Dopamine and dopamine D4 receptor activation: Signaling components, gene regulation, endogenous circadian systems, and visual function in mouse retina Público
Jackson, Chad Robert (2010)
Abstract
The retina is the primary neural structure responsible for image
formation. It functions
over a wide range of light intensities, in part due to its
endogenous rhythmic behavior.
Dopamine, a rhythmic neuromodulator, promotes light-adaptive
effects in the retina. Dopamine,
produced by a small subset of amacrine and interplexiform cells,
mediates photoreceptor input
signals, gap junction connectivity, contrast sensitivity, and
Na+/K+-ATPase activity in the mouse
retina. Also, it is involved in trophic actions, such as circadian
rhythms, retinal development,
and ocular growth. In the mammalian photoreceptor layer only
dopamine D2-like receptors are
expressed. In this layer, dopamine reduces a light-sensitive pool
of cyclic AMP, which is related
to photoreceptor Ca2+ influx. Mice lacking the D4R are unresponsive
to light and dopaminergic
agonists and display deficits in basal cAMP levels. In other
vertebrate retinas cAMP levels are
rhythmic, connecting this second messenger to the retina's
endogenous circadian clock(s). Thus,
I sought to investigate if the D4R is coupled to Ca2+-sensitive
adenylyl cyclase(s) controlling
retinal cAMP levels and determine if D4R activation affects retinal
gene expression and circadian
rhythms. Using molecular and biochemical assays it was determined
that the type 1 adenylyl
cyclase is the primary enzyme responsible for cAMP production in
mouse retina. Additionally,
we found that D4R modulates the expression of the Adcy1 gene
(encoding the type 1 adenylyl
cyclase). Knowing cAMP is rhythmic in vertebrate retina we assayed
the Adcy1 transcript,
cAMP, and Ca2+/CaM-stimulated adenylyl cyclase activity and found
each demonstrates a
significant rhythm. However, this rhythm was not detected in mice
lacking the D4R. We show
that antagonizing or abnormal stimulation of the D4R significantly
affects the normal Adcy1
retinal rhythm. Microarray analysis revealed that ~4.8% of the
measured retinal transcriptome is
affected by the D4R. Visual contrast sensitivity tests show mice
lacking the D4R have an
impaired ability to detect changes in contrast compared with WT
controls. These data
demonstrate that the D4R is essential for modulating rhythmic cAMP,
influencing basal and
circadian retinal gene expression, and visual function regulating
visual contrast sensitivity. This
investigation shows that the DA/D4R signaling system plays a
necessary role in retinal function.
Table of Contents
Table of Contents
Chapter I: Introduction............................................................................................................................................................................................................1
1.1 Project
overview.............................................................................................................................................................................................................2
1.2
Vision............................................................................................................................................................................................................................4
1.2.1 Neural retina: cellular structure and
functions...................................................................................................................................................................4
1.3 Retinal
dopamine............................................................................................................................................................................................................9
1.3.1 Importance of retinal
dopamine.....................................................................................................................................................................................9
1.3.2 Retinal dopamine production and
release.........................................................................................................................................................................9
1.3.3 Dopamine receptor subtype in the
retina.......................................................................................................................................................................13
1.3.3.1 Dopamine D4R
signaling...........................................................................................................................................................................................14
1.4 Circadian
rhythms..........................................................................................................................................................................................................16
1.4.1 Biological
clock..........................................................................................................................................................................................................16
1.4.2 Molecular
clock..........................................................................................................................................................................................................18
1.4.3 Retinal circadian
rhythms.............................................................................................................................................................................................20
1.4.4 Overall
hypothesis......................................................................................................................................................................................................23
Chapter II: Essential roles of dopamine D4 receptors and the type 1
adenylyl cyclase in photic control of cyclic AMP in photoreceptor
cells...................................................................................................................................................................................................................................25
2.1
Summary......................................................................................................................................................................................................................26
2.2
Introduction.................................................................................................................................................................................................................26
2.3 Materials and
methods....................................................................................................................................................................................................28
2.3.1
Animals....................................................................................................................................................................................................................28
