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• W2940727119 abstract "•Striatal MSNs release GABA to activate astrocyte Gi-coupled GABAB receptors•Astrocyte Gi pathway activation results in hyperactivity with disrupted attention•Astrocyte Gi pathway activation increases fast synaptic excitation and MSN firing•Behavioral and synaptic effects are due to reactivation of TSP1 in astrocytes Hyperactivity and disturbances of attention are common behavioral disorders whose underlying cellular and neural circuit causes are not understood. We report the discovery that striatal astrocytes drive such phenotypes through a hitherto unknown synaptic mechanism. We found that striatal medium spiny neurons (MSNs) triggered astrocyte signaling via γ-aminobutyric acid B (GABAB) receptors. Selective chemogenetic activation of this pathway in striatal astrocytes in vivo resulted in acute behavioral hyperactivity and disrupted attention. Such responses also resulted in upregulation of the synaptogenic cue thrombospondin-1 (TSP1) in astrocytes, increased excitatory synapses, enhanced corticostriatal synaptic transmission, and increased MSN action potential firing in vivo. All of these changes were reversed by blocking TSP1 effects. Our data identify a form of bidirectional neuron-astrocyte communication and demonstrate that acute reactivation of a single latent astrocyte synaptogenic cue alters striatal circuits controlling behavior, revealing astrocytes and the TSP1 pathway as therapeutic targets in hyperactivity, attention deficit, and related psychiatric disorders. Hyperactivity and disturbances of attention are common behavioral disorders whose underlying cellular and neural circuit causes are not understood. We report the discovery that striatal astrocytes drive such phenotypes through a hitherto unknown synaptic mechanism. We found that striatal medium spiny neurons (MSNs) triggered astrocyte signaling via γ-aminobutyric acid B (GABAB) receptors. Selective chemogenetic activation of this pathway in striatal astrocytes in vivo resulted in acute behavioral hyperactivity and disrupted attention. Such responses also resulted in upregulation of the synaptogenic cue thrombospondin-1 (TSP1) in astrocytes, increased excitatory synapses, enhanced corticostriatal synaptic transmission, and increased MSN action potential firing in vivo. All of these changes were reversed by blocking TSP1 effects. Our data identify a form of bidirectional neuron-astrocyte communication and demonstrate that acute reactivation of a single latent astrocyte synaptogenic cue alters striatal circuits controlling behavior, revealing astrocytes and the TSP1 pathway as therapeutic targets in hyperactivity, attention deficit, and related psychiatric disorders. Hyperactivity and disturbances of attention are common behavioral disorders (American Psychiatric Association, 2013American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders (DSM–5).Fifth Edition. American Psychiatric Association Publishing, 2013Crossref Google Scholar, Fayyad et al., 2007Fayyad J. De Graaf R. Kessler R. Alonso J. Angermeyer M. Demyttenaere K. De Girolamo G. Haro J.M. Karam E.G. Lara C. et al.Cross-national prevalence and correlates of adult attention-deficit hyperactivity disorder.Br. J. Psychiatry. 2007; 190: 402-409Crossref PubMed Scopus (990) Google Scholar, Polanczyk et al., 2007Polanczyk G. de Lima M.S. Horta B.L. Biederman J. Rohde L.A. The worldwide prevalence of ADHD: a systematic review and metaregression analysis.Am. J. Psychiatry. 2007; 164: 942-948Crossref PubMed Scopus (2241) Google Scholar) whose underlying causes are unknown and that lack adequate treatment (Curatolo et al., 2010Curatolo P. D’Agati E. Moavero R. The neurobiological basis of ADHD. Ital.J. Pediatr. 