Dopamine and dopamine D4 receptor activation: Signaling components, gene regulation, endogenous circadian systems, and visual function in mouse retina Open Access

Jackson, Chad Robert (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/0z708w82w?locale=en
Published

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

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Subfield / Discipline
Degree
Submission
Language
  • English
Research field
Keyword
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files