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Monoamine oxidase deficiency and emotional reactivity: neurochemical and developmental studies
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MONOAMINE OXIDASE DEFICIENCY AND EMOTIONAL REACTIVITY:
NEUROCHEMICAL AND DEVELOPMENTAL STUDIES
by
Anna Louise Scott
________________________________________________________________________
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
December 2010
Copyright 2010 Anna Louise Scot
ii
EPIGRAPH
“Emancipate yourself from mental slavery, none but ourselves can free our mind.”
------Bob Marley------
“Redemption Song.” Uprising, 1980
These lines were based on a speech given by Marcus Garvey in October 1937.
iii
DEDICATION
In loving memory
Betty Mills
1928-2008
iv
ACKNOWLEDGEMENTS
I offer my deepest personal gratitude to my parents, Greg and Susan Quirk, and my
husband, Matthew Scott, for their love, devotion, and good humor. This work would not
have been possible without my advisors, Dr. Marco Bortolato, Dr. Kevin Chen and Dr. Jean
Shih, who provided me with guidance and enthusiasm for research. I would like to thank
my dissertation committee members, Dr. Roger Duncan and Dr. Daniel Holschneider, for
their valuable scientific input. I am grateful to our collaborators, Dr. Aiwu Cheng, Dr. Igor
Rebrin, and Dr. Jassmine Ren, and my lab members, past and present, for the countless
hours they devoted to training me in their respective arts. I also wish to express my sincere
appreciation to Dr. Ronald Alkana and the USC Department of Pharmacology and
Pharmaceutical Sciences. Lastly, I offer my regards and blessings to my family, friends, and
Shady kitty, for their encouragement during the completion of this project.
Research Support
NIH National Institute of Mental Health R01 MH39085 (J. C. Shih)
Body and Elsie Welin Professorship (J. C. Shih)
American Foundation for Pharmaceutical Education Pre-doctoral Fellowship (A. L. Scott)
The USC School of Pharmacy
v
Statement of Contributions to Works in this Dissertation
This dissertation is composed of the author’s original work and contains no material
previously published or written by any other individual except where due reference is
made. All data contained herein was collected and analyzed by A. L. Scott. The authorship
on published manuscripts is described in greater detail below.
Authorships
Published and in press works by the Author incorporated into the Dissertation
Scott AL, Bortolato M, Chen K, Shih JC (2008). Novel monoamine oxidase A knock out mice
with human-like spontaneous mutation. NeuroReport 19: 739-743. A modified version of
this manuscript is incorporated in Chapter 2.
Scott AL, Bortolato M, Godar S, Chen K, Shih JC. Monoamine oxidase deficiency reverses the
emotional effects of nitric oxide synthesis inhibition. Und er review. A modified version of
this manuscript is incorporated in Chapter 3.
Cheng A, Scott AL, Ladenheim B, Chen K, Ouyang X, Lathia J, Mughal M, Cadet J, Mattson M,
Shih JC (2010). Monoamine Oxidases Regulate Telencephalic Neural Progenitors in Late
Embryonic and Early Postnatal Development. Journal of Neuroscience 30: 10752–10762. A
modified portion of this manuscript is incorporated in the Appendix.