2010; 36: 79Google Scholar, de la Peña et al., 2018de la Peña J.B. Dela Peña I.J. Custodio R.J. Botanas C.J. Kim H.J. Cheong J.H. Exploring the Validity of Proposed Transgenic Animal Models of Attention-Deficit Hyperactivity Disorder (ADHD).Mol. Neurobiol. 2018; 55: 3739-3754PubMed Google Scholar). Such disorders involve dysfunction in the striatum based on imaging studies in humans (Cubillo et al., 2012Cubillo A. Halari R. Smith A. Taylor E. Rubia K. A review of fronto-striatal and fronto-cortical brain abnormalities in children and adults with Attention Deficit Hyperactivity Disorder (ADHD) and new evidence for dysfunction in adults with ADHD during motivation and attention.Cortex. 2012; 48: 194-215Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, Riva et al., 2018Riva D. Taddei M. Bulgheroni S. The neuropsychology of basal ganglia.Eur. J. Paediatr. Neurol. 2018; 22: 321-326Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The striatum is the largest nucleus of the basal ganglia, a group of interconnected subcortical nuclei involved in movement, repetitive behavior, obsessions, habits, tics, and diverse neuropsychiatric conditions (Graybiel, 2008Graybiel A.M. Habits, rituals, and the evaluative brain.Annu. Rev. Neurosci. 2008; 31: 359-387Crossref PubMed Scopus (1121) Google Scholar). In the current study, we report the unexpected discovery that latent synaptogenic cues derived from striatal astrocytes drive behavioral hyperactivity with disrupted attention in adult mice. Initially documented over a century ago, astrocytes represent about 40% of all brain cells. They are the most numerous type of glia and tile the entire CNS (Barres, 2008Barres B.A. The mystery and magic of glia: a perspective on their roles in health and disease.Neuron. 2008; 60: 430-440Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar). During development, astrocytes provide important cues to regulate synapse formation and removal (Allen and Lyons, 2018Allen N.J. Lyons D.A. Glia as architects of central nervous system formation and function.Science. 2018; 362: 181-185Crossref PubMed Scopus (304) Google Scholar), whereas in adults, the finest astrocyte processes from these “bushy” cells continue to contact neurons, synapses, blood vessels, and other glial cells. In these locations, astrocytes mediate multiple active and homeostatic functions (Attwell et al., 2010Attwell D. Buchan A.M. Charpak S. Lauritzen M. Macvicar B.A. Newman E.A. Glial and neuronal control of brain blood flow.Nature. 2010; 468: 232-243Crossref PubMed Scopus (1612) Google Scholar, Khakh and Sofroniew, 2015Khakh B.S. Sofroniew M.V. Diversity of astrocyte functions and phenotypes in neural circuits.Nat. Neurosci. 2015; 18: 942-952Crossref PubMed Scopus (644) Google Scholar, Volterra et al., 2014Volterra A. Liaudet N. Savtchouk I. Astrocyte Ca2+ signalling: an unexpected complexity.Nat. Rev. Neurosci. 2014; 15: 327-335Crossref PubMed Scopus (276) Google Scholar). Astrocytes also display CNS area-specific properties and functions (Chai et al., 2017Chai H. Diaz-Castro B. Shigetomi E. Monte E. Octeau J.C. Yu X. Cohn W. Rajendran P.S. Vondriska T.M. Whitelegge J.P. et al.Neural circuit-specialized astrocytes: transcriptomic, proteomic, morphological, and functional evidence.Neuron. 2017; 95: 531-549.e9Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, Ben Haim and Rowitch, 2017Ben Haim L. Rowitch D.H. Functional diversity of astrocytes in neural circuit regulation.Nat. Rev. Neurosci. 2017; 18: 31-41Crossref PubMed Scopus (320) Google Scholar, Molofsky et al., 2014Molofsky A.V. Kelley K.W. Tsai H.H. Redmond S.A. Chang S.M. Madireddy L. Chan J.R. Baranzini S.E. Ullian E.M. Rowitch D.H. Astrocyte-encoded positional cues maintain sensorimotor circuit integrity.Nature. 