Our understanding of memory and learning has been largely overshadowed by neurocentric studies, leaving non-neuronal cells out of the equation. The cellular substrate for memory is thought to lie within engrams — ensembles of neurons that activate during learning, whose reactivation leads to recall of the acquired memory. Astrocytes are now taking centre stage in the modulation of memory and other cognitive functions. Contrary to widespread assumptions, these glial cells activate as sparse groups, or ensembles, and reactivation of astrocyte ensembles recruited during learning produces recall. Recent advances using activity-dependent tools to interrogate the roles of astrocytes in memory support a paradigm shift: engrams not only are composed of neurons but also include astrocyte ensembles that activate during learning, forming what we call ‘astroengrams’. Thus, the coordinated activity of neuronal and astrocytic engrams provides an integrated framework to orchestrate memory storage and recall. Recent evidence suggests that astrocytes, through coordinated activation in sparse ensembles, contribute to memory traces — termed ‘astro-neuronal engrams’. In this Perspective, Sánchez Romero and Navarrete discuss supporting evidence for astro-neuronal engrams and how these findings challenge traditional neurocentric models of memory.

ABSTRACT Astrocytes are active players in brain circuits, sensing and responding to neuronal activity, impacting behavior production. Activation of astrocytes triggers intracellular calcium elevations displaying complex spatiotemporal properties. Intracellular calcium activity is thought to underlie synaptic transmission, metabolism, and brain homeostasis modulation. However, the calcium-dependent signaling pathways involved in these processes are poorly understood, representing a critical knowledge gap in this field. To reveal calcium-dependent signaling pathways involved in circuit structure and function, we performed a multi-level analysis of the inositol 1,4,5-triphosphate receptor type 2 knockout (IP3R2 KO) mouse model which lacks somatic calcium elevations specifically in astrocytes. We focused on the hippocampus, a brain region responsible for cognitive function and emotional behaviors. The transcriptomic analysis of hippocampal tissue revealed that the lack of astrocytic somatic calcium causes the differential expression of hundreds of genes. Among these, 76 genes are regulated by the astrocyte-specific Foxo1 transcription factor. This transcription factor is over-expressed in the hippocampal astrocytes of this mouse model and regulates the expression of genes involved in spinogenesis and synaptic coverage. A detailed morphological analysis of hippocampal pyramidal neurons revealed dendrites with a shift to a more immature spine profile. This spine profile shift may underlie previously described a reduction of long-term depression and performance in fear memory tasks observed in this mouse model. Indeed, we confirmed that these mice lacking astrocytic somatic calcium display an enhancement of long-term fear memory. To verify a causal relationship between these structural, synaptic, and behavioral observations, we used a viral approach to induce the over-expression of Foxo1 in hippocampal astrocytes in naïve C57BL/6J mice. This viral-driven over-expression of Foxo1 in astrocytes of the stratum radiatum replicated the shift to an immature spine profile in dendrites of pyramidal neurons crossing the territory of these astrocytes and led to a reduction of long-term depression in the same region. Finally, this manipulation was sufficient to enhance long-term fear memory. The detailed characterization of the mouse model lacking astrocytic somatic calcium revealed that astrocytes modulate hippocampal circuit structure and function through Foxo1 signaling to enhance fear memory.

Cannabis is the most widely used illicit drug, particularly during adolescence. Its consumption disrupts glutamate homeostasis and synaptic plasticity in the nucleus accumbens (NAc). Here, we identify a functionally distinct accumbal astrocytic ensemble, associated with the ventral hippocampus–NAc (vHip→NAc) circuit, that is critical for tetrahydrocannabinol (THC)-induced synaptic and behavioral impairments. We employed the AstroLight tool to selectively manipulate this astrocytic ensemble. Our findings reveal that THC increases astrocytic calcium activity and glutamatergic tone within the NAc. These glutamatergic alterations are absent in astrocyte-specific p38α knockout mice, implicating p38α signaling in astrocyte-mediated plasticity. Astrocytic glutamate release within the vHip→NAc ensemble is required for THC-induced cognitive deficits, and targeted attenuation of THC-induced calcium activity in this ensemble prevents spatial learning and synaptic plasticity impairments. These results highlight a critical role for astrocytic ensembles in shaping behavior and emphasize their potential as therapeutic targets for mitigating the cognitive consequences of THC exposure. Cannabis exposure is a critical concern, particularly during adolescence. This study shows that tetrahydrocannabinol-induced cognitive deficits are mediated by a specific astrocytic ensemble in the nucleus accumbens whose manipulation prevents and reverses these effects.