vi
TABLE OF CONTENTS
Epigraph ii
Dedication iii
Acknowledgements iv
List of Tables viii
List of Figures ix
Abbreviations xi
Abstract xii
Chapter 1: Introduction 1
General Background 1
MAO A: Relevance for Brain and Behavior 3
MAO B: Relevance for Brain and Behavior 7
MAO A/B Knock out Mice 8
Human Disorders Resulting from MAO Deficiency 10
Substrates of Monoamine Oxidase 13
Nitric Oxide and Monoamine System: Evidence for Reciprocal Regulation 18
Current Gaps in Scientific Literature 20
Overarching Thesis Hypothesis 22
Subhypotheses 22
Chapter 2: Novel monoamine oxidase A knockout mice with human-like
spontaneous mutation 25
Chapter 2 Abstract 25
Introduction 26
Materials and Methods 27
Results 31
Identification and biochemical characterization of MAOA
A863T
KO mice 31
Physical and behavioral characterization of MAOA
A863T
KO mice 33
Brain-regional measurement of MAO A substrates and metabolites in WT,
MAO A
neo
, and MAOA
A863T
KO mice 35
Discussion 37
vii
Chapter 3: Monoamine oxidase deficiency reverses the emotional effects
of nitric oxide synthesis inhibition 43
Chapter 3 Abstract 43
Introduction 44
Materials and Methods 48
Results 55
Light-dark box test and brain regional monoamine levels in WT,
MAO A KO and MAO A/B KO mice 55
Home cage locomotor activity 59
Resident-intruder test 59
Tail suspension test 61
Emergence test 62
Biochemical studies 65
Discussion 67
Supplementary Results 74
Hearing and nociception 75
Object recognition 75
Fear learning 76
Chapter 4: Overall Discussion and Conclusions 80
Summary of overall findings 80
Relationship of the findings to the overarching hypothesis 82
Preclinical and clinical relevance 84
Bibliography 86
Appendix: Consequences of monoamine oxidase deficiency during
early development 108
Appendix Abstract 108
Introduction 109
Materials and Methods 112
Results 117
Brain monoamine levels in neonatal WT, MAOA
A863T
KO, MAO A/B KO mice 117
Effect of MAO deficiency on neurosphere cultures derived from
embryonic and neonatal neural stem cells 118
Ultrasonic vocalizations in neonatal WT and MAO A/B KO mice 121
Discussion 124
viii
LIST OF TABLES
Table 1.1 Neurochemical and behavioral features of MAO KO mice 10
Table 2.1 Catalytic activity of MAO A and MAO B in WT and
MAOA
A863T
KO mice 32
Table 2.2 Brain regional monoamine levels in 60-day old
WT, MAO A
neo
and MAOA
A863T
KO mice 36
Table 2.3 Neurochemical and behavioral features of MAO A-deficient mice 38
Table 3.1 Brain regional monoamine levels in adult WT, MAOA
A863T
KO
and MAO A/B KO mice 58
Table 3.2 Effects of predator urine and L-NAME on amygdala 5-HT
and NE levels in WT and MAO A/B KO mice 67
ix
LIST OF FIGURES
Figure 1.1 MAOA expression and function in MAOA
neo
mice 6
Figure 2.1 Genotyping of MAOA
A863T
KO mice 31
Figure 2.2 Sites of spontaneous mutation in mouse and human MAOA 32
Figure 2.3 Behavioral alterations in MAOA
A863T
KO mice 34
Figure 2.4 Open field activity in MAOA
A863T
KO and MAO A/B KO mice 40
Figure 2.5 Aggressive behaviors in WT, MAOA
neo
and MAOA
A863T
KO mice 41
Figure 3.1 Effects of L-NAME on locomotor and exploratory behaviors
in WT, MAO A KO, and MAO A/B KO mice in the light-dark box 56
Figure 3.2 Effects of L-NAME in WT and MAO A/B KO mice
in the resident-intruder test 60
Figure 3.3 Effects of L-NAME in WT and MAO A/B KO mice in the
tail suspension test 62
Figure 3.4 Effects of L-NAME in WT and MAO A/B KO mice in the
emergence test 63
Figure 3.5 Effects of fear induced by predator urine on brain regional
MAO A catalytic activity 66
Figure 3.6 Object recognition in WT and MAO A/B KO mice 76
Figure 3.7 Fear conditioning in WT and MAO A/B KO mice 77
Figure 4.1 MAO A/B KO pups exhibit diminished body weight 112
Figure 4.2 Brain monoamine levels in neonatal WT, MAOA
A863T
KO and
MAO A/B KO mice 117
Figure 4.3 Proliferative capacity of MAO A/B KO neural stem cells from the
developing telencephalon in vitro 119
Figure 4.4 Expression of epidermal growth factor receptor isoforms in WT
and MAO A/B KO neurosphere cultures 120
Figure 4.5 MAO A and MAO B activity in neural stem cells from the
developing telencephalon 121
Figure 4.6 Ultrasonic vocalizations in male WT and MAO A/B KO neonates 122
x
Figure 4.7 Ultrasonic vocalizations in female WT and MAO A/B KO neonates 123
Figure 4.8 Proliferation of MAO A/B KO neural progenitor cells in the
subventricular zone during embryonic development 127
xi