2014; 509: 189-194Crossref PubMed Scopus (192) Google Scholar). Despite these advances, the mechanisms of astrocyte-neuron signaling, its effects on the functions of intact neural circuits, their behavioral outputs, and their contributions to brain diseases remain to be fully elucidated. Replete with molecularly defined astrocytes, the striatum is an important circuit to explore astrocyte biology in adult mice (Chai et al., 2017Chai H. Diaz-Castro B. Shigetomi E. Monte E. Octeau J.C. Yu X. Cohn W. Rajendran P.S. Vondriska T.M. Whitelegge J.P. et al.Neural circuit-specialized astrocytes: transcriptomic, proteomic, morphological, and functional evidence.Neuron. 2017; 95: 531-549.e9Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, Kelley et al., 2018Kelley K.W. Nakao-Inoue H. Molofsky A.V. Oldham M.C. Variation among intact tissue samples reveals the core transcriptional features of human CNS cell classes.Nat. Neurosci. 2018; 21: 1171-1184Crossref PubMed Scopus (99) Google Scholar). As the major input nucleus of the basal ganglia, the striatum integrates converging excitatory and inhibitory signals from numerous parts of the brain and is involved in action selection and motor function (Graybiel, 2008Graybiel A.M. Habits, rituals, and the evaluative brain.Annu. Rev. Neurosci. 2008; 31: 359-387Crossref PubMed Scopus (1121) Google Scholar). We used several recently developed striatal astrocyte-selective genetic, transcriptomic, imaging, behavioral, and electrophysiology approaches (Bakhurin et al., 2016Bakhurin K.I. Mac V. Golshani P. Masmanidis S.C. Temporal correlations among functionally specialized striatal neural ensembles in reward-conditioned mice.J. Neurophysiol. 2016; 115: 1521-1532Crossref PubMed Scopus (38) Google Scholar, Chai et al., 2017Chai H. Diaz-Castro B. Shigetomi E. Monte E. Octeau J.C. Yu X. Cohn W. Rajendran P.S. Vondriska T.M. Whitelegge J.P. et al.Neural circuit-specialized astrocytes: transcriptomic, proteomic, morphological, and functional evidence.Neuron. 2017; 95: 531-549.e9Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, Srinivasan et al., 2016Srinivasan R. Lu T.Y. Chai H. Xu J. Huang B.S. Golshani P. Coppola G. Khakh B.S. New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.Neuron. 2016; 92: 1181-1195Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, Yu et al., 2018Yu X. Taylor A.M.W. Nagai J. Golshani P. Evans C.J. Coppola G. Khakh B.S. Reducing astrocyte calcium signaling in vivo alters striatal microcircuits and causes repetitive behavior.Neuron. 2018; 99: 1170-1187.e9Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) to interrogate the roles of bidirectional neuron-astrocyte interactions in the function of striatal microcircuits in vivo. We discovered an unexpected mechanism for astrocyte-neuron-mediated synaptic plasticity, a hitherto unknown role for astrocytes in hyperactivity and disrupted attention phenotypes, and potential therapeutic strategies targeting astrocytes to treat such psychiatric diseases. The results of statistical comparisons, n numbers, and p values are shown in the figures or figure legends with the relevant average data. When the average data are reported in the text, the statistics are also reported there. However, all statistical tests are reported in Table S1 for every experiment. We expressed the genetically encoded Ca2+ indicator GCaMP6f (Chen et al., 2013Chen T.W. Wardill T.J. Sun Y. Pulver S.R. Renninger S.L. Baohan A. Schreiter E.R. Kerr R.A. Orger M.B. Jayaraman V. et al.Ultrasensitive fluorescent proteins for imaging neuronal activity.Nature. 