Cannabis is the most widely used illicit drug, particularly during adolescence, and its consumption disrupts glutamate homeostasis and synaptic plasticity in the nucleus accumbens (NAc). While astrocytic cannabinoid receptors (CB1Rs) are known to modulate synaptic plasticity and behavior, the extent to which specific astrocytic ensembles drive tetrahydrocannabinol (THC)-induced alterations remains unclear. Here, we identify a functionally distinct accumbal astrocytic ensemble, related to ventral hippocampus-NAc (vHip->NAc) circuit, that is critical for THC-induced synaptic and behavioral impairments. Using fiber photometry, we monitored astrocytic calcium and glutamate dynamics within the NAc of wild-type and p38αMAPK−/− (Astrop38α) mice following chronic THC exposure, complemented by electrophysiological and behavioral assessments. Additionally, we employed AstroLight tool to selectively manipulate this astrocytic ensemble. Our findings reveal that: (1) THC increases astrocytic calcium activity and glutamatergic tone within the NAc; (2) the THC-induced glutamatergic alterations are absent in Astrop38α mice, implicating p38αMAPK signaling in astrocyte-mediated plasticity; (3) astrocytic glutamate release within the vHip->NAc ensemble is required for THC-induced cognitive deficits; and (4) targeted attenuation of THC-induced calcium activity in this ensemble prevents both spatial learning and synaptic plasticity impairments. These results highlight a critical role for astrocytic ensembles in shaping behavior and emphasize their potential as therapeutic targets for mitigating the cognitive consequences of THC exposure.

Astrocytes, dynamic cells crucial to brain function, have traditionally been overshadowed by the emphasis on neuronal activity in regulating behavior. Unlike neurons, which are organized into ensembles that encode different brain representations, astrocytes have long been considered a homogeneous population. This is partly because of the lack of tools available to map and manipulate specific subsets of astrocytes based on their functional activity, obscuring the extent of their specialization in circuits. Here, using AstroLight, a tool that translates astrocytic activity-mediated calcium signals into gene expression in a light-dependent manner, we have identified an astrocytic ensemble, a functionally specified subset of astrocytes that emerges upon activity during cue-motivated behaviors in the nucleus accumbens, an integrator hub in the reward system. Furthermore, through gain-of-function and loss-of-function manipulations, we demonstrate that this ensemble is essential for modulating cue–reward associations. These findings highlight the specialization of astrocytes into ensembles and their fine-tuning role in shaping salient behavior. Using the AstroLight system, the authors reveal that astrocytic ensembles in the nucleus accumbens regulate cue-motivated reward behavior, demonstrating that selective manipulation of tagged astrocytes can influence specific behavioral choices.

Astrocytes are active cells involved in brain function through the bidirectional communication with neurons, in which astrocyte calcium plays a crucial role. Synaptically evoked calcium increases can be localized to independent subcellular domains or expand to the entire cell, i.e., calcium surge. Because a single astrocyte may contact ~100,000 synapses, the control of the intracellular calcium signal propagation may have relevant consequences on brain function. Yet, the properties governing the spatial dynamics of astrocyte calcium remains poorly defined. Imaging subcellular responses of cortical astrocytes to sensory stimulation in mice, we show that sensory-evoked astrocyte calcium responses originated and remained localized in domains of the astrocytic arborization, but eventually propagated to the entire cell if a spatial threshold of >23% of the arborization being activated was surpassed. Using Itpr2 -/- mice, we found that type-2 IP 3 receptors were necessary for the generation of astrocyte calcium surge. We finally show using in situ electrophysiological recordings that the spatial threshold of the astrocyte calcium signal consequently determined the gliotransmitter release. Present results reveal a fundamental property of astrocyte physiology, i.e., a spatial threshold for astrocyte calcium propagation, which depends on astrocyte intrinsic properties and governs astrocyte integration of local synaptic activity and subsequent neuromodulation.

