Astrocytes and Behavior.

Animal behavior was classically considered to be determined exclusively by neuronal activity, whereas surrounding glial cells such as astrocytes played only supportive roles. However, astrocytes are as numerous as neurons in the mammalian brain, and current findings indicate a chemically based dialog between astrocytes and neurons. Activation of astrocytes by synaptically released neurotransmitters converges on regulating intracellular Ca2+ in astrocytes, which then can regulate the efficacy of near and distant tripartite synapses at diverse timescales through gliotransmitter release. Here, we discuss recent evidence on how diverse behaviors are impacted by this dialog. These recent findings support a paradigm shift in neuroscience, in which animal behavior does not result exclusively from neuronal activity but from the coordinated activity of both astrocytes and neurons. Decoding how astrocytes and neurons interact with each other in various brain circuits will be fundamental to fully understanding how behaviors originate and become dysregulated in disease.

Spatial organization of neuron-astrocyte interactions in the somatosensory cortex.

Microcircuits in the neocortex are functionally organized along layers and columns, which are the fundamental modules of cortical information processing. While the function of cortical microcircuits has focused on neuronal elements, much less is known about the functional organization of astrocytes and their bidirectional interaction with neurons. Here, we show that Cannabinoid type 1 receptor (CB1R)-mediated astrocyte activation by neuron-released endocannabinoids elevate astrocyte Ca2+ levels, stimulate ATP/adenosine release as gliotransmitters, and transiently depress synaptic transmission in layer 5 pyramidal neurons at relatively distant synapses (˃20 µm) from the stimulated neuron. This astrocyte-mediated heteroneuronal synaptic depression occurred between pyramidal neurons within a cortical column and was absent in neurons belonging to adjacent cortical columns. Moreover, this form of heteroneuronal synaptic depression occurs between neurons located in particular layers, following a specific connectivity pattern that depends on a layer-specific neuron-to-astrocyte signaling. These results unravel the existence of astrocyte-mediated nonsynaptic communication between cortical neurons and that this communication is column- and layer-specific, which adds further complexity to the intercellular signaling processes in the neocortex.

Endocannabinoid signaling in synaptic function.

In the last decades, astrocytes have emerged as important regulatory cells actively involved in brain function by exchanging signaling with neurons. The endocannabinoid (eCB) signaling is widely present in many brain areas, being crucially involved in multiple brain functions and animal behaviors. The present review presents and discusses current evidence demonstrating that astrocytes sense eCBs released during neuronal activity and subsequently release gliotransmitters that regulate synaptic transmission and plasticity. The eCB signaling to astrocytes and the synaptic regulation mediated by astrocytes activated by eCBs are complex phenomena that exhibit exquisite spatial and temporal properties, a wide variety of downstream signaling mechanisms, and a large diversity of functional synaptic outcomes. Studies investigating this topic have revealed novel regulatory processes of synaptic function, like the lateral regulation of synaptic transmission and the active involvement of astrocytes in the spike-timing dependent plasticity, originally thought to be exclusively mediated by the coincident activity of pre- and postsynaptic neurons, following Hebbian rules for associative learning. Finally, the critical influence of astrocyte-mediated eCB signaling on animal behavior is also discussed.

Altered calcium signaling in Bergmann glia contributes to spinocerebellar ataxia type-1 in a mouse model of SCA1.

