Microcircuitry in the entorhinal cortex [Elektronische Ressource] / by Prateep Sanker Beed

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• Publié par: freie_universitat_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 29
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Microcircuitry in the
Entorhinal Cortex


DISSERTATION

To obtain the academic degree
Doctor rerum naturalium (Dr. rer. nat.)


submitted to the
Department of Biology, Chemistry and Pharmacy
of Free University, Berlin


by
Prateep Sanker Beed
from Calcutta, India



January, 2010
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This experimental work of this thesis was completed under the supervision of Prof. Dr.
Dietmar Schmitz in the time period October 2006 – September 2009 at the Neuroscience
Research Centre, Charité, Berlin, Germany.


st1 Reviewer : Prof. Dr. Dietmar Schmitz
nd2 : Prof. Dr. Stephan Sigrist

thDate of Defence : 28 April, 2010
3 4 CONTENTS
Abreviatons 9
Abstract 11

1 Neuronal Microcircuits 15
1.1 Hierarchical organization
1.2 Establishment of microcircuits over development 16
1.3 Features of a microcircuit 17
1.3a Defining a mi
1.3b Structural stereotypy 18
1.3c Functional heterogeneity
1.4 The entorhinal cortex 19
1.4a Medial and lateral subdivisions 20
1.4b Layered structure 21
1.4c Extrinsic and intrinsic connectivity 22

2 Functional Microcircuitry of the Medial Entorhinal Cortex 27
2.1 Key functions of the medial entorhinal cortex 28
2.1a Spatial navigation 28
2.1b Excitability and synchrony 30
2.2 What can we learn from the microcircuitry of the medial entorhinal
cortex? 31
2.2a Layer 2 stellate and layer 2 pyramidal cells in spatial navigation 31
2.2b Layer 3 pyramidal cells in excitability and synchrony 32

3 Mapping Neuronal Microcircuitry by Photostimulation 35
3.1 Techniques to study neuronal microcircuitry 35
3.1a Anatomical studies 35
3.1b Fluorescent mice – GFP constructs 36
3.1c Focal flash photolysis of caged glutamate 36
2+3.1d Ca indicator methods 37
3.1e Activation of specific cell classes by a light-activated cation
channel and inhibition with a light-activated chloride pump 38
5 Contents
____________________________________________________________ ______________________________
3.1f Dual/multiple recordings of synaptically connected neurons 38
3.2 Our approach: Fast-scanning photostimulation (optimized
focal UV photolysis) to study neuronal microcircuits 39
3.3 Design of the control software 41
3.3a Navigation screen 41
3.3b Stimulation 43
3.4 Scanning process 44
3.5 Optical reference points
3.6 Two-objective method 44
3.7 A posteriori visual identification of optically stimulated targets 45
3.8 Effective resolution of the system: photo-induced spatial response profile 45
3.9 Mapping the functional connectivity of entorhinal layer 2 stellate cells 48
3.9a Stimulation protocol 49
3.9b Classification of signals: direct, indirect photo-induced
and indirect spontaneous PSCs 49
3.9c Specifying time windows 49
3.9d Quantification of point-specific synaptic input 52
3.9e Input maps 52
3.10 Inter-trial variability 54
3.11 Discussion and Outlook 55
3.11a One-photon versus two-photon uncaging 56
3.11b Comparison to other one-photon uncaging setups 56
3.11c Effective resolution of the system 58
3.11d Mapping of indirect responses 58

4 Cell-type Specific and Modular Organization of Microcircuitry in the
Medial Entorhinal Cortex 61
4.1 Calibration of spatial profiles of excitatory cells 62
4.2 Mapping excitatory layer 2 projection neurons 64
4.3 Synaptic inputs onto layer 2 stellate and pyramidal cells 66
4.4 Modular organization of deep layer inputs 68
4.5 Discussion and Outlook 69

5 Neuronal Synchrony and Excitability: Microcircuits to Receptors 75
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5.1 Role of kainate receptors in neuronal synchrony and excitability 76
5.2 Kainate receptors in the entorhinal cortex 77
5.3 GluK2 containing kainate receptors in neuronal synchrony 78
5.4 Kainate model of epilepsy 79
5.5 Neuronal loss following seizures in the entorhinal cortex 80
5.6 The layer 3 pyramidal cell in the medial entorhinal cortex 81
5.7 Kainate concentration dependent changes in holding current 81
5.8 Kainate receptor mediated currents 82
5.9 GluK2 is the major subunit mediating the kainate receptor current 85
5.10 Characterization of the kainate receptors 85
5.11 Synaptic activation of kainate receptors 87
5.12 Discussion and Outlook 90
5.13 Pathophysiological hyperexcitability 93


Summary 101

Appendices: Materials and Methods
A Mapping neuronal microcircuitry 106
B Cell-type specific and modular organization of microcircuitry 111
C Neuronal synchrony and excitability 112

References 115

Statement of Contribution 125

Deutsche Zusammenfassung 126
Resume 129
Publications 130
Conference Proceedings 131

Acknowledgements 132

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ABBREVIATIONS




ACSF - A rtifical Cerebro-Spinal Fluid P ction Potential
CA1-3 C ornu ammonis Area 1-3
DG D entate Gyrus
EC - E ntorhinal Cortex G lectroencephalogram / graphy
EPSC xcitatory Postsynaptic Current
m M edial Entorhinal Cortex
P - P ostnatal days
pA p icoAmpere
IUE in utero Electroporation
KO K nockout
L1–6 - Layers I,II, III,IV, Vand VI
L2S - L ayer II Stellate cell P ayer II Pyramidal cell
L3 ayer III Pyramidal cell PA ysophosphatidic Acid
L - ipid phosphate phosphatase
PRG P lasticity Related Gene
TLE T emporal Lobe Epilepsy
UV U ltraviolet
WT - W ildtype
9 10