ABBREVIATIONS
5-HT, serotonin
5-HIAA, 5-hydroxyindoleacetic acid
AMY, amygdala
BrdU, bromodeoxyuridine
DA, dopamine
DOPAC, dihydroxyphenylacetic acid
EGFR, epidermal growth factor receptor
HPLC, high pressure liquid chromatography
KO, knock-out
L-NAME, N
G
-nitro-L-arginine methyl ester
MAO A, monoamine oxidase A
MAO B, monoamine oxidase B
NE, norepinephrine
NO, nitric oxid e
NOS, nitric oxid e synthase
NSC, neural stem cell
NS, neurosphere
pCPA, parachlorophenylalanine
PEA, phenylethylamine
PFC, prefrontal cortex
SVZ, subventricular zone
USV, ultrasonic vocalization
WT, wild type
xii
ABSTRACT
Monoamine oxidases (MAOs) serve a crucial function in the regulation of mood and
behavior. The two isoenzymes, MAO A and MAO B, are expressed in a variety of brain and
peripheral tissues where they catalyze the oxidative deamination of neurotransmitter and
dietary monoamines. MAO A preferentially catabolizes serotonin (5-HT) and
norepinephrine (NE), MAO B prefers the trace amine phenylethylamine (PEA), and both
catabolize dopamine (DA). In the absence of MAO A, MAO B oxidizes MAO A’s preferred
substrates and vice versa, indicating partial functional redundancy.
Despite a wealth of evidence demonstrating that MAO mutations result in deficient
monoamine metabolism and maladaptive emotional reactivity, the specific mechanisms
underpinning this relationship remain highly elusive. Studies included in this dissertation
begin to fill this gap. This work has focused on MAO A and dual MAO A/B mutations that
result in deficient metabolism of 5-HT and NE. Knock out (KO) mice harboring these
mutations were evaluated using behavioral paradigms designed to explore different facets
of emotional responsiveness. These studies were carried out in conjunction with
biochemical and cellular assays to elucidate factors that contribute to the emotional
impairments exhibited by these lines. Collectively, these studies enhance our
understanding of the behavioral phenotypes displayed by MAO A KO and MAO A/B KO mice
and highlight novel neurochemical and developmental perturbations which may have a
causal role in the emotional disturbances displayed by these lines.
This dissertation (1) Describes a novel line of mice harboring a human-like mutation
in MAOA analogous to the cause of a rare disorder featuring impulsive aggressiveness; (2)
Provides strong evidence that the phenotype associated with total MAO d eficiency features a
general dysregyulation in the emotional processing of environmental cues; (3) Highlights
xiii
nitric oxid e as an interesting substrate for certain behavioral disturbances in MAO A KO and
MAO A/B KO mice and (4) Defines novel rol es for MAOs and 5-HT during embryonic and
early postnatal stages of d evel opment. Collectively, this work provides new models and
mechanisms to understand the consequences of MAO deficiency on the regulation of
emotional behaviors.
1
CHAPTER 1:
INTRODUCTION
General Background
Monoamine oxidases (MAOs) serve a crucial function in the regulation of mood and
behavior. Flavin-containing MAOs catalyze the oxidative deamination of both dietary and
neuroactive monoamines, generating the byproduct hydrogen peroxide (H 2O 2). The two
MAO isoenzymes, MAO A and MAO B, are both expressed on the outer mitochondrial
membrane in a variety of brain and peripheral tissues and (Shih and Thompson 1999).
Prior to their molecular characterization, the distinction between MAO A and MAO B was
defined on the basis of substrate and inhibitor sensitivity. MAO A preferentially catabolizes
serotonin (5-HT) and norepinephrine (NE), and MAO B prefers the trace amine
phenylethylamine (PEA) (Geha et al, 2001). Both catabolize dopamine (DA), although with
differing substrate specificities depending on the species; notably, DA is mainly degraded by
MAO A in the rodent brain, while MAO B plays a substantive role in this process in humans
and other primates (O'Carroll et al, 1983). Despite differences in substrate specificity,
enzymatic actions mediated by these two isoenzymes overlap to some degree, resulting in
the degradation of monoamines such as tryptamine and tyramine by both forms.
Nonetheless, the two isoenzymes are best distinguished based on pharmacological criteria:
MAO A is selectively inhibited by low doses of clorgyline (Johnston 1968) whereas MAO B is
blocked by low doses of deprenyl (selegiline) (Knoll and Magyar 1972).