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by an abnormal expansion of glutamine (Q) encoding CAG repeats in the ATAXIN1 (ATXN1) gene and characterized by progressive cerebellar ataxia, dysarthria, and eventual deterioration of bulbar functions. SCA1 shows severe degeneration of cerebellar Purkinje cells (PCs) and activation of Bergmann glia (BG), a type of cerebellar astroglia closely associated with PCs. Combining electrophysiological recordings, calcium imaging techniques, and chemogenetic approaches, we have investigated the electrical intrinsic and synaptic properties of PCs and the physiological properties of BG in SCA1 mouse model expressing mutant ATXN1 only in PCs. PCs of SCA1 mice displayed lower spontaneous firing rate and larger slow afterhyperpolarization currents (sIAHP) than wildtype mice, whereas the properties of the synaptic inputs were unaffected. BG of SCA1 mice showed higher calcium hyperactivity and gliotransmission, manifested by higher frequency of NMDAR-mediated slow inward currents (SICs) in PC. Preventing the BG calcium hyperexcitability of SCA1 mice by loading BG with the calcium chelator BAPTA restored sIAHP and spontaneous firing rate of PCs to similar levels of wildtype mice. Moreover, mimicking the BG hyperactivity by activating BG expressing Gq-DREADDs in wildtype mice reproduced the SCA1 pathological phenotype of PCs, i.e., enhancement of sIAHP and decrease of spontaneous firing rate. These results indicate that the intrinsic electrical properties of PCs, but not their synaptic properties, were altered in SCA1 mice and that these alterations were associated with the hyperexcitability of BG. Moreover, preventing BG hyperexcitability in SCA1 mice and promoting BG hyperexcitability in wildtype mice prevented and mimicked, respectively, the pathological electrophysiological phenotype of PCs. Therefore, BG plays a relevant role in the dysfunction of the electrical intrinsic properties of PCs in SCA1 mice, suggesting that they may serve as potential targets for therapeutic approaches to treat the spinocerebellar ataxia type 1.

Dysregulation of astrocytic Ca2+ signaling and gliotransmitter release in mouse models of α-synucleinopathies.

α-Synuclein is a major component of Lewy bodies (LB) and Lewy neurites (LN) appearing in the postmortem brain of Parkinson's disease (PD) and other α-synucleinopathies. While most studies of α-synucleinopathies have focused on neuronal and synaptic alterations as well as dysfunctions of the astrocytic homeostatic roles, whether the bidirectional astrocyte-neuronal communication is affected in these diseases remains unknown. We have investigated whether the astrocyte Ca2+ excitability and the glutamatergic gliotransmission underlying astrocyte-neuronal signaling are altered in several transgenic mouse models related to α-synucleinopathies, i.e., mice expressing high and low levels of the human A53T mutant α-synuclein (G2-3 and H5 mice, respectively) globally or selectively in neurons (iSyn mice), mice expressing human wildtype α-synuclein (I2-2 mice), and mice expressing A30P mutant α-synuclein (O2 mice). Combining astrocytic Ca2+ imaging and neuronal electrophysiological recordings in hippocampal slices of these mice, we have found that compared to non-transgenic mice, astrocytes in G2-3 mice at different ages (1-6 months) displayed a Ca2+ hyperexcitability that was independent of neurotransmitter receptor activation, suggesting that the expression of α-synuclein mutant A53T altered the intrinsic properties of astrocytes. Similar dysregulation of the astrocyte Ca2+ signal was present in H5 mice, but not in I2-2 and O2 mice, indicating α-synuclein mutant-specific effects. Moreover, astrocyte Ca2+ hyperexcitability was absent in mice expressing the α-synuclein mutant A53T selectively in neurons, indicating that the effects on astrocytes were cell-autonomous. Consistent with these effects, glutamatergic gliotransmission was enhanced in G2-3 and H5 mice, but was unaffected in I2-2, O2 and iSyn mice. These results indicate a cell-autonomous effect of pathogenic A53T expression in astrocytes that may contribute to the altered neuronal and synaptic function observed in α-synucleinopathies.

Autistic-like behavior and cerebellar dysfunction in Bmal1 mutant mice ameliorated by mTORC1 inhibition.

