Role of Actin-mediated Motility of Peripheral Astrocytic Processes in Synaptic Function

ABBREVIATIONS INTRODUCTION . 1 REVIEW OF THE LITERATURE . 1 1. Different glial cell types and their role in the mammalian brain . 1 1.1 Radial glia and glial progenitor cells . 2 1.2 Oligodendrocytes and Schwann cells . 3 1.3 Microglial cells . 3 1.4 Astroglial cells (a.k.a. astrocytes) . 4 2. Astroglia functions in the brain . 5 2.1 Metabolic support of neurons by astrocytes . 5 2.2 Blood-brain barrier (BBB) formation and regulation by astrocytes . 7 2.3 Astrocytic function in maintenance and regulation of extracellular matrix (ECM) . 9 2.4 Astrocytic coupling through gap junctions . 12 2.5 Ion channels, transporter and receptors expressed in astrocytic plasma membrane . 14 2.5. 1. Glutamate transporters . 14 2.5. 2. Connexin and pannexin hemichannels . 16 2.5. 3. Glutamate receptors . 17 2.5. 4. GABA receptors and transporters. 17 2.5. 5. Channels and transporters involved in D-serine release from astrocytes . 18 2.5. 6. Water and potassium channels . 18 2.5. 7. Purinoreceptors . 18 2.5. 8. Ephrin mediated reception . 19 2.6 Chemical excitability and calcium signaling in astrocytes . 19 2.7 Modulation of synaptic transmission by astrocytes: the concept of multipartite synapse . 21 2.7. 1. Presynaptic mechanisms of astrocyte action . 23 2.7. 1.1. Presynaptic action of purines released from astrocytes . 23 2.7. 1.2. Modulation of synaptic events by presynaptic glutamate . 24 2.7. 2. Postsynaptic mechanisms of astrocyte action . 25 2.7. 2.1. D-serine release from astrocytes . 25 2.7. 2.2. Postsynaptic action of Glycine . 26 2.7. 2.3. Postsynaptic action of astrocyte derived ATP . 26 2.7. 2.4. Neurotransmitter and ion clearance by astrocytic transporters and regulation of spillover . 27 3. Peripheral astrocytic process as a functional microdomain . 27 3.1 Ultrastructure of PAPs . 28 3.2 Receptors and transporters in PAPs . 29 3.3 Morphological changes of PAPs: two different mechanisms proposed . 30 3.3.1. Aquaporin mediated morphological changes . 30 3.3.2. Actin-dependent morphological changes . 30 4. Methodological approaches to study neuro-glial interactions . 32 4.1 Hypothalamus as a classical model of PAPs retraction . 32 4.2 Transgenic mouse models to study astrocytes . 32 4.3 Acute brain slices and organotypic cultures to study neuro-glial interactions . 34 4.4 In vivo methods to probe astrocytic morphological and functional changes . 35 4.4.1. Two-photon microscopy on living mouse brain . 35 4.4.1.1. Probes and dyes to label astroglia in vivo for TPEM . 36 4.4.1.2. In vivo microscopy on anaesthetized animals . 37 4.4.1.3. In vivo microscopy on awake animals . 38 4.5 Gene delivery to astrocytes . 39 4.5. 1. Specificity and efficiency of gene delivery to astrocytes . 39 4.5. 2. Gene-gun mediated gene delivery (biolistics) . 40 4.5. 3. In vivo electroporation . 41 4.5. 4. Viral gene delivery methods to get astrocyte specificity in vivo . 42 4.5. 4.1. Adeno-associated viral vectors for astrocytes . 42 4.5. 4.2. Lentiviral vectors for astrocytes . 43 4.5. 4.3. Adenoviral vectors for astrocytes . 44 AIMS . 46 METHODS . 47 Image analysis (I, II and III) . 47 Primary cortical astrocyte cultures (I) . 48 Photolysis of caged calcium and live cell imaging (I). 48 Postnatal in vivo electroporation (II) . 48 Preparation and in vivo application of adenoviral vectors (III) . 49 In vivo two-photon microscopy through the cranial window (III) . 49 Intrinsic BOLD signal optical imaging (III) . 50 Experimental animals (I, II and III) . 50 RESULTS AND DISCUSSION . 51 1. The role of Profilin-1 in the activity-dependent outgrowth of astrocytic peripheral processes (study I). 51 1.1 Actin turnover as the major mediator for Ca 2+ -dependent PAPs motility in vitro . 51 1.2 The novel tool to suppress activity-dependent PAPs motility . 53 2. Gene delivery to a postnatal rodent brain (studies II and III). 53 2.1 Stereotaxic plasmid microinjection and electroporation on neonatal rat brain (study II) . 53 2.2 Astrocyte-specific gene delivery using adenoviral vectors (study III). 55 3. Motility of cortical astrocytes and neuronal function (study III). 57 3.1 Astroglia spontaneous morphological changes are Profilin-1 dependent. 57 3.2 Structure-functional interplay: the effect of suppressed astrocytic motility on neuronal processing. 58 CONCLUSIONS . 61 ACKNOWLEDGEMENTS . 62 LIST OF REFERENCES . 63 LIST OF ORIGINAL PUBLICATIONS I. Molotkov D*. , Zobova S*. , Arcas JM. , Khiroug L. (2013) Calcium-induced outgrowth of astrocytic peripheral processes requires actin binding by Profilin-1. Cell Calcium 53: 338-348. The candidate substantially contributed to the experimental design, designed and performed molecular biology manipulations, performed microscopic experiments together with SZ, designed analysis approaches, analyzed the data together with SZ and wrote the manuscript together with SZ and LK. II. Molotkov D. , Yukin A. , Afzalov R. , Khiroug L. (2010) Gene delivery to postnatal rat brain by non-ventricular plasmid injection and electroporation. J of Vis Exp 43. The candidate designed experiments together with AY, performed experiments and analyzed the data, prepared the video together with RA, LK and AY, wrote the manuscript together with LK. III. Molotkov D. , Kislin M. , Zobova S. , Toptunov D. , Castren E. , Khiroug L. (2014) Suppression of astrocytic morphological changes does not affect BOLD signal during visual processing in anesthetized mice. Manuscript. The candidate substantially contributed to the experimental design, planed and performed molecular biology and viral work, performed BOLD signal acquisition experiments, participated in in vivo microscopy experiments, designed data analysis approaches together with DT and MK, analyzed the data and wrote the manuscript.