2013; 499: 295-300Crossref PubMed Scopus (3617) Google Scholar) in striatal astrocytes (Srinivasan et al., 2016Srinivasan R. Lu T.Y. Chai H. Xu J. Huang B.S. Golshani P. Coppola G. Khakh B.S. New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.Neuron. 2016; 92: 1181-1195Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) and depolarized medium spiny neurons (MSNs) to physiological upstate-like membrane potential transitions (Wilson and Kawaguchi, 1996Wilson C.J. Kawaguchi Y. The origins of two-state spontaneous membrane potential fluctuations of neostriatal spiny neurons.J. Neurosci. 1996; 16: 2397-2410Crossref PubMed Google Scholar) via whole-cell patch-clamping (Figures 1A–1C). MSN depolarization by ∼20–30 mV resulted in action potential (AP) firing and significantly increased the frequency of Ca2+ signals in nearby astrocytes (<50 μm away from MSN somata or dendrites) from 1.4 ± 0.2 to 2.4 ± 0.3 min−1 (Figures 1B and 1C; n = 24 astrocytes, 6 mice; p < 0.001). The amplitude of the Ca2+ signals was unaltered (0.3 ± 0.04 to 0.3 ± 0.04 dF/F; p > 0.05; n = 20 astrocytes, 5 mice), but their duration increased (2.8 ± 0.04 to 4.0 ± 0.4 s; p < 0.01; n = 20 astrocytes, 5 mice), likely reflecting merged events. No change in Ca2+ signals was observed by current injection via an open pipette, indicating that the astrocyte responses were not due to mechanical effects (Figures 1B and 1C; n = 20 astrocytes, 5 mice). Furthermore, astrocytes responded similarly when either D1 or D2 MSNs were depolarized (Figures S1A–S1C), likely reflecting developmental maturity in adult mice (Martín et al., 2015Martín R. Bajo-Grañeras R. Moratalla R. Perea G. Araque A. Circuit-specific signaling in astrocyte-neuron networks in basal ganglia pathways.Science. 2015; 349: 730-734Crossref PubMed Scopus (193) Google Scholar), consistent with anatomical data (Octeau et al., 2018Octeau J.C. Chai H. Jiang R. Bonanno S.L. Martin K.C. Khakh B.S. An Optical Neuron-Astrocyte Proximity Assay at Synaptic Distance Scales.Neuron. 2018; 98: 49-66.e9Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). MSN depolarization-evoked astrocyte Ca2+ signals were resistant to tetrodotoxin (TTX; 300 nM; Figure 1C; n = 21 astrocytes, 5 mice), which blocked all APs (Figure S1F). However, astrocyte Ca2+ signals were abolished by Cd2+ (50 μM; n = 20 astrocytes, 5 mice) and nimodipine (20 μM; n = 23 astrocytes, 4 mice), which both block MSN L-type Ca2+ channels (Bargas et al., 1994Bargas J. Howe A. Eberwine J. Cao Y. Surmeier D.J. Cellular and molecular characterization of Ca2+ currents in acutely isolated, adult rat neostriatal neurons.J. Neurosci. 1994; 14: 6667-6686Crossref PubMed Google Scholar, Carter and Sabatini, 2004Carter A.G. Sabatini B.L. State-dependent calcium signaling in dendritic spines of striatal medium spiny neurons.Neuron. 2004; 44: 483-493Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar; Figure 1C). The depolarization-evoked astrocyte Ca2+ signals were also blocked by MSN dialysis with the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA; 10 mM; n = 21 astrocytes, 4 mice) or with the light chain of tetanus toxin (LC-TeNT; 1 μM; Figure 1C; n = 24 astrocytes, 5 mice), which blocks vesicular release. Together with imaging of MSN activity during upstate-like heightened excitability (Figures S1D–S1G), these data show that MSN membrane potential transitions open high-voltage-activated Ca2+ channels and cause Ca2+-dependent vesicular release of a substance from MSNs that communicates to nearby astrocytes to cause intracellular Ca2+ elevations.Figure S1MSN Depolarization, which Induced Ca2+ Influx into MSNs, Activated Astrocyte Ca2+ Signaling Irrespective of MSN Subtypes, Related to Figure 