Electrical synapses [Elektronische Ressource] : cellular morphology and identification of connexins in the mouse retina / von Luis Pérez de Sevilla Müller

Electrical synapses [Elektronische Ressource] : cellular morphology and identification of connexins in the mouse retina / von Luis Pérez de Sevilla Müller

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Electrical synapses: Cellular morphology and identification of connexins in the mouse retina Von der Fakultät V, Mathematik und Naturwissenschaften der Carl von Ossietzky Universität Oldenburg zur Erlangung des Grades und Titels eines Doktors der Naturwissenschaften (Dr. rer. nat.) angenommene Dissertation Von Herrn Luis Pérez de Sevilla Müller, geboren am 14.07.1979 in Madrid, Spanien Gutachter: Prof. Dr. Reto Weiler Zweitgutachter: Prof. Dr. Henrik Mouritsen Tag der Disputation: 18.09.2008 „ My devotion to the retina is ancient history. The subject always fascinated me because, to my idea, life never succeeded in constructing a machine so subtilely devised and so perfectly adapted to an end as the visual apparatus…… I must not conceal the fact that in the study of this membrane I for the first time felt my faith in Darwinism weakened, being amazed and confounded by the supreme constructive ingenuity revealed not only in the retina and in the dioptric apparatus of the vertebrates but even in the meanest insect eye. There, in fine, I felt more profoundly than in any other subject of study the shuddering sensation of the unfathomable mystery of life.

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Electrical synapses: Cellular morphology
and identification of connexins in the
mouse retina




Von der Fakultät V, Mathematik und Naturwissenschaften
der Carl von Ossietzky Universität Oldenburg
zur Erlangung des Grades und Titels eines Doktors der
Naturwissenschaften (Dr. rer. nat.) angenommene
Dissertation





Von Herrn Luis Pérez de Sevilla Müller,
geboren am 14.07.1979
in Madrid, Spanien















































Gutachter: Prof. Dr. Reto Weiler

Zweitgutachter: Prof. Dr. Henrik Mouritsen

Tag der Disputation: 18.09.2008







„ My devotion to the retina is ancient history. The subject always
fascinated me because, to my idea, life never succeeded in constructing
a machine so subtilely devised and so perfectly adapted to an end as the
visual apparatus……
I must not conceal the fact that in the study of this membrane I for the
first time felt my faith in Darwinism weakened, being amazed and
confounded by the supreme constructive ingenuity revealed not only in
the retina and in the dioptric apparatus of the vertebrates but even in the
meanest insect eye. There, in fine, I felt more profoundly than in any
other subject of study the shuddering sensation of the unfathomable
mystery of life. “
(Cajal, 1937)



























Contents


Abbreviations IV
Acknowledgements VII
Zusammenfassung IX
Summary XII
1. The retina 1
2. Electrical synapses 3
2.1 Gap-junction proteins 4
2.1.1 The connexins 4
2.1.2 Gap junction structure 5
2.2 Gap junction in the mammalian retina 6
2.2.1 Horizontal cells 7
2.2.2 Amacrine cells form electrical networks 10
2.2.2.1 Rod amacrine cells 12
2.2.2.1.1 A17 amacrine cells 13
2.2.2.2 Displaced amacrine cells 15
2.2.3 Ganglion cells exhibit tracer coupling 16
2.2.3.1 Group RGA1 17
2.2.3.1.1 RGA1 18
2.2.3.1.2 RGA2 or alpha ganglion cells 19
3. Aims and objectives 21
3.1 Aim I: Classification of displaced amacrine cells 21
3.2 Aim II: Characterization of amacrine cells expressing Cx45 22
3.3 Aim III: Identification of retinal ganglion cells expressing Cx30.2 22
3.4 Aim IV: Morphology of horizontal cells and localization of Cx57 22
4. Materials and methods 23
4.1 Mouse strains and tissue preparation 23
4.2 Intracellular injections 23
4.3 Immunohistochemistry and confocal microscopy 25
4.4 ERG measurements 26
5. Results 27
5.1 Displaced amacrine cells of the mouse retina 27
I5.1.1 Classification of displaced amacrine cells 27
5.1.2 Neurotransmitter expression 28
5.2 Morphological, neurochemical and functional characterization of 30
amacrine cell types expressing Cx45 in the mouse retina
5.2.1 Classification of Cx45-expressing amacrine cells 30
5.2.2 Coupling patterns of the Type One cells 34
5.2.3 Coupling patterns off A17 cells 36
5.2.4 Coupling of EGFP amacrine cells in Cx45-deficient mice 37
5.2.5 Neurotransmitter of Cx45-expressing amacrine cells 38
5.3 Morphological and functional characterization of ganglion cell 39
types expressing connexin30.2 in the mouse retina
5.4 Localization of Cx57 in horizontal cells of the mouse retina 40
5.5 Contributions of photoreceptor inputs to the light responses of the 41
mouse retina

