HCN2 channels in local hippocampal inhibitory interneurons constrain temporoammonic LTP [Elektronische Ressource] / Lucas M. A. Matt

Luc Gwiazdzinski

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• Biologie

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 26
• Langue: English
• Poids de l'ouvrage: 3 Mo

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TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Zoologie


HCN2 channels in local hippocampal inhibitory interneurons
constrain temporoammonic LTP

Lucas M. A. Matt


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt der Technischen Universität München
zur Erlangung des akademischen Grades eines


Doktors der Naturwissenschaften


genehmigten Dissertation.





Vorsitzender: Univ.-Prof. Dr. M. Schemann
Prüfer der Dissertation:
1. Univ.-Prof. Dr. H. Luksch
2. apl. Prof. Dr. Th. Kleppisch
3. r. H. Adelsberger





Die Dissertation wurde am 19.04.2010 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 07.07.2010 angenommen.
Index
I Index
II FIGURES III
1 INTRODUCTION 1
1.1 The hippocampus and its role in learning and memory 1
1.1.1 Anatomy of the hippocampus 1
1.1.2 Function of the hippocampus 5
1.1.3 Learning and synaptic plasticity 6
1.2 HCN channels 8
1.2.1 Structure 8
1.2.2 Physiology of I 9 h
1.3 Conditional mutagenesis of genes using the Cre-loxP system in mice 12
1.4 Aim of this work 14
2 MATERIALS AND METHODS 15
2.1 Experimental Animals 15
2.1.1 Animal welfare 15
2.1.2 Transgenic mouse lines 15
2.1.3 Genotyping of experimental animals 17
2.2 Western blot analysis 22
2.2.1 Protein extraction from whole tissue 22
2.2.2 Protein quantification assay after Lowry 22
2.2.3 Immunoblotting 23
2.3 Immunohistochemistry 27
2.3.1 Cryo sectioning of mouse brains 27
2.3.2 Immunohistochemical staining 27
2.4 Electrophysiology 30
2.4.1 Preparation of acute slices 30
2.4.2 Field EPSP (fEPSP) recordings in hippocampal slices 31
2.4.3 Whole-cell patch-clamp recording 32
2.4.4 Data analysis 37
3 RESULTS 39
3.1 Expression of HCN1 and HCN2 channels in the hippocampus 39
3.2 The pyramidal neuron specific conditional knockout 40
3.3 LTP in the PP is not influenced by HCN2 in CA1 pyramidal cells 42
-/-3.3.1 LTP is enhanced in the PP of HCN1 mice 42
3.3.2 Basal synaptic transmission in HCN mutants is not impaired 44
-/- PyrKO3.3.3 an of HCN2 but not of HCN2 46
3.4 HCN2 is expressed in somatostatin-positive stratum oriens interneurons 48
-/-3.5 HCN2 mice show impaired inhibition of the PP 50
+/+ -/-3.5.1 Disinhibition enhances LTP in the PP of HCN2 but not HCN2 50
i Index
-/-3.5.2 Basal inhibition of the PP is impaired in HCN2 mice 51
3.5.3 HCN2 increases the frequency of sIPSCs in CA1 pyramidal cells 54
3.5.4 O-LM cells contribute to sIPSCs in CA1 pyramidal cells 56
3.6 Electrophysiological properties of O-LM cells in HCN mutants 57
3.6.1 Identification of O-LM cells 57
3.6.2 I currents in O-LM cells are mediated by HCN1 and HCN2 59 h
3.6.3 HCN channels modulate the resting membrane potential in O-LM interneurons 61
-/-3.6.4 Spontaneous activity in O-LM interneurons of HCN2 is not affected by zatebradine 63
4 DISCUSSION 64
5 SUMMARY 71
6 APPENDIX 73
6.1 Abbreviations 73
6.2 Antibodies 75
6.2.1 Primary antibodies 75
6.2.2 Secondary antibodies 75
6.3 Primers 75
7 REFERENCES 76
8 ACKNOWLEDGEMENTS 82
9 CURRICULUM VITAE 84

