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Regulation of Mammalian Spinal Locomotor Networks by Glial Cells

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Regulation of Mammalian Spinal Locomotor Networks by Glial Cells Regulation of Mammalian Spinal Locomotor Networks by Glial Cells David Acton This thesis is submitted in partial fulfilment for the degree of PhD at the University of St Andrews September 2016 i ii Acknowledgements I would like to express my sincere gratitude to my supervisor Dr Gareth Miles and the members of his lab during my time there. I would also like to acknowledge the other members of the Spinal Cord and Movement group, from whom I have learnt a great deal. I wish to thank the Wellcome Trust for funding my studentship and the University of St Andrews for all the assistance it has provided. The research presented here would not have been possible without the technical assistance of John Macintyre and the staff of St Mary’s Animal Unit, for which I am also very grateful. Finally, I would like to thank my family for their enormous support and encouragement throughout my studies. i Abstract Networks of interneurons within the spinal cord coordinate the rhythmic activation of muscles during locomotion. These networks are subject to extensive neuromodulation, ensuring appropriate behavioural output. Astrocytes are proposed to detect neuronal activity via Gαq- linked G-protein coupled receptors and to secrete neuromodulators in response. However, there is currently a paucity of evidence that astrocytic information processing of this kind is important in behaviour. Here, it is shown that protease-activated receptor-1 (PAR1), a Gαq- linked receptor, is preferentially expressed by glia in the spinal cords of postnatal mice. During ongoing locomotor-related network activity in isolated spinal cords, PAR1 activation stimulates release of adenosine triphosphate (ATP), which is hydrolysed to adenosine extracellularly. Adenosine then activates A1 receptors to reduce the frequency of locomotor-related bursting recorded from ventral roots. This entails inhibition of D1 dopamine receptors, activation of which enhances burst frequency. The effect of A1 blockade scales with network activity, consistent with activity-dependent production of adenosine by glia. Astrocytes also regulate activity by controlling the availability of D-serine or glycine, both of which act as co-agonists of glutamate at N-methyl-D-aspartate receptors (NMDARs). The importance of NMDAR regulation for locomotor-related activity is demonstrated by blockade of NMDARs, which reduces burst frequency and amplitude. Bath-applied D-serine increases the frequency of locomotor-related bursting but not intense synchronous bursting produced by blockade of inhibitory transmission, implying activity-dependent regulation of co-agonist availability. Depletion of endogenous D-serine increases the frequency of locomotor-related but not synchronous bursting, indicating that D-serine is required at a subset of NMDARs expressed by inhibitory interneurons. Blockade of the astrocytic glycine transporter GlyT1 increases the frequency of locomotor-related activity, but application of glycine has no effect, indicating that GlyT1 regulates glycine at excitatory synapses. These results indicate that glia play an important role in regulating the output of spinal locomotor networks. ii Contents Acknowledgements ......................................................................................................................... i Abstract ............................................................................................................................................... ii Chapter 1: General introduction ............................................................................................... 1 Spinal locomotor networks in mammals ....................................................................................... 1 Excitatory transmission in mammalian spinal locomotor networks ...................................... 6 Neuromodulation of spinal locomotor networks .......................................................................... 7 Dopaminergic modulation ......................................................................................................... 11 Adenosinergic modulation ......................................................................................................... 14 Modulation of NMDARs via the co-agonist binding site ....................................................... 20 Information processing by astrocytes.......................................................................................... 25 Astrocyte identity ........................................................................................................................ 35 Outline of study ............................................................................................................................... 36 Chapter 2: Stimulation of glia reveals modulation of mammalian spinal locomotor networks by adenosine .......................................................................... 38 Introduction ...................................................................................................................................... 38 Methods ........................................................................................................................................... 41 Ethics Statement ........................................................................................................................ 41 Tissue preparation ..................................................................................................................... 41 Ventral root recordings from whole spinal cords ................................................................... 41 Ventral root recordings from hemicords .................................................................................. 42 Whole-cell patch-clamp recordings ......................................................................................... 43 Biosensor recordings ................................................................................................................. 43 Data analysis ............................................................................................................................... 44 Immunohistochemistry ............................................................................................................... 45 Drug and Solution Preparation ................................................................................................. 46 iii Results ............................................................................................................................................. 46 Stimulation of glia modulates locomotor network output ...................................................... 46 Network modulation following PAR1 activation is mediated by adenosine derived from ATP .................................................................................................... 51 Glial cell-derived adenosine does not modulate excitatory components of the locomotor circuitry .................................................................................... 54 Glial cell-derived adenosine mediates feedback inhibition of locomotor network activity ......................................................................................................... 57 Three-enzyme microelectrode biosensors for the detection of adenosine are not suitable for use in a mouse spinal cord preparation ............................ 59 Adenosine modulates the frequency of synaptic inputs onto interneurons during locomotor-related activity in hemicords ............................................... 62 Discussion ....................................................................................................................................... 70 Chapter 3: Adenosine derived from glia activates A1 receptors to inhibit signalling by excitatory D1-like dopamine receptors ...................................... 79 Introduction ...................................................................................................................................... 79 Methods ........................................................................................................................................... 82 Ethics Statement ........................................................................................................................ 82 Tissue preparation ..................................................................................................................... 82 Ventral root recordings .............................................................................................................. 82 Data analysis ............................................................................................................................... 83 Solution, drug and enzyme preparation .................................................................................. 83 Results ............................................................................................................................................. 84 Selective activation of D1-like receptors increases the frequency of locomotor-related network activity ....................................................................................... 84 Activation of D1-like receptors is required for the modulation of locomotor frequency by glial cell-derived adenosine ............................................................ 84 Glial-cell derived adenosine modulates locomotor-related activity in a PKA-dependent manner ...................................................................................................
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