Dysfunctional serotonergic neuron-astrocyte signaling in depressive-like states.

Astrocytes play crucial roles in brain homeostasis and are regulatory elements of neuronal and synaptic physiology. Astrocytic alterations have been found in Major Depressive Disorder (MDD) patients; however, the consequences of astrocyte Ca2+ signaling in MDD are poorly understood. Here, we found that corticosterone-treated juvenile mice (Cort-mice) showed altered astrocytic Ca2+ dynamics in mPFC both in resting conditions and during social interactions, in line with altered mice behavior. Additionally, Cort-mice displayed reduced serotonin (5-HT)-mediated Ca2+ signaling in mPFC astrocytes, and aberrant 5-HT-driven synaptic plasticity in layer 2/3 mPFC neurons. Downregulation of astrocyte Ca2+ signaling in naïve animals mimicked the synaptic deficits found in Cort-mice. Remarkably, boosting astrocyte Ca2+ signaling with Gq-DREADDS restored to the control levels mood and cognitive abilities in Cort-mice. This study highlights the important role of astrocyte Ca2+ signaling for homeostatic control of brain circuits and behavior, but also reveals its potential therapeutic value for depressive-like states.

Reversible silencing of endogenous receptors in intact brain tissue using 2-photon pharmacology.

The physiological activity of proteins is often studied with loss-of-function genetic approaches, but the corresponding phenotypes develop slowly and can be confounding. Photopharmacology allows direct, fast, and reversible control of endogenous protein activity, with spatiotemporal resolution set by the illumination method. Here, we combine a photoswitchable allosteric modulator (alloswitch) and 2-photon excitation using pulsed near-infrared lasers to reversibly silence metabotropic glutamate 5 (mGlu5) receptor activity in intact brain tissue. Endogenous receptors can be photoactivated in neurons and astrocytes with pharmacological selectivity and with an axial resolution between 5 and 10 µm. Thus, 2-photon pharmacology using alloswitch allows investigating mGlu5-dependent processes in wild-type animals, including synaptic formation and plasticity, and signaling pathways from intracellular organelles.

A roadmap to integrate astrocytes into Systems Neuroscience.

Systems neuroscience is still mainly a neuronal field, despite the plethora of evidence supporting the fact that astrocytes modulate local neural circuits, networks, and complex behaviors. In this article, we sought to identify which types of studies are necessary to establish whether astrocytes, beyond their well-documented homeostatic and metabolic functions, perform computations implementing mathematical algorithms that sub-serve coding and higher-brain functions. First, we reviewed Systems-like studies that include astrocytes in order to identify computational operations that these cells may perform, using Ca2+ transients as their encoding language. The analysis suggests that astrocytes may carry out canonical computations in a time scale of subseconds to seconds in sensory processing, neuromodulation, brain state, memory formation, fear, and complex homeostatic reflexes. Next, we propose a list of actions to gain insight into the outstanding question of which variables are encoded by such computations. The application of statistical analyses based on machine learning, such as dimensionality reduction and decoding in the context of complex behaviors, combined with connectomics of astrocyte-neuronal circuits, is, in our view, fundamental undertakings. We also discuss technical and analytical approaches to study neuronal and astrocytic populations simultaneously, and the inclusion of astrocytes in advanced modeling of neural circuits, as well as in theories currently under exploration such as predictive coding and energy-efficient coding. Clarifying the relationship between astrocytic Ca2+ and brain coding may represent a leap forward toward novel approaches in the study of astrocytes in health and disease.

GABAergic-astrocyte signaling: A refinement of inhibitory brain networks.

Interneurons play a critical role in precise control of network operation. Indeed, higher brain capabilities such as working memory, cognitive flexibility, attention, or social interaction rely on the action of GABAergic interneurons. Evidence from excitatory neurons and synapses has revealed astrocytes as integral elements of synaptic transmission. However, GABAergic interneurons can also engage astrocyte signaling; therefore, it is tempting to speculate about different scenarios where, based on particular interneuron cell type, GABAergic-astrocyte interplay would be involved in diverse outcomes of brain function. In this review, we will highlight current data supporting the existence of dynamic GABAergic-astrocyte communication and its impact on the inhibitory-regulated brain responses, bringing new perspectives on the ways astrocytes might contribute to efficient neuronal coding.