The medial prefrontal cortex (mPFC) is involved in cognitive functions such as working memory. Astrocytic cannabinoid type 1 receptor (CB1R) induces cytosolic calcium (Ca2+) concentration changes with an impact on neuronal function. mPFC astrocytes also express adenosine A1 and A2A receptors (A1R, A2AR), being unknown the crosstalk between CB1R and adenosine receptors in these cells. We show here that a further level of regulation of astrocyte Ca2+ signaling occurs through CB1R-A2AR or CB1R-A1R heteromers that ultimately impact mPFC synaptic plasticity. CB1R-mediated Ca2+ transients increased and decreased when A1R and A2AR were activated, respectively, unveiling adenosine receptors as modulators of astrocytic CB1R. CB1R activation leads to an enhancement of long-term potentiation (LTP) in the mPFC, under the control of A1R but not of A2AR. Notably, in IP3R2KO mice, that do not show astrocytic Ca2+ level elevations, CB1R activation decreases LTP, which is not modified by A1R or A2AR. The present work suggests that CB1R has a homeostatic role on mPFC LTP, under the control of A1R, probably due to physical crosstalk between these receptors in astrocytes that ultimately alters CB1R Ca2+ signaling.

While neurons have traditionally been considered the primary players in information processing, the role of astrocytes in this mechanism has largely been overlooked due to experimental constraints. In this review, we propose that astrocytic ensembles are active working groups that contribute significantly to animal conduct and suggest that studying the maps of these ensembles in conjunction with neurons is crucial for a more comprehensive understanding of behavior. We also discuss available methods for studying astrocytes and argue that these ensembles, complementarily with neurons, code and integrate complex behaviors, potentially specializing in concrete functions.

Astrocytes, dynamic cells that play a crucial role in brain function, have traditionally been overshadowed by the emphasis on neuronal activity in regulating behavior. This is in part due to the lack of tools to experimentally map and manipulate specific subsets of astrocytes based on their functional activity, which hampers our understanding of how astrocyte networks contribute to the organization of behavior. Here, using a tool that can translate astrocytic activity–mediated calcium signals into gene expression in a light-dependent manner, we have identified an active astrocytic ensemble -which refers to active astrocytes working together- associated with cue-motivated behavior in the Nucleus Accumbens (NAc), a central hub in the reward system. We mapped behavior-related astrocytic ensemble activity to the ventral and posterior regions of the NAc. Furthermore, we demonstrated that direct activation of this specific astrocytic ensemble through optogenetics was sufficient to retrieve the direction of labeled reward behavior. Overall, these findings uncover the fine-tuning role of astrocytic ensembles in shaping salient behavior.

Drug use is a growing problem in actual society. Usually, the first experience with drugs takes place during adolescence, being cannabis the most used illicit drug. Although cannabis could be considered a harmless drug, we are beginning to appreciate its consequences. Chronic exposure to addictive drugs has shown to imbalance glutamate homeostasis in Nucleus Accumbens (NAc), altering plasticity mechanisms such as long-term depression. Therefore, it is crucial to elucidate the mechanisms underlying these alterations and how to reverse them. It is known the activation of cannabinoid receptors in astrocytes modulate synaptic plasticity and could be involved in glutamate homeostasis. However, the functional role of astrocytes in alterations derived from chronic drug exposure is not fully understood. In this study, we analyzed how astrocytes contribute to alterations produced by tetrahydrocannabinol (THC). Using fiber photometry in vivo we analyzed astrocytic activity (Ca2+ and glutamate dynamics) in NAc after 1mg/kg THC chronic administration in wildtype and p38αMAPK-/- (Astrop38α) mice4 and we performed electrophysiology experiments to analyze synaptic plasticity. Moreover, we performed behavioral tests to assess whether THC had reinforcing properties or affected learning and memory. Furthermore, using a chemogenetic approaches (DREADDs) we activated NAc astrocytes to analyze their behavioral implications. We observed: 1)THC increases astrocytic calcium activity; 2)THC induces glutamate release in NAc in wildtype, but not Astrop38α; 3)NAc astrocytes are involved in learning; 4)Removal of p38αMAPK in NAc astrocytes restores THC-related impairments. Altogether, our results reveal astrocytes as critical elements for the maintenance of glutamate signaling, with a significant role in drug-use-related alterations.