Although circadian and sleep disorders are frequently associated with autism spectrum disorders (ASD), it remains elusive whether clock gene disruption can lead to autistic-like phenotypes in animals. The essential clock gene Bmal1 has been associated with human sociability and its missense mutations are identified in ASD. Here we report that global Bmal1 deletion led to significant social impairments, excessive stereotyped and repetitive behaviors, as well as motor learning disabilities in mice, all of which resemble core behavioral deficits in ASD. Furthermore, aberrant cell density and immature morphology of dendritic spines were identified in the cerebellar Purkinje cells (PCs) of Bmal1 knockout (KO) mice. Electrophysiological recordings uncovered enhanced excitatory and inhibitory synaptic transmission and reduced firing rates in the PCs of Bmal1 KO mice. Differential expression of ASD- and ataxia-associated genes (Ntng2, Mfrp, Nr4a2, Thbs1, Atxn1, and Atxn3) and dysregulated pathways of translational control, including hyperactivated mammalian target of rapamycin complex 1 (mTORC1) signaling, were identified in the cerebellum of Bmal1 KO mice. Interestingly, the antidiabetic drug metformin reversed mTORC1 hyperactivation and alleviated major behavioral and PC deficits in Bmal1 KO mice. Importantly, conditional Bmal1 deletion only in cerebellar PCs was sufficient to recapitulate autistic-like behavioral and cellular changes akin to those identified in Bmal1 KO mice. Together, these results unveil a previously unidentified role for Bmal1 disruption in cerebellar dysfunction and autistic-like behaviors. Our findings provide experimental evidence supporting a putative role for dysregulation of circadian clock gene expression in the pathogenesis of ASD.

Astrocyte-neuronal network interplay is disrupted in Alzheimer's disease mice.

Alzheimer's disease (AD) is associated with senile plaques of beta-amyloid (Aß) that affect the function of neurons and astrocytes. Brain activity results from the coordinated function of neurons and astrocytes in astroglial-neuronal networks. However, the effects of Aß on astroglial and neuronal network function remains unknown. Simultaneously monitoring astrocyte calcium and electric neuronal activities, we quantified the impact of Aß on sensory-evoked cortical activity in a mouse model of AD. At rest, cortical astrocytes displayed spontaneous hyperactivity that was related to Aß density. Sensory-evoked astrocyte responsiveness was diminished in AD mice, depending on the density and distance of Aß, and the responses showed altered calcium dynamics. Hence, astrocytes were spontaneously hyperactive but hypo-responsive to sensory stimulation. Finally, AD mice showed sensory-evoked electrical cortical hyperresponsiveness associated with altered astrocyte-neuronal network interplay. Our findings suggest dysfunction of astrocyte networks in AD mice may dysregulate cortical electrical activity and contribute to cognitive decline.

Astrocyte Signaling Gates Long-Term Depression at Corticostriatal Synapses of the Direct Pathway.

Despite extensive research into understanding synaptic mechanisms of striatal plasticity, the functional role played by astrocytes in this region remains to be fully elucidated. It was recently demonstrated that high-frequency stimulation (HFS) of cortical inputs induced long-term depression (LTD) mediated by adenosine A1 receptor (A1R) activation at corticostriatal synapses of the direct pathway [cortico-striatal projection neuron (dSPN)] in the dorsolateral striatum (DLS). Because astrocyte-derived adenosine has been shown to regulate synaptic transmission in several brain areas, we investigated whether this form of neuron-astrocyte signaling contributes to synaptic plasticity in the DLS of male and female mice. We found that cortical HFS increases calcium (Ca2+) levels in striatal astrocytes through activation of metabotropic glutamate receptor type 5 (mGluR5) signaling and that this astrocyte-mediated response is necessary for A1R-mediated LTD. Consistent with this, astrocyte activation with Gq designer receptors exclusively activated by designer drugs (DREADDs) induced A1R-mediated synaptic depression at cortico-dSPN synapses. Together, these results indicate that astrocytes are integral elements of striatal A1R-mediated LTD.SIGNIFICANCE STATEMENT Abnormal striatal circuit function is implicated in several disorders such as Parkinson's disease and Huntington's disease. Thus, there is a need to better understand the mechanisms supporting proper striatal activity. While extensive work has revealed the many important contributions from neurons in striatal function, far less is known about the role of astrocytes in this brain area. We show that long-term depression (LTD) at corticostriatal synapses of the direct pathway is not strictly a neuronal phenomenon; astrocytes respond to corticostriatal stimulation and this astrocyte response is necessary for LTD. This research adds to the accumulating evidence that astrocytes are active and integral players in synaptic communication, and that neuron-astrocyte interactions are key cellular processes involved in brain function.