Melanopsin for precise optogenetic activation of astrocyte-neuron networks.

Optogenetics has been widely expanded to enhance or suppress neuronal activity and it has been recently applied to glial cells. Here, we have used a new approach based on selective expression of melanopsin, a G-protein-coupled photopigment, in astrocytes to trigger Ca2+ signaling. Using the genetically encoded Ca2+ indicator GCaMP6f and two-photon imaging, we show that melanopsin is both competent to stimulate robust IP3-dependent Ca2+ signals in astrocyte fine processes, and to evoke an ATP/Adenosine-dependent transient boost of hippocampal excitatory synaptic transmission. Additionally, under low-frequency light stimulation conditions, melanopsin-transfected astrocytes can trigger long-term synaptic changes. In vivo, melanopsin-astrocyte activation enhances episodic-like memory, suggesting melanopsin as an optical tool that could recapitulate the wide range of regulatory actions of astrocytes on neuronal networks in behaving animals. These results describe a novel approach using melanopsin as a precise trigger for astrocytes that mimics their endogenous G-protein signaling pathways, and present melanopsin as a valuable optical tool for neuron-glia studies.

Neuron-astrocyte signaling is preserved in the aging brain.

Astrocytes play crucial roles in brain homeostasis and are emerging as regulatory elements of neuronal and synaptic physiology by responding to neurotransmitters with Ca2+ elevations and releasing gliotransmitters that activate neuronal receptors. Aging involves neuronal and astrocytic alterations, being considered risk factor for neurodegenerative diseases. Most evidence of the astrocyte-neuron signaling is derived from studies with young animals; however, the features of astrocyte-neuron signaling in adult and aging brain remain largely unknown. We have investigated the existence and properties of astrocyte-neuron signaling in physiologically and pathologically aging mouse hippocampal and cortical slices at different lifetime points (0.5 to 20 month-old animals). We found that astrocytes preserved their ability to express spontaneous and neurotransmitter-dependent intracellular Ca2+ signals from juvenile to aging brains. Likewise, resting levels of gliotransmission, assessed by neuronal NMDAR activation by glutamate released from astrocytes, were largely preserved with similar properties in all tested age groups, but DHPG-induced gliotransmission was reduced in aged mice. In contrast, gliotransmission was enhanced in the APP/PS1 mouse model of Alzheimer's disease, indicating a dysregulation of astrocyte-neuron signaling in pathological conditions. Disruption of the astrocytic IP3 R2 mediated-signaling, which is required for neurotransmitter-induced astrocyte Ca2+ signals and gliotransmission, boosted the progression of amyloid plaque deposits and synaptic plasticity impairments in APP/PS1 mice at early stages of the disease. Therefore, astrocyte-neuron interaction is a fundamental signaling, largely conserved in the adult and aging brain of healthy animals, but it is altered in Alzheimer's disease, suggesting that dysfunctions of astrocyte Ca2+ physiology may contribute to this neurodegenerative disease. GLIA 2017 GLIA 2017;65:569-580.

Endocannabinoids Induce Lateral Long-Term Potentiation of Transmitter Release by Stimulation of Gliotransmission.

Endocannabinoids (eCBs) play key roles in brain function, acting as modulatory signals in synaptic transmission and plasticity. They are recognized as retrograde messengers that mediate long-term synaptic depression (LTD), but their ability to induce long-term potentiation (LTP) is poorly known. We show that eCBs induce the long-term enhancement of transmitter release at single hippocampal synapses through stimulation of astrocytes when coincident with postsynaptic activity. This LTP requires the coordinated activity of the 3 elements of the tripartite synapse: 1) eCB-evoked astrocyte calcium signal that stimulates glutamate release; 2) postsynaptic nitric oxide production; and 3) activation of protein kinase C and presynaptic group I metabotropic glutamate receptors, whose location at presynaptic sites was confirmed by immunoelectron microscopy. Hence, while eCBs act as retrograde signals to depress homoneuronal synapses, they serve as lateral messengers to induce LTP in distant heteroneuronal synapses through stimulation of astrocytes. Therefore, eCBs can trigger LTP through stimulation of astrocyte-neuron signaling, revealing novel cellular mechanisms of eCB effects on synaptic plasticity.