2.3.2 Ex vivo retinal
incubations...........................................................................................................................................................................................28
2.3.3 Adenylyl cyclase
assay...............................................................................................................................................................................................29
2.3.4 Acute and sub-chronic drug
treatment...........................................................................................................................................................................30
2.3.5 Western
blotting........................................................................................................................................................................................................30
2.3.6 RNA isolation and quantitative real-time
PCR.........................................................31
2.3.7 Laser capture
microdissection...................................................................................31
2.3.8 Statistical
analysis.....................................................................................................33
2.4
Results......................................................................................................................................34
2.4.1 Identification of the adenylyl cyclase isoform regulated by
light and D4R
activation............................................................................................................................34
2.4.2 Expression of AC1 in Drd4-/- mouse
retina..............................................................40
2.4.3 Effect of D4R antagonism on Adcy1
expression......................................................43
2.5
Discussion................................................................................................................................46
Chapter III: Dopamine D4 receptor activation controls circadian
timing of the adenylyl cyclase
1/cyclic AMP signaling system in mouse
retina........................................................53
3.1
Summary..................................................................................................................................54
3.2
Introduction..............................................................................................................................55
3.3 Materials and
methods.............................................................................................................56
3.3.1
Animals.....................................................................................................................56
3.3.2 RNA isolation, quantification, and quantitative real-time
PCR...............................57
3.3.3 In vivo agonist and antagonist
experiments..............................................................57
3.3.4 Retinal cyclic AMP
accumulation............................................................................58
3.3.5 Adenylyl cyclase
assay.............................................................................................59
3.3.6 Statistical
analysis.....................................................................................................59
3.4
Results......................................................................................................................................59
3.4.1 Daily rhythms of Drd4 transcript levels in mouse
retina..........................................59
3.4.2 Dopamine D4 receptors and circadian cyclic AMP synthesis in
retina....................59
3.4.3 Dopamine D4 receptor activation mediates rhythmic behavior of
Adcy1
expression.................................................................................................................69
3.5
Discussion................................................................................................................................72
3.5.1 D4R control of the rhythmic expression of
Adcy1....................................................72
3.5.2 D4R control of rhythmic adenylyl cyclase enzyme
activity......................................74
3.5.3 Dopamine/D4R signaling system controls cyclic AMP in mouse
retina..................75
3.5.4 Is the DA/D4R signaling system an entrainment mechanism in
retina?...................75
Chapter IV: Dopamine D4 receptor signaling in mouse retina controls
gene regulation and visual
function............................................................................................................77
4.1
Summary..................................................................................................................................78
4.2
Introduction..............................................................................................................................79
4.3 Materials and
methods.............................................................................................................80
4.3.1 Animal care/usage and tissue
collection...................................................................80
4.3.2 Microarray
analysis...................................................................................................81
4.3.3 GeneNetwork
analysis..............................................................................................82
4.3.4 Ingenuity Pathway
analysis.......................................................................................82
4.3.5 Rhythmic gene
expression........................................................................................82
4.3.6 Optokinetic tracking
(OKT)......................................................................................83
4.4
Results......................................................................................................................................84
4.4.1 Microarray analysis gene expression changes: WT vs.
Drd4-/-................................84
4.4.2 qRT-PCR confirmation
analysis...............................................................................85
4.4.3 Rhythmic gene
regulation.........................................................................................85
4.4.4 GeneNetwork
analysis..............................................................................................95
4.4.5 Ingenuity pathway
analysis.......................................................................................95
4.5 Retinal function in Drd4-/-
mice.............................................................................................100
4.5.1 Visual Acuity and Contrast Sensitivity
assessment................................................100
4.6
Discussion..............................................................................................................................103
Chapter V: Conclusions and future
directions.............................................................................106
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