Dysregulation of Astrocyte-Neuronal Communication in Alzheimer's Disease.

Recent studies implicate astrocytes in Alzheimer's disease (AD); however, their role in pathogenesis is poorly understood. Astrocytes have well-established functions in supportive functions such as extracellular ionic homeostasis, structural support, and neurovascular coupling. However, emerging research on astrocytic function in the healthy brain also indicates their role in regulating synaptic plasticity and neuronal excitability via the release of neuroactive substances named gliotransmitters. Here, we review how this "active" role of astrocytes at synapses could contribute to synaptic and neuronal network dysfunction and cognitive impairment in AD.

Gi/o protein-coupled receptors inhibit neurons but activate astrocytes and stimulate gliotransmission.

G protein-coupled receptors (GPCRs) play key roles in intercellular signaling in the brain. Their effects on cellular function have been largely studied in neurons, but their functional consequences on astrocytes are less known. Using both endogenous and chemogenetic approaches with DREADDs, we have investigated the effects of Gq and Gi/o GPCR activation on astroglial Ca2+ -based activity, gliotransmitter release, and the functional consequences on neuronal electrical activity. We found that while Gq GPCR activation led to cellular activation in both neurons and astrocytes, Gi/o GPCR activation led to cellular inhibition in neurons and cellular activation in astrocytes. Astroglial activation by either Gq or Gi/o protein-mediated signaling stimulated gliotransmitter release, which increased neuronal excitability. Additionally, activation of Gq and Gi/o DREADDs in vivo increased astrocyte Ca2+ activity and modified neuronal network electrical activity. Present results reveal additional complexity of the signaling consequences of excitatory and inhibitory neurotransmitters in astroglia-neuron network operation and brain function.

Glial Cell Calcium Signaling Mediates Capillary Regulation of Blood Flow in the Retina.

UNLABELLED: The brain is critically dependent on the regulation of blood flow to nourish active neurons. One widely held hypothesis of blood flow regulation holds that active neurons stimulate Ca(2+) increases in glial cells, triggering glial release of vasodilating agents. This hypothesis has been challenged, as arteriole dilation can occur in the absence of glial Ca(2+) signaling. We address this controversy by imaging glial Ca(2+) signaling and vessel dilation in the mouse retina. We find that sensory stimulation results in Ca(2+) increases in the glial endfeet contacting capillaries, but not arterioles, and that capillary dilations often follow spontaneous Ca(2+) signaling. In IP3R2(-/-) mice, where glial Ca(2+) signaling is reduced, light-evoked capillary, but not arteriole, dilation is abolished. The results show that, independent of arterioles, capillaries actively dilate and regulate blood flow. Furthermore, the results demonstrate that glial Ca(2+) signaling regulates capillary but not arteriole blood flow. SIGNIFICANCE STATEMENT: We show that a Ca(2+)-dependent glial cell signaling mechanism is responsible for regulating capillary but not arteriole diameter. This finding resolves a long-standing controversy regarding the role of glial cells in regulating blood flow, demonstrating that glial Ca(2+) signaling is both necessary and sufficient to dilate capillaries. While the relative contributions of capillaries and arterioles to blood flow regulation remain unclear, elucidating the mechanisms that regulate capillary blood flow may ultimately lead to the development of therapies for treating diseases where blood flow regulation is disrupted, including Alzheimer's disease, stroke, and diabetic retinopathy. This finding may also aid in revealing the underlying neuronal activity that generates BOLD fMRI signals.

Calcium signaling in astrocytes and gliotransmitter release.