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 Ca²âº 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 Ca²âº 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 Ca²âº elevations and LTP are absent in IP3R2 knock-out mice. Downregulating astrocyte Ca²âº signal by loading astrocytes with BAPTA or GDPßS also prevents LTP, which is restored by simultaneous astrocyte Ca²âº uncaging and postsynaptic depolarization. Therefore, cholinergic-induced LTP requires astrocyte Ca²âº 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 Ca²âº signal simultaneously evoked by cholinergic activity. Therefore, the astrocyte Ca²âº signal is necessary for cholinergic-induced synaptic plasticity, indicating that astrocytes are directly involved in brain storage information.

Nucleus basalis-enabled stimulus-specific plasticity in the visual cortex is mediated by astrocytes.

Although cholinergic innervation of the cortex by the nucleus basalis (NB) is known to modulate cortical neuronal responses and instruct cortical plasticity, little is known about the underlying cellular mechanisms. Using cell-attached recordings in vivo, we demonstrate that electrical stimulation of the NB, paired with visual stimulation, can induce significant potentiation of visual responses in excitatory neurons of the primary visual cortex in mice. We further show with in vivo two-photon calcium imaging, ex vivo calcium imaging, and whole-cell recordings that this pairing-induced potentiation is mediated by direct cholinergic activation of primary visual cortex astrocytes via muscarinic AChRs. The potentiation is absent in conditional inositol 1,4,5 trisphosphate receptor type 2 KO mice, which lack astrocyte calcium activation, and is stimulus-specific, because pairing NB stimulation with a specific visual orientation reveals a highly selective potentiation of responses to the paired orientation compared with unpaired orientations. Collectively, these findings reveal a unique and surprising role for astrocytes in NB-induced stimulus-specific plasticity in the cerebral cortex.

Astrocyte calcium signal and gliotransmission in human brain tissue.

Brain function is recognized to rely on neuronal activity and signaling processes between neurons, whereas astrocytes are generally considered to play supportive roles for proper neuronal function. However, accumulating evidence indicates that astrocytes sense and control neuronal and synaptic activity, indicating that neuron and astrocytes reciprocally communicate. While this evidence has been obtained in experimental animal models, whether this bidirectional signaling between astrocytes and neurons occurs in human brain remains unknown. We have investigated the existence of astrocyte-neuron communication in human brain tissue, using electrophysiological and Ca(2+) imaging techniques in slices of the cortex and hippocampus obtained from biopsies from epileptic patients. Cortical and hippocampal human astrocytes displayed spontaneous Ca(2+) elevations that were independent of neuronal activity. Local application of transmitter receptor agonists or nerve electrical stimulation transiently elevated Ca(2+) in astrocytes, indicating that human astrocytes detect synaptic activity and respond to synaptically released neurotransmitters, suggesting the existence of neuron-to-astrocyte communication in human brain tissue. Electrophysiological recordings in neurons revealed the presence of slow inward currents (SICs) mediated by NMDA receptor activation. The frequency of SICs increased after local application of ATP that elevated astrocyte Ca(2+). Therefore, human astrocytes are able to release the gliotransmitter glutamate, which affect neuronal excitability through activation of NMDA receptors in neurons. These results reveal the existence of reciprocal signaling between neurons and astrocytes in human brain tissue, indicating that astrocytes are relevant in human neurophysiology and are involved in human brain function.

Early cortical GABAergic interneurons determine the projection patterns of L4 excitatory neurons.