6. Discussion 43
6.1 Displaced amacrine cells 43
6.2 Expression of Cx45 in the mouse retina 45
6.3 Localization of Cx57 in horizontal cells of the mouse retina 47
6.4 Cx30.2 is expressed in the mouse retina 47
6.4.1 Cx30.2 is expressed in RG cells 48 A1
6.4.2 Gap-junction protein of the displaced amacrine cells 49

7. Publications 51
7.1 Luis Pérez de Sevilla Müller, Jennifer Shelley, and Reto Weiler 51
(2007). Displaced amacrine cells of the mouse retina. J Comp Neurol
505:177-189

7.2 Jennifer Trümpler, Karin Dedek, Timm Schubert, Luis Pérez de 52
Sevilla Müller, Mathias Seeliger, Peter Humphries, Martin Biel and
Reto Weiler (2007). Rod and cone contributions to horizontal cell
light responses in the mouse retina (in press).

7.3 Luis Pérez de Sevilla Müller, Karin Dedek, Ulrike Janssen- 53
Bienhold, Maria M. Kreuzberg, Susanne Lorenz, Klaus Willecke, and
Reto Weiler. Expression and modulation of Connexin30.2, a novel
gap junction protein in the mammalian retina. (Submitted)

II7.4 Ulrike Janssen-Bienhold , Jennifer Trümpler, Gerrit Hilgen, 54
Konrad Schultz, Luis Pérez de Sevilla Müller, Stephan Sonntag, Karin
Dedek, Petra Dirks, Klaus Willecke, and Reto Weiler, Connexin57 is
expressed in dendro-dendritic and axo-axonal gap junctions of mouse
horizontal cells and its distribution is modulated by light. (submitted in
J. Comp. Neurol.)

8. Literature 55
9. Contribution of Collaborators 77
80 10. Curriculum Vitae







































III

Abbreviations


Amacrine cells ACs

cytoplasmic N-terminal AT


BPC Bipolar cell

c-AMP Adenosin-3’,5’-cyclic phosphate
ChAt Choline acetyltranferase
CNS
Central nervous system

Cx
Connexin

CL
Intracellular loop

CT
Cytoplasmic carboxy-terminal

DHT 5,7-dihydroxytryptamine

Enhanced green fluorescent protein
EGFP

EL
Extracellular loop

ERG
Electroretinogram

FDG
Fluorescein di-beta-D galactopyranoside

GABA
y-aminobutyric acid

IVGCL
Ganglion cell layer

GCs
Ganglion cells

GluR Glutamate receptor
HCs
Horizontal cells

INL
Inner nuclear layer

IPL
Inner plexiform layer

kDa
kilodalton

KO Knock out

Nestin-Cre
Cre-recombinase expression under nestin promoter control

NGS
Normal goat serum

Nm
Nanometer

ONL
Outer nuclear layer

OPL
Outer plexiform layer

Phosphate buffer
PB


PFA Paraformaldehyde


TM Transmembrane domains

V
WFAC Wide-field amacrine cell


WT Wild type


µm Micrometer











































VI



Acknowledgments

First of all I have to thank my supervisor Prof. Dr. Reto Weiler who patiently guided my
work and helped me to improve my research skills with suggestions and ideas.
Despite of being a very busy man, he always could find time to speak about my
experiments. Without him, this study would not have been possible.

Big thanks go to Prof. Dr. Ulrike Janssen-Bienhold who gave me the opportunity to
work with her in a fascinating project. It was always a pleasure to discuss work with
her.

I am thankful to Prof. Dr. Josef Ammermüller who patiently taught me the ERG method
and who always found time to discuss with me all my data.

I am thankful to Dr. Jennifer Trümpler who helped me to improve my work and my
dissertation.

Dr. Konrad Schultz introduced me to the immunohistochemistry world and showed me
the incredible world of the microscopes. I really appreciate all the time he spent
helping me with the confocal microscopy and I enjoyed the nice atmosphere he gives
in the lab.

Many thanks go to Dr. Karin Dedek and Dr. Timm Schubert who helped me with my
experiments.

I express my grateful to all the members of the lab; Josef Meier, Bettina Kewitz, Dr.
Petra Dirks, Susanne Wallenstein, Dr. Andreas Feigenspan, Nicole Iben, Tobias
Dallenga, Susanne Lorenz, Gerrit Hilgen, Petra Bolte, and Mario Pieper.

Thanks go to Dr. Stephan Maxeiner, Dr. Maria Kreuzberg, and Prof. Dr. Klaus
Willecke in Bonn University for providing me the transgenic mice.

VII