Glia are as numerous in the brain as neurons and widely known to serve supportive roles such as structural scaffolding, extracellular ionic and neurotransmitter homeostasis, and metabolic support. However, over the past two decades, several lines of evidence indicate that astrocytes, which are a type of glia, play active roles in neural information processing. Astrocytes, although not electrically active, can exhibit a form of excitability by dynamic changes in intracellular calcium levels. They sense synaptic activity and release neuroactive substances, named gliotransmitters, that modulate neuronal activity and synaptic transmission in several brain areas, thus impacting animal behavior. This "dialogue" between astrocytes and neurons is embodied in the concept of the tripartite synapse that includes astrocytes as integral elements of synaptic function. Here, we review the recent work and discuss how astrocytes via calcium-mediated excitability modulate synaptic information processing at various spatial and time scales.

A spatial threshold for astrocyte calcium surge.

Astrocytes are active cells involved in brain function through the bidirectional communication with neurons, in which the astrocyte calcium signal 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. In turn, astrocytes may regulate individual synapses by calcium-dependent release of gliotransmitters. Because a single astrocyte may contact ~100,000 synapses, the control of the intracellular calcium signal propagation may have relevant consequences on brain function by regulating the spatial range of astrocyte neuromodulation of synapses. Yet, the properties governing the spatial dynamics of the astrocyte calcium signal 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 transgenic IP3R2-/- mice, we found that type-2 IP3 receptors were necessary for the generation of the 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 calcium physiology, i.e., a spatial threshold for the astrocyte intracellular calcium signal propagation, which depends on astrocyte intrinsic properties and governs the astrocyte integration of local synaptic activity and the subsequent neuromodulation.

Differential cone pathway influence on intrinsically photosensitive retinal ganglion cell subtypes.

A small subset of ganglion cells in the mammalian retina express the photopigment melanopsin and are intrinsically photosensitive (ipRGCs). These cells are the primary conduits through which photic information is relayed to non-image-forming visual centers that mediate behaviors such as the pupillary light reflex and circadian entrainment. M1 and M2 cells comprise distinct morphological subpopulations of ipRGC, and possess physiological diversity in their intrinsic membrane properties and intrinsic light responses. Additionally, evidence now indicates that all ipRGCs receive photic information from rods/cones via synaptic signaling. It has recently been reported that Off-stratifying M1 cells paradoxically receive input from the On pathway within the Off sublamina of the inner plexiform layer. The purpose of the current study was to examine the functional consequences of cone pathway signaling to M1 and M2 cells. Using pharmacological tools and single-cell recordings of synaptic responses in wild-type and melanopsin-null mice, we found that the On pathway forms the primary excitatory synaptic input to both M1 and M2 cells. This input was much more influential in shaping the light-evoked responses and resting membrane properties of M2 cells than M1 cells. These findings indicate a surprising differential reliance upon cone-mediated phototransduction by ipRGC subpopulations. These findings also suggest that ipRGC subtypes signal diverse photic information to various non-image-forming visual centers.

Intrinsic phototransduction persists in melanopsin-expressing ganglion cells lacking diacylglycerol-sensitive TRPC subunits.

In mammals, intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate various non-image-forming photic responses, such as circadian photoentrainment, pupillary light reflex and pineal melatonin suppression. ipRGCs directly respond to environmental light by activation of the photopigment melanopsin followed by the opening of an unidentified cation-selective channel. Studies in heterologous expression systems and in the native retina have strongly implicated diacylglycerol-sensitive transient receptor potential channels containing TRPC3, TRPC6 and TRPC7 subunits in melanopsin-evoked depolarization. Here we show that melanopsin-evoked electrical responses largely persist in ipRGCs recorded from early postnatal (P6-P8) and adult (P22-P50) mice lacking expression of functional TRPC3, TRPC6 or TRPC7 subunits. Multielectrode array (MEA) recordings performed at P6-P8 stages under conditions that prevent influences from rod/cone photoreceptors show comparable light sensitivity for the melanopsin-evoked responses in these mutant mouse lines in comparison to wild-type (WT) mice. Patch-clamp recordings from adult mouse ipRGCs lacking TRPC3 or TRPC7 subunits show intrinsic light-evoked responses equivalent to those recorded in WT mice. Persistence of intrinsic light-evoked responses was also noted in ipRGCs lacking TRPC6 subunits, although with significantly smaller magnitudes. These results demonstrate that the melanopsin-evoked depolarization in ipRGCs is not mediated by either TRPC3, TRPC6 or TRPC7 channel subunits alone. They also suggest that the melanopsin signaling pathway includes TRPC6-containing heteromeric channels in mature retinas.