During cerebral cortex development, excitatory pyramidal neurons (PNs) establish specific projection patterns while receiving inputs from GABAergic inhibitory interneurons (INs). Whether these inhibitory inputs can shape PNs' projection patterns is, however, unknown. While layer 4 (L4) PNs of the primary somatosensory (S1) cortex are all born as long-range callosal projection neurons (CPNs), most of them acquire local connectivity upon activity-dependent elimination of their interhemispheric axons during postnatal development. Here, we demonstrate that precise developmental regulation of inhibition is key for the retraction of S1L4 PNs' callosal projections. Ablation of somatostatin INs leads to premature inhibition from parvalbumin INs onto S1L4 PNs and prevents them from acquiring their barrel-restricted local connectivity pattern. As a result, adult S1L4 PNs retain interhemispheric projections responding to tactile stimuli, and the mice lose whisker-based texture discrimination. Overall, we show that temporally ordered IN activity during development is key to shaping local ipsilateral S1L4 PNs' projection pattern, which is required for fine somatosensory processing.

Mechanical and Functional Responses in Astrocytes under Alternating Deformation Modes Using Magneto-Active Substrates.

This work introduces NeoMag, a system designed to enhance cell mechanics assays in substrate deformation studies. NeoMag uses multidomain magneto-active materials to mechanically actuate the substrate, transmitting reversible mechanical cues to cells. The system boasts full flexibility in alternating loading substrate deformation modes, seamlessly adapting to both upright and inverted microscopes. The multidomain substrates facilitate mechanobiology assays on 2D and 3D cultures. The integration of the system with nanoindenters allows for precise evaluation of cellular mechanical properties under varying substrate deformation modes. The system is used to study the impact of substrate deformation on astrocytes, simulating mechanical conditions akin to traumatic brain injury and ischemic stroke. The results reveal local heterogeneous changes in astrocyte stiffness, influenced by the orientation of subcellular regions relative to substrate strain. These stiffness variations, exceeding 50% in stiffening and softening, and local deformations significantly alter calcium dynamics. Furthermore, sustained deformations induce actin network reorganization and activate Piezo1 channels, leading to an initial increase followed by a long-term inhibition of calcium events. Conversely, fast and dynamic deformations transiently activate Piezo1 channels and disrupt the actin network, causing long-term cell softening. These findings unveil mechanical and functional alterations in astrocytes during substrate deformation, illustrating the multiple opportunities this technology offers.

The CB1 receptor interacts with cereblon and drives cereblon deficiency-associated memory shortfalls.

Cereblon/CRBN is a substrate-recognition component of the Cullin4A-DDB1-Roc1 E3 ubiquitin ligase complex. Destabilizing mutations in the human CRBN gene cause a form of autosomal recessive non-syndromic intellectual disability (ARNSID) that is modelled by knocking-out the mouse Crbn gene. A reduction in excitatory neurotransmission has been proposed as an underlying mechanism of the disease. However, the precise factors eliciting this impairment remain mostly unknown. Here we report that CRBN molecules selectively located on glutamatergic neurons are necessary for proper memory function. Combining various in vivo approaches, we show that the cannabinoid CB1 receptor (CB1R), a key suppressor of synaptic transmission, is overactivated in CRBN deficiency-linked ARNSID mouse models, and that the memory deficits observed in these animals can be rescued by acute CB1R-selective pharmacological antagonism. Molecular studies demonstrated that CRBN interacts physically with CB1R and impairs the CB1R-Gi/o-cAMP-PKA pathway in a ubiquitin ligase-independent manner. Taken together, these findings unveil that CB1R overactivation is a driving mechanism of CRBN deficiency-linked ARNSID and anticipate that the antagonism of CB1R could constitute a new therapy for this orphan disease.

Brain IGF-I regulates LTP, spatial memory, and sexual dimorphic behavior.