Mice with insulin receptor (IR)–deficient astrocytes (GFAP-IR knockout [KO] mice) show blunted responses to insulin and reduced brain glucose uptake, whereas IR-deficient astrocytes show disturbed mitochondrial responses to glucose. While exploring the functional impact of disturbed mitochondrial function in astrocytes, we observed that GFAP-IR KO mice show uncoupling of brain blood flow with glucose uptake. Since IR-deficient astrocytes show higher levels of reactive oxidant species (ROS), this leads to stimulation of hypoxia-inducible factor-1α and, consequently, of the vascular endothelial growth factor angiogenic pathway. Indeed, GFAP-IR KO mice show disturbed brain vascularity and blood flow that is normalized by treatment with the antioxidant N -acetylcysteine (NAC). NAC ameliorated high ROS levels, normalized angiogenic signaling and mitochondrial function in IR-deficient astrocytes, and normalized neurovascular coupling in GFAP-IR KO mice. Our results indicate that by modulating glucose uptake and angiogenesis, insulin receptors in astrocytes participate in neurovascular coupling.

Astrocytes are key elements of brain circuits that are involved in different aspects of the neuronal physiology relevant to brain functions. Although much effort is being made to understand how the biology of astrocytes affects brain circuits, astrocytic network heterogeneity and plasticity is still poorly defined. Here, we have combined structural and functional imaging of astrocyte activity recorded in mice using the Ca2+-modulated photoactivatable ratiometric integrator and specific optostimulation of glutamatergic pathways to map the functional neuron-astrocyte circuitries in the nucleus accumbens (NAc). We showed pathway-specific astrocytic responses induced by selective optostimulation of main inputs from the prefrontal cortex, basolateral amygdala, and ventral hippocampus. Furthermore, co-stimulation of glutamatergic pathways induced non-linear Ca2+-signaling integration, revealing integrative properties of NAc astrocytes. All these results demonstrate the existence of specific neuron-astrocyte circuits in the NAc, providing an insight to the understanding of how the NAc integrates information.

Neuregulin 1 (NRG1) and its receptor ERBB4 are schizophrenia (SZ) risk genes that control the development of both excitatory and inhibitory cortical circuits. Most studies focused on the characterization ErbB4 deficient mice. However, ErbB4 deletion concurrently perturbs the signaling of Nrg1 and Neuregulin 3 (Nrg3), another ligand expressed in the cortex. In addition, NRG1 polymorphisms linked to SZ locate mainly in non-coding regions and they may partially reduce Nrg1 expression.Here, to study the relevance of Nrg1 partial loss-of-function in cortical circuits we characterized a recently developed haploinsufficient mouse model of Nrg1 (Nrg1tm1Lex). These mice display SZ-like behavioral deficits. The cellular and molecular underpinnings of the behavioral deficits in Nrg1tm1Lex mice remain to be established.With multiple approaches including Magnetic Resonance Spectroscopy (MRS), electrophysiology, quantitative imaging and molecular analysis we found that Nrg1 haploinsufficiency impairs the inhibitory cortical circuits. We observed changes in the expression of molecules involved in GABAergic neurotransmission, decreased density of Vglut1 excitatory buttons onto Parvalbumin interneurons and decreased frequency of spontaneous inhibitory postsynaptic currents. Moreover, we found a decreased number of Parvalbumin positive interneurons in the cortex and altered expression of Calretinin. Interestingly, we failed to detect other alterations in excitatory neurons that were previously reported in ErbB4 null mice suggesting that the Nrg1 haploinsufficiency does not entirely phenocopies ErbB4 deletions.Altogether, this study suggests that Nrg1 haploinsufficiency primarily affects the cortical inhibitory circuits in the cortex and provides new insights into the structural and molecular synaptic impairment caused by NRG1 hypofunction in a preclinical model of SZ.

Activity-dependent long-term changes in synaptic transmission known as synaptic plasticity are fundamental processes in brain function and are recognized as the cellular basis of learning and memory. While the neuronal mechanisms underlying synaptic plasticity have been largely identified, the involvement of astrocytes in these processes has been less recognized. However, astrocytes are emerging as important cells that regulate synaptic function by interacting with neurons at tripartite synapses. In this review, we discuss recent evidence suggesting that astrocytes are necessary elements in long-term synaptic depression (LTD). We highlight the mechanistic heterogeneity of astrocyte contribution to this form of synaptic plasticity and propose that astrocytes are integral participants in LTD.