The Role of RGS14 in Learning, Memory, and Synaptic Plasticity Public
Lee, Sarah Emerson (2012)
Published
Abstract
The hippocampus is crucial for converting new experiences into
long-
term memories following initial learning. Learning and memory are
closely
linked to synaptic plasticity, which involves altering the strength
of connections
between of neurons especially within the dentate gyrus
(DG)-CA3-CA1
trisynaptic circuit of the hippocampus. Conspicuously absent from
this circuit
is the intervening CA2 whose existence as a distinct region has
been subject
to debate. The CA2 only recently been implicated in learning and
memory.
CA2 neurons have a striking lack of synaptic long-term potentiation
(LTP).
RGS14 is differentially expressed during postnatal development and
is highly
enriched in CA2 pyramidal neurons. RGS14 is critically important
for
suppressing synaptic plasticity in these cells and hippocampal
learning and
memory. RGS14 is an unusual scaffolding protein that integrates G
protein
and MAP kinase signaling pathways making it well positioned to
suppress
plasticity in CA2 neurons. Supporting this idea, we find that
deletion of exons
2-7 of the RGS14 gene yields mice that lack RGS14 (RGS14-KO) that
also
express robust LTP following high frequency stimulation of Schaffer
collateral
synapses, but with no impact on synaptic plasticity in CA1 neurons.
When
tested behaviorally, RGS14-KO mice exhibited marked enhancement in
the
acquisition of spatial learning and of object recognition memory
compared with
their wild type littermates, but showed no differences in their
performance on
tests of non-hippocampal-dependent behaviors. These results
demonstrate
that RGS14 is a key regulator of signaling pathways linking
synaptic plasticity
in CA2 pyramidal neurons to hippocampal-based learning and memory,
and
RGS14 may serve as a memory filter that could be a pharmacological
target to
provide general cognitive enhancement in patients with
neurodegenerative
disorders.
Table of Contents
Table of Contents
CHAPTER 1: Introduction
1
1.1 Learning and Memory
2
1.2 The role of the Hippocampus in Memory
2
1.3 Neuroanatomy and Circuitry of the Hippocampus
5
1.4 The hippocampus in Human Disease
8
1.5 Cellular and Molecular Mechanisms of Memory hippocampal based memory
11
1.6 G protein Coupled Receptors
20
1.7 G Protein Coupled Receptor Signal Transduction
21
1.8 The Regulators of G Protein Signaling
23
1.9 Structure and interactions of RGS14
24
1.10 Objective of this dissertation
27
CHAPTER 2: Differential Expression of RGS14 during postnatal mouse brain development
29
2.1 Introduction
30
2.2 Materials and Methods
32
2.2.1 Plasmids and Cell lysates
32
2.2.2 Antibodies
33
2.2.3 Preparation of mouse brain lysates
33
2.2.4 Western Blot Analysis
34
2.2.5 Immunohistochemistry
34
2.2.6 Electron Microscopy
35
2.3 Results
36
2.3.1 RGS14 Monoclonal Antibody Characterization
33
2.3.2 Specific antibody for RGS14 protein
39
2.3.3 RGS14 antibody staining in RGS14-KO mice
39
2.3.4 Temporal Expression of RGS14 during postnatal brain development
42
2.3.5 Regional expression ofRGS14 in the developing mouse brain
44
2.3.6 RGS14 Protein Expression in Adult Mouse Brain
47
2.4 Discussion
53
CHAPTER 3: RGS14 is a natural suppressor of Learning, Memory, and Synaptic Plasticity
55
3.1 Introduction
57
3.2 Materials and Methods
58
3.2.1 Generation, genotyping, and RT-PCR of RGS14-KO mutant mice
58
3.2.3 Hippocampal Slice Preparation
59
3.2.4 Whole-cell patch recordings
59
3.2.5 Field potential recordings
60
3.2.6 Novel-object recognition
61
3.2.7 Morris water maze
61
3.2.8 Locomotor Activity
62
3.2.9 Startle Response
62
3.2.10 Elevated-Plus Maze Test
64
3.3 Results
65
3.3.1 Generation of an RGS14 Knock-out Mouse Model
65
3.3.2 Determining the function of RGS14 in synaptic activity
68
3.3.3 LTP in CA2 is blocked by MEK inhibition
70
3.3.4 RGS14-KO mice exhibit enhanced learning and memory
74
3.3.5 RGS14-KO mouse performance in non-hippocampus dependent tasks
78
3.4 Discussion
82
CHAPTER 4: Seizure susceptibility and extinction of learning in RGS14-KO Mice
85
4.1 Introduction
86
4.2 Materials and Methods.
88
4.2.1 Pilocarpine-induced seizures
88
4.2.2 Immunohistochemistry
88
4.2.3 Morris water maze
89
4.2.4 HeLa cells expressing EGFP-RGS14 Lentivirus
90
4.3 Results
91
4.3.1 RGS14-KO Mice are resistant to Pilocarpine induced seizures
91
4.3.2 Reversal and Extinction in the Morris water maze
95
4.3.3 Lentivirus expression of RGS14
95
4.4 Discussion
100
4.4.1 Pilocarpine induced seizure resistance in RGS14-KO mice
100
4.4.2 Extinction and Reversal in Morris water maze
101
4.4.3 Lentivirus vector expression of RGS14
102
CHAPTER 5: Conclusion
104
5.1 Roles for RGS14 during postnatal development
105
5.2 RGS14 is important for the acquisition of hippocampal-based spatial learning and object memory
106
5.3 RGS14 is a natural suppressor of synaptic plasticity in CA2 neurons
107
5.4 Possible mechanistic roles for RGS14 and its binding partners in synaptic plasticity.
110
5.5 Future Studies
116
5.6 Pharmacological Relevance
118
5.7 Summary
119
REFERENCES
121
Document Outline- Circulation documents
- Table of Contents
- List of Figures and Tables
- Sarah Lee Thesis FINAL
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