Abstract
Several studies have contributed to our understanding of astrocytes, especially Bergmann glia, in the cerebellum; but, until recently, none has looked at their function in vivo. Multicell bolus loading of fluorescent calcium indicators in combination with the astrocytic marker SR101 has allowed imaging of up to hundreds of astrocytes at once in the intact cerebellum. In addition, the selective targeting of astrocytes with fluorescent calcium indicator proteins has enabled the study of their function in vivo without the confounding effects of other neuropil signals and with a resolution that surpasses multicell bolus loading and SR101 staining. The two astrocyte types of the cerebellar cortex, Bergmann glia, and velate protoplasmic astrocytes display a diverse signaling repertoire in vivo, which ranges from localized calcium elevations in subcellular processes to waves, triggered by the release of purines and mediated by purinergic receptors that span multiple processes and can involve tens of astrocytes. During locomotor behavior, even larger numbers of astrocytes display calcium increases that are driven by neuronal activity and correlate with global changes in blood flow. In this review, we give an overview of our current understanding of the function of Bergmann glia and velate protoplasmic astrocytes and the promise of the tools used to study their calcium dynamics and function in vivo.
This is a preview of subscription content,
to check access.

References
Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science (New York, NY) 248(4951):73–76
Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci U S A 100(12):7319–7324
Wang X, Lou N, Xu Q, Tian GF, Peng WG, Han X et al (2006) Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo. Nat Neurosci 9(6):816–823
Schummers J, Yu H, Sur M (2008) Tuned responses of astrocytes and their influence on hemodynamic signals in the visual cortex. Science (New York, NY) 320(5883):1638–1643
Ding S, Fellin T, Zhu Y, Lee SY, Auberson YP, Meaney DF et al (2007) Enhanced astrocytic Ca2+ signals contribute to neuronal excitotoxicity after status epilepticus. J Neurosci 27(40):10674–10684
Kuchibhotla KV, Lattarulo CR, Hyman BT, Bacskai BJ (2009) Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice. Science (New York, NY) 323(5918):1211–1215
Halassa MM, Florian C, Fellin T, Munoz JR, Lee SY, Abel T et al (2009) Astrocytic modulation of sleep homeostasis and cognitive consequences of sleep loss. Neuron 61(2):213–219
Prolo LM, Takahashi JS, Herzog ED (2005) Circadian rhythm generation and entrainment in astrocytes. J Neurosci 25(2):404–408
Suh J, Jackson FR (2007) Drosophila ebony activity is required in glia for the circadian regulation of locomotor activity. Neuron 55(3):435–447
Fellin T, Halassa MM, Terunuma M, Succol F, Takano H, Frank M et al (2009) Endogenous nonneuronal modulators of synaptic transmission control cortical slow oscillations in vivo. Proceedings of the National Academy of Sciences of the United States of America, Aug 17
Chan-Palay V, Palay SL (1972) The stellate cells of the rat's cerebellar cortex. Z Anat Entwicklungsgesch 136(2):224–248
Rakic P (1971) Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electronmicroscopic study in Macacus Rhesus. J Comp Neurol 141(3):283–312
Reichenbach A, Siegel A, Rickmann M, Wolff JR, Noone D, Robinson SR (1995) Distribution of Bergmann glial somata and processes: implications for function. J Hirnforsch 36(4):509–517
Halassa MM, Fellin T, Takano H, Dong JH, Haydon PG (2007) Synaptic islands defined by the territory of a single astrocyte. J Neurosci 27(24):6473–6477
Grosche J, Matyash V, Moller T, Verkhratsky A, Reichenbach A, Kettenmann H (1999) Microdomains for neuron-glia interaction: parallel fiber signaling to Bergmann glial cells. Nat Neurosci 2(2):139–143
Grosche J, Kettenmann H, Reichenbach A (2002) Bergmann glial cells form distinct morphological structures to interact with cerebellar neurons. J Neurosci Res 68(2):138–149
Castejon OJ, Dailey ME, Apkarian RP, Castejon HV (2002) Correlative microscopy of cerebellar Bergmann glial cells. J Submicrosc Cytol Pathol 34(2):131–142
Svoboda K, Tank DW, Denk W (1996) Direct measurement of coupling between dendritic spines and shafts. Science (New York, NY) 272(5262):716–719
Regan MR, Huang YH, Kim YS, Dykes-Hoberg MI, Jin L, Watkins AM et al (2007) Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the developing and mature CNS. J Neurosci 27(25):6607–6619