Insulin-like growth factor-I (IGF-I) exerts multiple actions, yet the role of IGF-I from different sources is poorly understood. Here, we explored the functional and behavioral consequences of the conditional deletion of Igf-I in the nervous system (Igf-I Δ/Δ), and demonstrated that long-term potentiation was impaired in hippocampal slices. Moreover, Igf-I Δ/Δ mice showed spatial memory deficits in the Morris water maze, and the significant sex-dependent differences displayed by Igf-I Ctrl/Ctrl mice disappeared in Igf-I Δ/Δ mice in the open field and rota-rod tests. Brain Igf-I deletion disorganized the granule cell layer of the dentate gyrus (DG), and it modified the relative expressions of GAD and VGLUT1, which are preferentially localized to inhibitory and excitatory presynaptic terminals. Furthermore, Igf-I deletion altered protein modules involved in receptor trafficking, synaptic proteins, and proteins that functionally interact with estrogen and androgen metabolism. Our findings indicate that brain IGF-I is crucial for long-term potentiation, and that it is involved in the regulation of spatial memory and sexual dimorphic behaviors, possibly by maintaining the granule cell layer structure and the stability of synaptic-related protein modules.

Protocol to downregulate GABAergic-astrocyte signaling via astrocyte-selective ablation of GABAB receptor in adult mice.

Here, we present a protocol to selectively downregulate GABAB receptor (GABABR) expression in astrocytes of mouse medial prefrontal cortex (mPFC). We first describe the procedure of surgeries and viral injections. We then detail genetic, histological, and functional characterizations of astrocytic GABABR ablation using RT-PCR, imaging, and behavioral assays. The use of GABAB flox mice can be easily adapted to generate astrocyte-selective GABABR ablation in different brain areas and postnatal stages, leading to local downregulation of GABAergic-astrocyte signaling without developmental issues. For complete details on the use and execution of this protocol, please refer to Mederos et al. (2021).

Glutamate released spontaneously from astrocytes sets the threshold for synaptic plasticity.

Astrocytes exhibit spontaneous calcium oscillations that could induce the release of glutamate as gliotransmitter in rat hippocampal slices. However, it is unknown whether this spontaneous release of astrocytic glutamate may contribute to determining the basal neurotransmitter release probability in central synapses. Using whole-cell recordings and Ca(2+) imaging, we investigated the effects of the spontaneous astrocytic activity on neurotransmission and synaptic plasticity at CA3-CA1 hippocampal synapses. We show here that the metabolic gliotoxin fluorocitrate (FC) reduces the amplitude of evoked excitatory postsynaptic currents and increases the paired-pulse facilitation, mainly due to the reduction of the neurotransmitter release probability and the synaptic potency. FC also decreased intracellular Ca(2+) signalling and Ca(2+) -dependent glutamate release from astrocytes. The addition of glutamine rescued the effects of FC over the synaptic potency; however, the probability of neurotransmitter release remained diminished. The blockage of group I metabotropic glutamate receptors mimicked the effects of FC on the frequency of miniature synaptic responses. In the presence of FC, the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N ',N '-tetra-acetate or group I metabotropic glutamate receptor antagonists, the excitatory postsynaptic current potentiation induced by the spike-timing-dependent plasticity protocol was blocked, and it was rescued by delivering a stronger spike-timing-dependent plasticity protocol. Taken together, these results suggest that spontaneous glutamate release from astrocytes contributes to setting the basal probability of neurotransmitter release via metabotropic glutamate receptor activation, which could be operating as a gain control mechanism that regulates the threshold of long-term potentiation. Therefore, endogenous astrocyte activity provides a novel non-neuronal mechanism that could be critical for transferring information in the central nervous system.

Metabolic Changes in Brain Slices over Time: a Multiplatform Metabolomics Approach.

Brain slice preparations are widely used for research in neuroscience. However, a high-quality preparation is essential and there is no consensus regarding stable parameters that can be used to define the status of the brain slice preparation after its collection at different time points. Thus, it is critical to fully characterize the experimental conditions for ex vivo studies using brain slices for electrophysiological recording. In this study, we used a multiplatform (LC-MS and GC-MS) untargeted metabolomics-based approach to shed light on the metabolome and lipidome changes taking place at different time intervals during the brain slice preparation process. We have found significant modifications in the levels of 300 compounds, including several lipid classes and their derivatives, as well as metabolites involved in the GABAergic pathway and the TCA cycle. All these preparation-dependent changes in the brain biochemistry related to the time interval should be taken into consideration for future studies to facilitate non-biased interpretations of the experimental results.