1Show full caption(A) Representative images showing tdTomato-positive (i, D1) and tdTomato-negative (ii, D2) MSNs from Drd1a-tdTomato mice before and after whole-cell patching (dialyzed with Alexa 568). (B) Depolarization of D1 or D2 MSNs to upstate like levels (96 ± 21 action potentials in i and 130 ± 6 action potentials in ii) increased the frequency of astrocyte Ca2+ signals (3 representative cells for each). (C) Astrocyte Ca2+ signal frequency before and after D1 or D2 MSN depolarization (n = 4 mice per group). ≥ 20% increase in Ca2+ signal frequency was observed in 7 out of 9 astrocytes (i) and 7 out of 10 astrocytes (ii). (D-E) Simultaneous electrophysiological recording and “fast” line scan intracellular Ca2+ imaging from MSNs filled with Fluo-4 via the patch pipette. (F) Representative traces from a MSN during depolarizing current injections (400 pA). MSNs displayed no action potentials (AP) in the presence of 300 nM TTX (with or without Cd2+) in the bath. However, intracellular BAPTA dialysis did not block MSN APs. (G) Representative line scan data of MSN intracellular Ca2+ levels before, during and after somatic depolarization (400 pA current injections). (H) The left graph shows representative traces for MSN intracellular Ca2+ line scan imaging data under the various conditions shown. The right bar graphs summarize average data from such experiments for basal and peak Fluo-4 intensity (n = 5-6 MSNs from 3-5 mice). Overall, in these experiments MSN-depolarization evoked intracellular Ca2+ elevations in MSNs were not abolished by TTX, but were abolished by bath application of Cd2+ and by BAPTA dialysis. Paired t test between before (basal) and after MSN depol (C). One-way ANOVA test (F). Scale bars, 20 μm (A) and 40 μm in (E). Data are shown as mean ± s.e.m. Full details of n numbers, precise P values and statistical tests are reported in Table S1. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ∗∗∗∗ indicates p < 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Representative images showing tdTomato-positive (i, D1) and tdTomato-negative (ii, D2) MSNs from Drd1a-tdTomato mice before and after whole-cell patching (dialyzed with Alexa 568). (B) Depolarization of D1 or D2 MSNs to upstate like levels (96 ± 21 action potentials in i and 130 ± 6 action potentials in ii) increased the frequency of astrocyte Ca2+ signals (3 representative cells for each). (C) Astrocyte Ca2+ signal frequency before and after D1 or D2 MSN depolarization (n = 4 mice per group). ≥ 20% increase in Ca2+ signal frequency was observed in 7 out of 9 astrocytes (i) and 7 out of 10 astrocytes (ii). (D-E) Simultaneous electrophysiological recording and “fast” line scan intracellular Ca2+ imaging from MSNs filled with Fluo-4 via the patch pipette. (F) Representative traces from a MSN during depolarizing current injections (400 pA). MSNs displayed no action potentials (AP) in the presence of 300 nM TTX (with or without Cd2+) in the bath. However, intracellular BAPTA dialysis did not block MSN APs. (G) Representative line scan data of MSN intracellular Ca2+ levels before, during and after somatic depolarization (400 pA current injections). (H) The left graph shows representative traces for MSN intracellular Ca2+ line scan imaging data under the various conditions shown. The right bar graphs summarize average data from such experiments for basal and peak Fluo-4 intensity (n = 5-6 MSNs from 3-5 mice). Overall, in these experiments MSN-depolarization evoked intracellular Ca2+ elevations in MSNs were not abolished by TTX, but were abolished by bath application of Cd2+ and by BAPTA dialysis. Paired t test between before (basal) and after MSN depol (C). One-way ANOVA test (F). Scale bars, 20 μm (A) and 40 μm in (E). Data are shown as mean ± s.e.m. Full details of n numbers, precise P values and statistical tests are reported in Table S1. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ∗∗∗∗ indicates p < 0.0001. Since MSNs are GABAergic, we explored roles for γ-aminobutyric acid (GABA) in MSN-to-astrocyte signaling. A role for GABA was supported by RNA sequencing (RNA-seq) (Chai et al., 2017Chai H. Diaz-Castro B. Shigetomi E. Monte E. Octeau J.C. Yu X. Cohn W. Rajendran P.S. Vondriska T.M. Whitelegge J.P. et al.Neural circuit-specialized astrocytes: transcriptomic, proteomic, morphological, and functional evidence.Neuron. 2017; 95: 531-549.e9Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar) and qPCR data showing enrichment of GABAB receptor Gabbr1 and Gabbr2 mRNAs in striatal astrocytes (Figure 1D; 4 mice). GABAB receptor type 1 (GB1R) proteins (gene: Gabbr1) were abundant in striatal astrocytes isolated by fluorescence-activated cell sorting (FACS) (Chai et al., 2017Chai H. Diaz-Castro B. Shigetomi E. Monte E. Octeau J.C. Yu X. Cohn W. Rajendran P.S. Vondriska T.M. Whitelegge J.P. et al.Neural circuit-specialized astrocytes: transcriptomic, proteomic, morphological, and functional evidence.Neuron. 2017; 95: 531-549.e9Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar) from Aldh1l1-EGFP mice (Figure 1E; n = 6, 20 mice). Furthermore, consistent with functional expression of GABA receptors in astrocytes, bath application of GABA (300 μM; n = 24 astrocytes, 5 mice) and the GABAB receptor agonist baclofen (50 μM; n = 20 astrocytes, 5 mice) increased astrocyte Ca2+ signals (Figure 1F; p < 0.01). The effect of baclofen was blocked by the GABAB receptor antagonist CGP55845 (10 μM; n = 15 astrocytes, 4 mice; Figure 1F), which also blocked the MSN depolarization-evoked astrocyte Ca2+ signals (Figure 1F; n = 18 astrocytes, 4 mice). Astrocyte Ca2+ responses evoked by baclofen and by MSN depolarization were abolished (Figure 1G) in mice in which GB1Rs were deleted from the striatum (Figures 1G, S2A, and S2B). We explored whether MSN depolarization stimulated astrocytes via GABA in vivo. We expressed ChR2(H134R) in MSNs and assessed immediate-early gene (c-Fos) expression in astrocytes following optical stimulation. We detected GB1R-dependent c-Fos expression in astrocytes following MSN ChR2(H134R) stimulation. The optically stimulated increase in c-Fos expression in astrocytes was significantly reduced when GB1R was deleted (Figures 1H and 1I; 4 mice). Ca2+ signals are a readout of diverse astrocyte G-protein-coupled receptors (GPCRs) (Porter and McCarthy, 1997Porter J.T. McCarthy K.D. Astrocytic neurotransmitter receptors in situ and in vivo.Prog. Neurobiol. 1997; 51: 439-455Crossref PubMed Scopus (405) Google Scholar). GB1Rs couple to Gi proteins, which in astrocytes (Haustein et al., 2014Haustein M.D. Kracun S. Lu X.H. Shih T. Jackson-Weaver O. Tong X. Xu J. Yang X.W. O’Dell T.J. Marvin J.S. et al.Conditions and constraints for astrocyte calcium signaling in the hippocampal mossy fiber pathway.Neuron. 2014; 82: 413-429Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) leads to Ca2+ elevation by activation of phospholipase C, which we confirmed for the GABAB receptor responses (Figures S2C and S2D). Furthermore, MSNs intermingled extensively with astrocytes (Chai et al., 2017Chai H. Diaz-Castro B. Shigetomi E. Monte E. Octeau J.C. Yu X. Cohn W. Rajendran P.S. Vondriska T.M. Whitelegge J.P. et al.Neural circuit-specialized astrocytes: transcriptomic, proteomic, morphological, and functional evidence.Neuron. 