Ultrastructural Contributions to Desensitization at the Cerebellar Mossy Fiber to Granule Cell Synapse

Transcription

1 Ultrastructural Contributions to Desensitization at the Cerebellar Mossy Fiber to Granule Cell Synapse Matthew A.Xu-Friedman and Wade G. Regehr Department of Neurobiology, Harvard Medical School, Boston, MA Pittt 2003 Bridgett Payne June 5,

2 Overview Neuroscience Terminology Introduction to the Paper Materials and Methods Results- electrophysiology, visualization, and modeling Results Discussion and Summary 2

3 Neuroscience Terms Neurons- dendrites, cell body, axon, presynaptic terminals Synapses- active zone neurotransmitter, PSD Mossy Fiber to Granule Cell; Glutamate Neurons Synapses Receptors Receptors- gate ion channels AMPA Glia- surround and support neurons, uptake glutamate Images taken from 3

4 Synaptic Transmission 4

5 Introduction to Paper: Isolated Synapses Synapses Spatially Interacting in Extracellular Space Hypothesis: Ultrastructure Influences Short-term term Depression at the Mossy Fiber to Granule Cell Synapse 1. Is desensitization a contributor to short-term term plasticity? Electrophysiology 2. Does the ultrastructure contribute to desensitization via spillover? Anatomy and Modeling 5

6 UltraStructure Ultrastructure : Synaptic Architecture Glomerular Structure Small diffusion space Implicated in Spillover, and Glutamate Pooling Spillover: : Glutamate released at one synapse affects neighboring synapes Pooling: : The build up of extracellular glutamate Mossy fiber terminal Glial Contact Postsynaptic densities on granule cell dendrites Mossy fiber terminal 6

7 Nerve Synapses in the Cerebellum Cerebellar neurons are organized in layers Mossy fibers carry input to granule cells Mossy fiber to granule cell synapses occur the glomerulus Image: Eagleman, D.M; Bartol, T.M., Cerebellar Glomerli, Proceedings of the 8th Annual Joint Symposium on Neural Computation 7

8 Short-Term Synaptic Plasticity and Desensitization: Synaptic plasticity: Use-dependent change in synaptic strength Facilitation : Increase Depression: Decrease Postsynaptic Response Depression may be monitored through the application of paired pulses Desensitization: Decreased sensitivity to neurotransmitter following prolonged exposure When receptors enter a desensitized state, they do not respond to the presence of glutamate and reactivation may be delayed Post-synaptic synaptic AMPA receptor desensitization may be a contributor to paired-pulse pulse depression 8

9 Materials and Methods to evaluate the role of desensitization Short-term term Plasticity Electrophysiology Postsynaptic Whole Cell Voltage-clamp recordings Granule Cell Cells voltage clamped to -70 mv, presynaptic mossy fibers were stimulated and EPSC recorded. Ultrastucture Electron Microscopy Ultrastucture Slices of rat cerebellum were prepared, photographed and processed ed to give 3D reconstructions. Simulations Modeling Glutamate Diffusion and AMPA receptor desensitization Diffusion of single vesicle from single release site on infinite plane surface 2 Models for AMPA receptor desensitization 9

10 Electrophysiology Results Paired Pulse Depression & AMPA Receptor Desensitization To Quantify Depression: PPR=Paired Pulse Ratio =EPSC 2 /EPSC 1 Effects of Cyclothiazide Blocks Desensitization Reduces Depression Ca concentrations reflect different conditions of initial release probability (p r ) 2 Ca e = 2mM Ca 3 Ca e = 3mM Ca Desensitization is a factor Does Spillover contribute? 10

11 Mossy Fiber Glomerulus Ultrastructure Single Electron Microscopic Section Mossy Fiber Axon: Blue Granule Cell Dendrites: Pink Glial Processes: Yellow Serial Sections through one release site On the order of nm (~60 nm) Presynaptic vesicles (~50 nm) Widening of Synaptic Cleft (~20 nm) Postsynaptic Density 1µm 11

12 Synaptic Visualization Serial Electron Microscopic Reconstructions 3D Morphology Mossy Fibers Climbing Fibers Postsynaptic Densities Glial Contact Mossy fiber Visualize the density of release sites Stereopairs Much less dense on climbing fibers Location and abundance of glia on nerve terminals Result: : Average Distance Between Release Sites : 0.46 µm Average of 7.1 neighbors within 1 µm 12

13 Describing the Dynamics of Glutamate Diffusion of Glutamate Concentration Geometry of Extracellular Space Approximated as flat plane Glutamate Pumps and Buffers Not included in model due to their minor role at the mossy fiber glomerulus 13

14 Glutamate Dynamics: Modeling Glutamate Diffusion C(r, t) =? C 3 2 r 4Dt Setting Parameters 0 3tD? e Equation for a Gaussian Distribution Assumption of radial symmetry allows the solution of the differential equation for concentration Electron micrographs: Vesicle Radius? = 25 nm Cleft width (approximate)? = 20 nm Ranges from Literature Vesicle Glutamate Concentration C o ~ 100 mm Diffusion Constant D = 0.4 µm 2 /msec 14

15 Glutamate Diffusion Results Glutamate Concentration Decreases with the distance from release site at specific times Varies in time since release at specific distances- a wave of glutamate concentration passes at a fixed radial distance (Log scale on the y-axis) 15

16 Modeling AMPA Receptor Desensitization Do the calculated levels of Glutamate Induce Significant AMPA Desensitization? Characterizing granule cell AMPA receptors directly is not feasible: Granule cell AMPA receptors are not expressed on somata unless held in culture Instead: 2 Different AMPA Receptor Models 1. Purkinje Cells : Slowly Desensitizing Receptors 2. Chick Magnocellularis neurons: Rapidly Desensitizing Receptors Model 1 Glutamate Kinetics for a single vesicle from a single release site were passed through the models to determine extent of desensitization site were passed through the models to determine extent 16 of

17 Modeling Results A Significant Proportion of AMPA receptors are desensitized by 10 msec After 10 msec at 1.0µm from release site: 1. Slowly Desensitizing: 19 % 2. Rapidly Desensitizing: >60 % There are an Spillover from a single release site can desensitize receptors at many adjacent sites. 17

18 Summary for the Mossy Fiber to Granule Cell Synapse Electrophysiology Synapse Exhibits Paired-Pulse Pulse Depression A portion of this depression is caused by postsynaptic receptor desensitization Ultrastructure There is a very high density of release sites The ultrastructure suggests that spillover and pooling from many release sites is likely Simulations There is a significant desensitization of receptors with the release of a single vesicle Receptors with a high affinity for glutamate and fast desensitization rates show heightened desensitization Spillover may account for desensitization even when the probability of release (p r ) is low 18

19 Critique Short-term term depression is only partially described Receptor desensitization is a part of the depression - Vesicle Depletion - Saturation of Receptors Extent of Desensitization is Inconclusive Actual Convolutions of Extracellular Space A more precise ultrastructural model would better describe diffusion and spillover 19

20 Acknowledgements Many thanks to: The members of my Journal Club Group Paul Gera David Sivakoff The Students of the Stiles Lab John Pattillo, Will Ford, and Jack Chang Dr. Joel R. Stiles BBSI 20