The role of hippocampal GluA1-containing AMPA receptors in learning and memory [Elektronische Ressource] / presented by Florian Freudenberg

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences





















Presented by
Diplom-Biologist Florian Freudenberg
Born in Twistringen
Oral examination: 29.06.2009 12:00 The role of hippocampal GluA1-containing AMPA
receptors in learning and memory




























Referees: Prof. Dr. P. H. Seeburg
Prof. Dr. H. Monyer






















Erklärung gemäß § 8 (3) b) und c) der Promotionsordnung:
Ich erkläre hiermit, dass ich die vorgelegte Dissertation selbst verfasst und mich dabei
keiner anderen als der von mir ausdrücklich bezeichneten Quellen und Hilfen bedient
habe. Des Weiteren erkläre ich, dass ich an keiner anderen Stelle ein
Prüfungsverfahren beantragt bzw. die Dissertation in dieser oder anderer Form bereits
anderweitig als Prüfungsarbeit verwendet oder einer anderen Fakultät als Dissertation
vorgelegt habe.


Heidelberg, 30. April 2009






Für StephanieTable of contents I

Table of Contents
Summary ................................................................................................................. 1
Zusammenfassung... 2
1 Introduction.......... 3
1.1 Hippocampus (HPC)........................................................................................ 3
1.1.1 Anatomy of the hippocampal formation.................... 4
1.1.2 Cell types and intrinsic connections.......................... 5
1.1.2.1 DG..................................................................................................... 5
1.1.2.2 HPC (CA1, CA2 and CA3) 6
1.1.2.3 Interneurons of the hippocampus........................................................ 7
1.1.3 Extrinsic connections................................................ 8
1.1.3.1 Intrahippocampal connections............................ 8
1.1.3.2 Neocortex .......................................................................................... 9
1.1.3.3 Amygdala 9
1.1.3.4 Subcortical structures........................................................................10
1.1.3.5 Thalamus and Hypothalamus............................10
1.1.3.6 Brain stem.........................................................................................10
1.1.4 Hippocampal physiology .........................................................................10
1.2 Ionotropic glutamate receptors (iGluRs).........................12
1.2.1 AMPA receptors......................................................................................14
-/-1.3 GluA1 knock-out (GluA1 ) mice...................................18
-/-1.3.1 Behavioral changes in GluA1 mice........................20
1.3.1.1 Locomotor activity............................................................................20
1.3.1.2 General cognitive abilities.20
1.3.1.3 Spatial working memory (SWM).......................................................21
1.3.1.4 Pavlovian fear conditioning...............................23
1.7.1.5 Porsolt forced swim test (FST)..........................24
1.4 Viral gene transfer................................................................25
1.5 Aim of the thesis.............................26
2 Materials and Methods.......................28
2.1 Mice...............................................................................................................28
2.2 Viruses...........28
2.2.1 Viral vectors ............................................................................................28
Table of contents II

2.2.2 Virus production......................................................................................29
2.2.3 Primary hippocampal cultures..................................30
2.2.4 Virus injection.........................30
2.3 Immunohistochemistry ...................................................................................31
2.3.1 Fluorescent staining of primary hippocampal cultures..............................31
2.3.2 Fluorescent immunostaining of brain slices..............31
2.3.3 Diaminobenzidine (DAB) immunohistochemistry of brain slices .............32
2.3.4 Microscopy and image analysis................................................................33
2.4 Immunoblotting..............................................................34
2.4.1 Preparation of synaptoneurosomes...........................34
2.4.2 Quantitative immunoblotting ...................................................................34
2.5 Behavioral testing................................35
2.5.1 Groups and tests assessed.........35
2.5.2 Tests for activity and general cognitive abilities.......................................36
2.5.2.1 Locomotor activity in the open field..................36
2.5.2.2 General cognitive abilities in the puzzle box......36
2.5.3 Tests for spatial working memory............................................................37
2.5.3.1 Rewarded alternation on the T-maze.................37
2.5.3.2 Novel arm exploration on the Y-maze...............................................38
2.5.4 Tests for emotionally motivated learning.................40
2.5.4.1 Pavlovian fear conditioning...............................................................40
2.5.4.2 FST...................................................................41
2.6 Statistical analysis..........................42
3 Results..................................................................................................................44
3.1 Viruses and virus infection.............44
3.1.1 GluA1 expressing viruses ........................................................................44
3.1.1.1 Quality of virus purification and virus titers......44
3.1.1.2 Immunoblotting of virus infected HPCs............45
3.1.1.3 Efficiency of virus injections.............................................................46
3.1.2 Cre-expressing virus ................................................49
3.1.2.1 Quality of virus purification and virus titers ......................................49
3.1.2.2 Efficiency of virus injections.............................................................50
3.2 Behavior of mice from the knock-in approach................52
3.2.1 Rescue of hyperactivity in the open field..................................................53
Table of contents III

3.2.2 Hippocampal GluA1-expression does not change general cognitive
abilities in the puzzle box.........................................................................55
3.2.3 SWM is not rescued by hippocampal expression of GluA1 ......................57
3.2.4 Pavlovian fear conditioning is not rescued by hippocampal expression
of GluA1..................................................................................................61
3.3 Behavior of mice from the knock-out approach..............63
3.3.1 Lack of GluA1 in HPC partially impairs SWM........64
3.3.2 Pavlovian fear conditioning is not dependent on GluA1 in dorsal or ventral
HPC .........................................................................................................65
3.3.3 GluA1 in dorsal and ventral HPC is required for the expression of
behavioral despair in FST.........67
4 Discussion ............................................................................................................69
4.1 Stereotaxic injections of rAAVs induce efficient transduction of
hippocampal neurons ......................................................................................70
-/-4.2 Hyperactivity of GluA1 mice is abolished by hippocampal expression of
GluA1.............................................................................................................71
4.3 Anxiety-related behaviors in the open field are increased by expression of
-/-GluA1 in complete HPC of GluA1 mice........................................................73
-/-4.4 General cognitive abilities are not altered in GluA1 mice..............................74
4.5 SWM is not solely dependent on GluA1-containing AMPA receptors
in HPC............................................................................................................75
4.6 The acquisition of Pavlovian fear conditioning does not depend on GluA1-
containing AMPA receptors in HPC................................................................77
4.7 Experience-dependent expression of behavioral despair requires GluA1-
containing AMPA receptors in HPC79
4.8 Conclusions....................................................................................................80
5 Abbreviations......81
6 References............83
7 Scientific contributions.........................................................