2017; 95: 531-549.e9Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, Octeau et al., 2018Octeau J.C. Chai H. Jiang R. Bonanno S.L. Martin K.C. Khakh B.S. An Optical Neuron-Astrocyte Proximity Assay at Synaptic Distance Scales.Neuron. 2018; 98: 49-66.e9Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and their dendrites were closely juxtaposed with astrocyte somata and processes (Figures S2E and S2F; n = 26 images, 4 mice), providing the proximity for MSN-released GABA to stimulate astrocyte GABA receptors. We hypothesize that MSNs release GABA from their dendrites to mediate astrocyte responses; dendritic release of neurotransmitters, including GABA, is known (Waters et al., 2005Waters J. Schaefer A. Sakmann B. Backpropagating action potentials in neurones: measurement, mechanisms and potential functions.Prog. Biophys. Mol. Biol. 2005; 87: 145-170Crossref PubMed Scopus (125) Google Scholar). It has also been suggested that hippocampal astrocytes respond to glutamate, ATP, and/or endocannabinoid release from dendrites (Bernardinelli et al., 2011Bernardinelli Y. Salmon C. Jones E.V. Farmer W.T. Stellwagen D. Murai K.K. Astrocytes display complex and localized calcium responses to single-neuron stimulation in the hippocampus.J. Neurosci. 2011; 31: 8905-8919Crossref PubMed Scopus (48) Google Scholar, Navarrete and Araque, 2008Navarrete M. Araque A. Endocannabinoids mediate neuron-astrocyte communication.Neuron. 2008; 57: 883-893Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar) via release mechanisms that are not yet delineated. Taken together, our data provide strong evidence for MSN-to-astrocyte signaling mediated by neuronal GABA release acting on astrocyte GABAB receptors (Figures 1, S1, and S2). We comment on our use of mice carrying a floxed (f/f) Gabbr1 allele and the use of adeno-associated viruses (AAVs). In the preceding sections, we deleted GB1Rs from astrocytes using striatal AAV2/5 GfaABC1D-Cre microinjections. We could identify astrocytes based on their bushy morphologies as well as by marker expression (Figures S2A and S2B), and we could therefore easily monitor the consequences of deleting GB1Rs in single-cell evaluations (Figures 1G and 1I). However, as reported in Figures S3A–S3D and the associated legend, we could not use Gabbr1 f/f mice for astrocyte-selective evaluations of more complex phenomena, such as animal behavior. To explore the consequences of GB1R Gi pathway activation in astrocytes, we used chemogenetic approaches that were fully validated for astrocyte selectivity (Adamsky et al., 2018Adamsky A. Kol A. Kreisel T. Doron A. Ozeri-Engelhard N. Melcer T. Refaeli R. Horn H. Regev L. Groysman M. et al.Astrocytic Activation Generates De Novo Neuronal Potentiation and Memory Enhancement.Cell. 2018; 174: 59-71.e14Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, Chai et al., 2017Chai H. Diaz-Castro B. Shigetomi E. Monte E. Octeau J.C. Yu X. Cohn W. Rajendran P.S. Vondriska T.M. Whitelegge J.P. et al.Neural circuit-specialized astrocytes: transcriptomic, proteomic, morphological, and functional evidence.Neuron. 2017; 95: 531-549.e9Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). GABAB receptors exist in multiple brain cells, including neurons; therefore, GABAB receptor agonists cannot be used in vivo to interrogate astrocyte GABAB receptor-mediated physiology. Furthermore, currently available genetic strategies cannot selectively delete GABAB receptors only from striatal astrocytes in the adult brain (Figures S3A–S3D). Hence, to specifically explore the consequences of striatal astrocyte GABAB Gi pathway activation in vivo, which is necessary to interpret behavioral effects, we expressed human M4 muscarinic (hM4) receptor (hM4Di) designer receptors exclusively activated by designer drugs (DREADDs) (Roth, 2016Roth B.L. DREADDs for Neuroscientists.Neuron. 