Rothstein JD, Martin L, Levey AI, Dykes-Hoberg M, Jin L, Wu D et al (1994) Localization of neuronal and glial glutamate transporters. Neuron 13(3):713–725
Bergles DE, Dzubay JA, Jahr CE (1997) Glutamate transporter currents in bergmann glial cells follow the time course of extrasynaptic glutamate. Proc Natl Acad Sci U S A 94(26):14821–14825
Matsui K, Jahr CE, Rubio ME (2005) High-concentration rapid transients of glutamate mediate neural-glial communication via ectopic release. J Neurosci 25(33):7538–7547
Clark BA, Barbour B (1997) Currents evoked in Bergmann glial cells by parallel fibre stimulation in rat cerebellar slices. J Physiol 502(Pt 2):335–350
Matsui K, Jahr CE (2004) Differential control of synaptic and ectopic vesicular release of glutamate. J Neurosci 24(41):8932–8939
Bellamy TC, Ogden D (2005) Short-term plasticity of Bergmann glial cell extrasynaptic currents during parallel fiber stimulation in rat cerebellum. Glia. 52(4):325–335
Iino M, Goto K, Kakegawa W, Okado H, Sudo M, Ishiuchi S et al (2001) Glia-synapse interaction through Ca2+-permeable AMPA receptors in Bergmann glia. Science (New York, NY) 292(5518):926–929
Piet R, Jahr CE (2007) Glutamatergic and purinergic receptor-mediated calcium transients in Bergmann glial cells. J Neurosci 27(15):4027–4035
Ishiuchi S, Tsuzuki K, Yamada N, Okado H, Miwa A, Kuromi H et al (2001) Extension of glial processes by activation of Ca2+-permeable AMPA receptor channels. Neuroreport 12(4):745–748
Riquelme R, Miralles CP, De Blas AL (2002) Bergmann glia GABA(A) receptors concentrate on the glial processes that wrap inhibitory synapses. J Neurosci 22(24):10720–10730
Hoogland TM, Kuhn B, Gobel W, Huang W, Nakai J, Helmchen F et al (2009) Radially expanding transglial calcium waves in the intact cerebellum. Proc Natl Acad Sci U S A 106(9):3496–3501
Nimmerjahn A, Mukamel EA, Schnitzer MJ (2009) Motor behavior activates Bergmann glial networks. Neuron 62(3):400–412
Kirischuk S, Scherer J, Kettenmann H, Verkhratsky A (1995) Activation of P2-purinoreceptors triggered Ca2+ release from InsP3-sensitive internal stores in mammalian oligodendrocytes. J Physiol 483(Pt 1):41–57
Beierlein M, Regehr WG (2006) Brief bursts of parallel fiber activity trigger calcium signals in bergmann glia. J Neurosci 26(26):6958–6967
Jung S, Pfeiffer F, Deitmer JW (2000) Histamine-induced calcium entry in rat cerebellar astrocytes: evidence for capacitative and non-capacitative mechanisms. J Physiol 527(Pt 3):549–561
Guthrie PB, Knappenberger J, Segal M, Bennett MV, Charles AC, Kater SB (1999) ATP released from astrocytes mediates glial calcium waves. J Neurosci 19(2):520–528
Bowser DN, Khakh BS (2004) ATP excites interneurons and astrocytes to increase synaptic inhibition in neuronal networks. J Neurosci 24(39):8606–8620
Hoogland TM, Civillico EF, Kuhn B (2007) Molecular layer interneurons relay cerebellar cortical activity to Bergmann glial cells. J Neurosci 27(42):11167–11169
Newman EA (1986) Regional specialization of the membrane of retinal glial cells and its importance to K+ spatial buffering. Ann NY Acad Sci 481:273–286
Kurth-Nelson ZL, Mishra A, Newman EA (2009) Spontaneous glial calcium waves in the retina develop over early adulthood. J Neurosci 29(36):11339–11346
Takano T, Tian GF, Peng W, Lou N, Libionka W, Han X et al (2006) Astrocyte-mediated control of cerebral blood flow. Nat Neurosci 9(2):260–267
Dombeck DA, Khabbaz AN, Collman F, Adelman TL, Tank DW (2007) Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron 56(1):43–57
Kuhn B, Hoogland TM, Nakai J, Flint J, Wang SS (2006) In vivo visualization of synaptically evoked glial calcium signals using G-cAMP-2. Program No 43311 2006 Neuroscience Meeting Planner Atlanta, GA: Society for Neuroscience, 2006 Online
Chan-Palay V, Palay SL (1972) The form of velate astrocytes in the cerebellar cortex of monkey and rat: high voltage electron microscopy of rapid Golgi preparations. Z Anat Entwicklungsgesch 138(1):1–19
Garaschuk O, Milos RI, Konnerth A (2006) Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo. Nat Protoc 1(1):380–386
Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2(12):932–940
Sullivan MR, Nimmerjahn A, Sarkisov DV, Helmchen F, Wang SS (2005) In vivo calcium imaging of circuit activity in cerebellar cortex. J Neurophysiol 94(2):1636–1644
Hirase H, Qian L, Bartho P, Buzsaki G (2004) Calcium dynamics of cortical astrocytic networks in vivo. PLoS Biol 2(4):E96
Ozden I, Sullivan MR, Lee HM, Wang SS (2009) Reliable coding emerges from coactivation of climbing fibers in microbands of cerebellar Purkinje neurons. J Neurosci 29(34):10463–10473
Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F (2004) Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 1(1):31–37
Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M et al (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388(6645):882–887
Regehr WG, Atluri PP (1995) Calcium transients in cerebellar granule cell presynaptic terminals. Biophys J 68(5):2156–2170
Mank M, Santos AF, Direnberger S, Mrsic-Flogel TD, Hofer SB, Stein V et al (2008) A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat Methods 5(9):805–811
Hasan MT, Friedrich RW, Euler T, Larkum ME, Giese G, Both M et al (2004) Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control. PLoS Biol 2(6):e163
Heim N, Garaschuk O, Friedrich MW, Mank M, Milos RI, Kovalchuk Y et al (2007) Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor. Nat Methods 4(2):127–129
Mao T, O'Connor DH, Scheuss V, Nakai J, Svoboda K (2008) Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators. PLoS ONE 3(3):e1796
Judkewitz B, Rizzi M, Kitamura K, Hausser M (2009) Targeted single-cell electroporation of mammalian neurons in vivo. Nat Protoc 4(6):862–869
Wallace DJ, Meyer zum Alten Borgloh S, Astori S, Yang Y, Bausen M, Kugler S et al (2008) Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor. Nat Methods 5(9):797–804
Burger C, Nguyen FN, Deng J, Mandel RJ (2005) Systemic mannitol-induced hyperosmolality amplifies rAAV2-mediated striatal transduction to a greater extent than local co-infusion. Mol Ther 11(2):327–331
Wang CY, Wang S (2006) Astrocytic expression of transgene in the rat brain mediated by baculovirus vectors containing an astrocyte-specific promoter. Gene Ther 13(20):1447–1456
Shevtsova Z, Malik JM, Michel U, Bahr M, Kugler S (2005) Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo. Exp Physiol 90(1):53–59
Lowery RL, Zhang Y, Kelly EA, Lamantia CE, Harvey BK, Majewska AK (2009) Rapid, long-term labeling of cells in the developing and adult rodent visual cortex using double-stranded adeno-associated viral vectors. Developmental Neurobiology, Jun 23
Colin A, Faideau M, Dufour N, Auregan G, Hassig R, Andrieu T et al (2009) Engineered lentiviral vector targeting astrocytes in vivo. Glia 57(6):667–679
Croci C, Fasano S, Superchi D, Perani L, Martellosio A, Brambilla R et al (2006) Cerebellar neurons and glial cells are transducible by lentiviral vectors without decrease of cerebellar functions. Dev Neurosci 28(3):216–221
Le Gal La Salle G, Robert JJ, Berrard S, Ridoux V, Stratford-Perricaudet LD, Perricaudet M et al (1993) An adenovirus vector for gene transfer into neurons and glia in the brain. Science (New York, NY) 259(5097):988–990
He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B (1998) A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 95(5):2509–2514
Nakai J, Ohkura M, Imoto K (2001) A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein. Nat Biotechnol 19(2):137–141
Tallini YN, Ohkura M, Choi BR, Ji G, Imoto K, Doran R et al (2006) Imaging cellular signals in the heart in vivo: cardiac expression of the high-signal Ca2+ indicator GCaMP2. Proc Natl Acad Sci U S A 103(12):4753–4758
Sato Y, Shiraishi Y, Furuichi T (2004) Cell specificity and efficiency of the Semliki forest virus vector- and adenovirus vector-mediated gene expression in mouse cerebellum. J Neurosci Methods 137(1):111–121
Boulos S, Meloni BP, Arthur PG, Bojarski C, Knuckey NW (2006) Assessment of CMV, RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures. Brain Res 1102(1):27–38
Palmer AE, Giacomello M, Kortemme T, Hires SA, Lev-Ram V, Baker D et al (2006) Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem Biol 13(5):521–530
Pascual O, Casper KB, Kubera C, Zhang J, Revilla-Sanchez R, Sul JY et al (2005) Astrocytic purinergic signaling coordinates synaptic networks. Science (New York, NY) 310(5745):113–116
Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8(9):1263–1268
Acknowledgments
We thank Drs. I. Ozden, E.F. Civillico, and S.S.-H. Wang for their valuable feedback on this manuscript. Parts of the work described in this review was supported by grants from NIH (RO1 NS 045193-05A2) and the New Jersey Governor’s Council for Autism.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hoogland, T.M., Kuhn, B. Recent Developments in the Understanding of Astrocyte Function in the Cerebellum In Vivo. Cerebellum 9, 264–271 (2010). https://doi.org/10.1007/s12311-009-0139-z
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12311-009-0139-z