ii Figures
II Figures
Figure 1: Anatomical location of the hippocampus. 1
Figure 2: Hippocampal cytoarchitecture. 2
Figure 3: Hippocampal wiring. 3
Figure 4: Schematic drawing of an inhibitory feedback circuit involving an
oriens-lacunosum moleculare interneuron 4
Figure 5: Schematic representation of an HCN channel subunit. 8
Figure 6: Structural relationship between the HCN channel subtypes. 9
Figure 7: Cre/loxP mediated excision of DNA. 13
Figure 8: Schematic representation of the mutant HCN2 alleles. 16
Figure 9: Figure depicting the position of recording and stimulating electrodes
during fEPSP experiments. 31
Figure 10: Expression of the HCN1 and HCN2 channel subunits in the
hippocampal CA1-region. 39
Figure 11: Hippocampal expression of HCN2 is strongly reduced in the
conditional knockout 41
Figure 12: Mutant mice lacking the HCN1 channel show enhanced LTP in the
direct perforant but not in the Schaffer collateral pathway when
compared to littermate controls. 43
Figure 13: None of the HCN channel mutants displays changes in the I/O
relation in either TA or PP inputs. 44
Figure 14: None of the HCN channel mutants displays changes in the paired-
pulse facilitation in either TA or PP inputs. 46
Figure 15: LTP in the Schaffer collateral and direct perforant path inputs of
mutant mice lacking the HCN2 channel. 47
Figure 16: High magnification of confocal fluorescence images from
PyrKOinterneurons in the stratum oriens of WT and HCN2 mice. 49
Figure 17: Wild type mice and HCN2 null mutants show equivalent LTP in the
direct perforant path under conditions of disinhibition. 51
Figure 18: The HCN2 channel is critical for the inhibition of basal synaptic
transmission in the PP, but not the SC pathway. 53
Figure 19: The HCN2 channel supports spontaneous inhibitory currents in
CA1 pyramdial cells. 55
Figure 20: The metabotropic glutamate receptor subtype 1 agonist S-(3,5)-
dihydroxyphenylglycin stimulates spontaneous inhibitory currents in
CA1 pyramidal cells. 57
Figure 21: Visual identification of O-LM interneurons. 58
Figure 22: The HCN2 channel subunit mediates a major portion of I currents h
in O-LM interneurons. 59
Figure 23: The HCN2 channel regulates resting membrane potential and
spontaneous activity of O-LM interneurons. 62
Figure 24: Zatebradine does not influence the spontaneous activity of O-LM
-/-cells in HCN2 mice. 63
iii Introduction
1 Introduction
1.1 The hippocampus and its role in learning and memory
Perception, cognition and consciousness are commonly considered as fundamen-
tal human qualities. However, all three properties could not exist without the ability
of the brain to reliably retain information over extended periods of time. For a long
time, scholars have been wondering how this remarkable accomplishment is
achieved. When neuroscientists successfully started to tackle the problem in the
th20 century, one of their most important models for the study of memory was a
region of the brain called hippocampus. Up until now, a lot has been learned about
the mechanics of information storage in the brain, but there are still many open
questions. To date, the hippocampus continues to serve as an outstanding model
to study the cellular and molecular events that establish and/or erase memory.
1.1.1 Anatomy of the hippocampus

Figure 1: Anatomical location of the hippocampus. This drawing shows the location of the
hippocampus in the temporal lobe of the rat brain (modified after Cheung and Cardinal, 2005).
1 Introduction
The hippocampal formation is named after the Greek "seahorse" (hippos = horse,
kampos = sea monster). It is located in the medial temporal lobe (Figure 1) and
contains cells and neuronal connections that are highly conserved in all mammals.
The hippocampal formation can be subdivided into three sections: the subiculum,
the dentate gyrus (DG) and the hippocampus proper. The latter is usually referred
to as 'the hippocampus' and consists of the four subfields CA1 to CA4 (cornu
ammonis, the ram's horn, named after its curved shape). Pyramidal cells represent
the major population of neuronal cells in the hippocampus proper. Their somas are
tightly packed in the stratum pyramidale (sp) while their dendrites extend through
the stratum radiatum (sr) to the stratum lacunosum moleculare (slm).

Figure 2: Hippocampal cytoarchitecture. Schematic section transversal to the longitudinal axis of
the hippocampus. The major subfields, layers and neuronal connections are indicated. Pyramidal
cells of the CA1 are blue, of the CA3 green and granule cells of the DG are black. CA1/CA3: cornu
ammonis; DG: dentate gyrus; so, sp, sr, slm, sm: strata oriens, pyramidale, radiatum, lacunosum
moleculare, and moleculare; TP: tractus perforans, PP: perforant (temporoammonic) pathway, SC:
Schaffer collaterals.
The pyramidal cells of the area CA1 (Figure 2, blue cells) represent the final target
of intrahippocampal excitatory connections producing the major glutamatergic
output from the hippocampus to the cortex. These cells receive two main excitato-
ry inputs (Figure 2 and Figure 3) from the entorhinal cortex (EC). The first is the tri-
synaptic pathway. Originating in layer II of the EC (ECII) it relays through the
2 Introduction
granule cells of the DG and the CA3 pyramidal neurons. The axons of the latter
finally terminate at the proximal dendrites of the CA1 pyramidal cells in the sr
constituting the Schaffer collateral fibers (SC; Figure 2 and Figure 3, green). The
second input, the direct perforant or temporoammonic pathway (PP; Figure 2 and
Figure 3, red) directly connects from layer III of the entorhinal cortex (ECIII) to the
slm, where the distal dendr