positive vesicles, provide a molecular basis for regulated exocytosis in astrocyte.

Glutamate-mediated cytosolic calcium oscillations regulate a pulsatile prostaglandin release from cultured rat astrocytes.

The synaptic release of glutamate evokes in astrocytes periodic increases in [Ca2+]i, due to the activation of metabotropic glutamate receptors (mGluRs). The frequency of these [Ca2+]i oscillations is controlled by the level of neuronal activity, indicating that they represent a specific, frequency-coded signalling system of neuron-to-astrocyte communication. We recently found that neuronal activity-dependent [Ca2+]i oscillations in astrocytes are the main signal that regulates the coupling between neuronal activity and blood flow, the so-called functional hyperaemia. Prostaglandins play a major role in this fundamental phenomenon in brain function, but little is known about a possible link between [Ca2+]i oscillations and prostaglandin release from astrocytes. To investigate whether [Ca2+]i oscillations regulate the release of vasoactive prostaglandins, such as the potent vasodilator prostaglandin E2 (PGE2), from astrocytes, we plated wild-type human embryonic kidney (HEK)293 cells, which respond constitutively to PGE2 with [Ca2+]i elevations, onto cultured astrocytes, and used them as biosensors of prostaglandin release. After loading the astrocyte-HEK cell co-cultures with the calcium indicator Indo-1, confocal microscopy revealed that mGluR-mediated [Ca2+]i oscillations triggered spatially and temporally coordinated [Ca2+]i increases in the sensor cells. This response was absent in a clone of HEK cells that are unresponsive to PGE2, and recovered after transfection with the InsP3-linked prostanoid receptor EP1. We conclude that [Ca2+]i oscillations in astrocytes regulate prostaglandin releases that retain the oscillatory behaviour of the [Ca2+]i changes. This finely tuned release of PGE2 from astrocytes provides a coherent mechanistic background for the role of these glial cells in functional hyperaemia.

Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation.

The cellular mechanisms underlying functional hyperemia--the coupling of neuronal activation to cerebral blood vessel responses--are not yet known. Here we show in rat cortical slices that the dilation of arterioles triggered by neuronal activity is dependent on glutamate-mediated [Ca(2+)](i) oscillations in astrocytes. Inhibition of these Ca(2+) responses resulted in the impairment of activity-dependent vasodilation, whereas selective activation--by patch pipette--of single astrocytes that were in contact with arterioles triggered vessel relaxation. We also found that a cyclooxygenase product is centrally involved in this astrocyte-mediated control of arterioles. In vivo blockade of glutamate-mediated [Ca(2+)](i) elevations in astrocytes reduced the blood flow increase in the somatosensory cortex during contralateral forepaw stimulation. Taken together, our findings show that neuron-to-astrocyte signaling is a key mechanism in functional hyperemia.

Calcium oscillations encoding neuron-to-astrocyte communication.

The observation that the excitatory neurotransmitter glutamate released from presynaptic terminals can activate, beside the post-synaptic neuron, the glial cell astrocyte, stimulated glial cell research like no other event since the recognition in the 1980s that astrocytes can express on their membrane many receptors for classical neurotransmitters. The properties and the functional role(s) of such a neuron-to-astrocyte signaling have now become the focus of intense research in neurobiology. Indeed, a growing body of evidence has recently highlighted the ability of astrocytes to work as sophisticated detectors of synaptic activity: by changing the frequency of [Ca(2+)](i) oscillations evoked by the synaptic release of glutamate, these cells display the remarkable capacity to discriminate between different levels and patterns of synaptic activity. Furthermore, the observation that astrocytes increase the frequency of [Ca(2+)](i) oscillations in response to repetitive episodes of high neuronal activity challenges the common concept that memory function in the brain is an exclusive property of neuronal cells. Glutamate-mediated [Ca(2+)](i) elevations can also trigger in astrocytes the release of glutamate that can ultimately affect neuronal transmission. Given the wide role played by glutamate in brain physiology, our view on how the brain operates needs now to be revised taking into account the bi-directional, glutamatergic communication between neurons and astrocytes.