2016; 89: 683-694Abstract Full Text Full Text PDF PubMed Scopus (794) Google Scholar) using established methods that result in selective expression within 84% ± 3% of striatal astrocytes (Chai et al., 2017Chai H. Diaz-Castro B. Shigetomi E. Monte E. Octeau J.C. Yu X. Cohn W. Rajendran P.S. Vondriska T.M. Whitelegge J.P. et al.Neural circuit-specialized astrocytes: transcriptomic, proteomic, morphological, and functional evidence.Neuron. 2017; 95: 531-549.e9Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, Yu et al., 2018Yu X. Taylor A.M.W. Nagai J. Golshani P. Evans C.J. Coppola G. Khakh B.S. Reducing astrocyte calcium signaling in vivo alters striatal microcircuits and causes repetitive behavior.Neuron. 2018; 99: 1170-1187.e9Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) using AAVs (Figures S3E–S3G; 4 mice). hM4Di and GCaMP6f were also co-expressed so that the consequences of hMD4i activation could be imaged (Figure 2A; n = 34 mice). We confirmed that intrastriatal microinjection of AAV2/5-delivered cargo was astrocyte-selective and restricted to the striatum, although there was a little expression proximal to the needle tract in astrocytes of the cortex and, sometimes, of the corpus callosum (Figure S3E). We suspect that such expression occurred in all past studies employing viruses because it is impossible to reach subcortical brain structures without advancing the needle through the overlying tissue; all studies employing microinjections (including ours) need to be interpreted with this anatomical caveat in mind. In brain slices from control mice, the hM4Di agonist clozapine-N-oxide (CNO; 1 μM) had no effect on astrocyte Ca2+ signals (Figures 2B and 2C; Video S1; n = 14 astrocytes, 4 mice). However, in brain slices from mice expressing hM4Di in striatal astrocytes, CNO evoked significant astrocyte Ca2+ elevations (Figures 2B and 2C; Video S2; n = 11 astrocytes, 4 mice; p < 0.0001). These were similar to those mediated by GABAB receptors (Figure 1F) and other endogenous GPCRs (the CNO-evoked response area was 52.1 ± 8.4 dF/F.sec, whereas that for phenylephrine (Srinivasan et al., 2016Srinivasan R. Lu T.Y. Chai H. Xu J. Huang B.S. Golshani P. Coppola G. Khakh B.S. New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.Neuron. 2016; 92: 1181-1195Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) acting on α1 receptors was 62.5 ± 8.8 dF/F.sec; n = 11 and 12 astrocytes, n = 4 and 3 mice). Furthermore, 2 h after acute in vivo administration of CNO (Alexander et al., 2009Alexander G.M. Rogan S.C. Abbas A.I. Armbruster B.N. Pei Y. Allen J.A. Nonneman R.J. Hartmann J. Moy S.S. Nicolelis M.A. et al.Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors.Neuron. 2009; 63: 27-39Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar), striatal astrocytes in brain slices displayed significantly elevated spontaneous Ca2+ signals (Figures 2E and 2F; n = 21 and 28 astrocytes, n = 3 and 4 mice). In vivo hM4Di activation by CNO increased c-Fos expression in striatal astrocytes (Figure 2F; n = 4 mice). Thus, CNO stimulated hM4Di-expressing striatal astrocytes to a level similar to that" @default.
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• W2940727119 date "2019-05-01" @default.
• W2940727119 modified "2023-10-16" @default.
• W2940727119 title "Hyperactivity with Disrupted Attention by Activation of an Astrocyte Synaptogenic Cue" @default.
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• W2940727119 doi "https://doi.org/10.1016/j.cell.2019.03.019" @default.
• W2940727119 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6526045" @default.
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