GABAergic signaling to astrocytes in the prefrontal cortex sustains goal-directed behaviors.

GABA interneurons play a critical role in higher brain functions. Astrocytic glial cells interact with synapses throughout the whole brain and are recognized as regulatory elements of excitatory synaptic transmission. However, it is largely unknown how GABAergic interneurons and astrocytes interact and contribute to stable performance of complex behaviors. Here, we found that genetic ablation of GABAB receptors in medial prefrontal cortex astrocytes altered low-gamma oscillations and firing properties of cortical neurons, which affected goal-directed behaviors. Remarkably, working memory deficits were restored by optogenetic stimulation of astrocytes with melanopsin. Furthermore, melanopsin-activated astrocytes in wild-type mice enhanced the firing rate of cortical neurons and gamma oscillations, as well as improved cognition. Therefore, our work identifies astrocytes as a hub for controlling inhibition in cortical circuits, providing a novel pathway for the behaviorally relevant midrange time-scale regulation of cortical information processing and consistent goal-directed behaviors.

Melanopsin for Time-Controlling Activation of Astrocyte -Neuron Networks.

Melanopsin, a mammalian G-protein-coupled photopigment, is a novel optical tool which enables studying astrocyte-neuron networks. Here, we describe the required guidelines to take advantage of this promising optical tool for functional neuron-glia studies. The selective expression of melanopsin in astrocytes allows triggering astrocytic Ca2+ signaling, changes in synaptic transmission, and modifying behavioral responses.

Sex-dependent calcium hyperactivity due to lysosomal-related dysfunction in astrocytes from APOE4 versus APOE3 gene targeted replacement mice.

BACKGROUND: The apolipoprotein E (APOE) gene exists in three isoforms in humans: APOE2, APOE3 and APOE4. APOE4 causes structural and functional alterations in normal brains, and is the strongest genetic risk factor of the sporadic form of Alzheimer's disease (LOAD). Research on APOE4 has mainly focused on the neuronal damage caused by defective cholesterol transport and exacerbated amyloid-ß and Tau pathology. The impact of APOE4 on non-neuronal cell functions has been overlooked. Astrocytes, the main producers of ApoE in the healthy brain, are building blocks of neural circuits, and Ca2+ signaling is the basis of their excitability. Because APOE4 modifies membrane-lipid composition, and lipids regulate Ca2+ channels, we determined whether APOE4 dysregulates Ca2+signaling in astrocytes. METHODS: Ca2+ signals were recorded in astrocytes in hippocampal slices from APOE3 and APOE4 gene targeted replacement male and female mice using Ca2+ imaging. Mechanistic analyses were performed in immortalized astrocytes. Ca2+ fluxes were examined with pharmacological tools and Ca2+ probes. APOE3 and APOE4 expression was manipulated with GFP-APOE vectors and APOE siRNA. Lipidomics of lysosomal and whole-membranes were also performed. RESULTS: We found potentiation of ATP-elicited Ca2+responses in APOE4 versus APOE3 astrocytes in male, but not female, mice. The immortalized astrocytes modeled the male response, and showed that Ca2+ hyperactivity associated with APOE4 is caused by dysregulation of Ca2+ handling in lysosomal-enriched acidic stores, and is reversed by the expression of APOE3, but not of APOE4, pointing to loss of function due to APOE4 malfunction. Moreover, immortalized APOE4 astrocytes are refractory to control of Ca2+ fluxes by extracellular lipids, and present distinct lipid composition in lysosomal and plasma membranes. CONCLUSIONS: Immortalized APOE4 versus APOE3 astrocytes present: increased Ca2+ excitability due to lysosome dysregulation, altered membrane lipidomes and intracellular cholesterol distribution, and impaired modulation of Ca2+ responses upon changes in extracellular lipids. Ca2+ hyperactivity associated with APOE4 is found in astrocytes from male, but not female, targeted replacement mice. The study suggests that, independently of Aß and Tau pathologies, altered astrocyte excitability might contribute to neural-circuit hyperactivity depending on APOE allele, sex and lipids, and supports lysosome-targeted therapies to rescue APOE4 phenotypes in LOAD.