G-Protein-Coupled Receptors in Astrocyte-Neuron Communication.

Astrocytes, a major type of glial cell, are known to play key supportive roles in brain function, contributing to ion and neurotransmitter homeostasis, maintaining the blood-brain barrier and providing trophic and metabolic support for neurons. Besides these support functions, astrocytes are emerging as important elements in brain physiology through signaling exchange with neurons at tripartite synapses. Astrocytes express a wide variety of neurotransmitter transporters and receptors that allow them to sense and respond to synaptic activity. Principal among them are the G-protein-coupled receptors (GPCRs) in astrocytes because their activation by synaptically released neurotransmitters leads to mobilization of intracellular calcium. In turn, activated astrocytes release neuroactive substances called gliotransmitters, such as glutamate, GABA, and ATP/adenosine that lead to synaptic regulation through activation of neuronal GPCRs. In this review we will present and discuss recent evidence demonstrating the critical roles played by GPCRs in the bidirectional astrocyte-neuron signaling, and their crucial involvement in the astrocyte-mediated regulation of synaptic transmission and plasticity.

Astrocyte and neuron cooperation in long-term depression.

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.

Functional and morphological differences among intrinsically photosensitive retinal ganglion cells.

A subset of ganglion cells in the mammalian retina express the photopigment melanopsin and are intrinsically photosensitive (ipRGCs). These cells are implicated in non-image-forming visual responses to environmental light, such as the pupillary light reflex, seasonal adaptations in physiology, photic inhibition of nocturnal melatonin release, and modulation of sleep, alertness, and activity. Morphological studies have confirmed the existence of at least three distinct subpopulations of ipRGCs, but studies of the physiology of ipRGCs at the single cell level have focused mainly on M1 cells, the dendrites of which stratify solely in sublamina a (OFF sublamina) of the retinal inner plexiform layer (IPL). Little work has been done to compare the functional properties of M1 cells to those of M2 cells, the dendrites of which stratify solely in sublamina b (ON sublamina) of the IPL. The goal of the current study was to compare the morphology, intrinsic light response, and intrinsic membrane properties of M1 and M2 cells in the mouse retina. Here we demonstrate additional morphological differences between M1 and M2 cells as well as distinct physiological characteristics of both the intrinsic light responses and intrinsic membrane properties. M2 cells displayed a more complex dendritic arborization and higher input resistance, yet showed lower light sensitivity and lower maximal light responses than M1 cells. These data indicate morphological and functional heterogeneity among ipRGCs.

Variable loss of Kir4.1 channel function in SeSAME syndrome mutations.

SeSAME syndrome is a complex disease characterized by seizures, sensorineural deafness, ataxia, mental retardation and electrolyte imbalance. Mutations in the inwardly rectifying potassium channel Kir4.1 (KCNJ10 gene) have been linked to this condition. Kir4.1 channels are weakly rectifying channels expressed in glia, kidney, cochlea and possibly other tissues. We determined the electrophysiological properties of SeSAME mutant channels after expression in transfected mammalian cells. We found that a majority of mutations (R297C, C140R, R199X, T164I) resulted in complete loss of Kir4.1 channel function while two mutations (R65P and A167V) produced partial loss of function. All mutant channels were rescued upon co-transfection of wild-type Kir4.1 but not Kir5.1 channels. Cell-surface biotinylation assays indicate significant plasma membrane expression of all mutant channels with exception of the non-sense mutant R199X. These results indicate the differential loss of Kir channel function among SeSAME syndrome mutations.