MAO A and MAO B are encoded by two independent genes that are closely linked on
the X chromosome (Xp11.23) (Lan et al, 1989), in opposite direction with tail-to-tail
orientation, and display identical number of exons (15) and intron–exon organization
(Grimsby et al, 1991). These similarities, in conjunction with the fact that the primary
2
amino acid sequences of the two isoenzymes have 70% identity (Bach et al, 1988), suggest
that MAO A and MAO B arose from a common ancestral gene.
Despite these similarities, studies of the transcriptional regulation of MAO A and
MAO B (Shih et al, 1999a) indicate that these genes are activated and repressed by different
transcription factors, which may account for differences in the expression of these two
isoenzymes in brain and peripheral tissues (Grimsby et al, 1990) and throughout
development and aging (Nicotra et al, 2004). Although they are both expressed to some
extent in most peripheral tissues and organs, MAO A is the predominant form in the gut
(Anderson et al, 1993) and is prevalent in fibroblasts and placental tissue (Egashira and
Yamanaka 1981). MAO B is the only isoform in platelets and lymphocytes (Bond and
Cundall 1977; Donnelly and Murphy 1977). Both forms are found in the liver (Thorpe et al,
1987). In the brain, MAO A is primarily expressed in catecholaminergic neurons, whereas
MAO B is expressed in serotonergic and histaminergic neurons and glial cells (Westlund et
al, 1985; Shih 1991). The underlying reasons for the apparent mismatch of MAO B in
serotonergic neurons, however, remain to be elucidated.
3
MAO A: Relevance for Brain and Behavior
MAO A emerges earlier than MAO B in the developing brain but shows little
variation during adulthood (Nicotra et al, 2004). In rodent brain, MAO A activity initially
predominates over MAO B; at birth, the MAO A/MAO B ratio is 3:1, but 8 weeks later it is 1:3
(Rao et al, 1995). Brain regional studies indicated that MAO A activity is high in most
regions by postnatal day 5, and adult levels are attained around day 20 (Leung et al, 1993).
In human brain, MAO A activity is high at birth, decreases markedly during the first 2 years,
and then persists at a constant level through adulthood (Kornhuber et al, 1989). The
decline in MAO A in the early postnatal years is believed to result from the reduction in
neuronal density that occurs during the phase of development (Huttenlocher 1979).
MAO inhibitors were the first antidepressant medications developed and are
believed to enhance mood primarily through inhibition of MAO A, countering the reduction
in 5-HT and NE (and, to a more limited extent, DA) that characterizes depression (Bortolato
et al, 2008). Although highly effective in treating depression, MAO inhibitors are generally
not the first choice among clinicians, due to the risk of the so-called “cheese reaction,”
consisting in severe, potentially lethal hypertensive crises following the consumption of
tyramine-rich foods (Anderson et al, 1993). The risk of this side effect spurred the
development of novel antidepressant medications with different mechanisms of action,
including the tricyclic antidepressants and serotonin selective reuptake inhibitors (SSRIs).
However, the development of reversible inhibitors of MAO A (RIMAs) (Amrein et al, 1993),
which have proven effective in treatment-resistant (Amsterdam and Shults 2005) and
atypical depression (Nierenberg et al, 1998), may spur the development of novel MAO
inhibitors with enhanced safety and effectiveness.
4
Although the antidepressant effects of MAO A-selective inhibitors highlight the
important contributions of this enzyme to the regulation of mood and behavior in the
mature brain, knock out (KO) mice with mutated MAOA have revealed crucial roles for this
enzyme in the developing brain. The first line of MAO KO mice (“Tg8”) was generated by
the inadvertent insertion of an IFN-β minigene into exon 2 of the MAOA coding region of one
cell embryos of C3H/HeJ mice (Cases et al, 1995). MAO A enzyme activity was undetectable
throughout all tissues in Tg8 mice, whereas the expression and activity of MAO B was
equivalent with WT C3H/HeJ mice.
The phenotype of Tg8 mice was characterized by deficient metabolism of 5-HT and
NE, resulting in markedly higher levels of these monoamines in the brain. Brain 5-HT levels
were increased most dramatically in neonatal Tg8 pups, when MAO B expression is lowest,
and were gradually reduced to levels almost equivalent with WT mice in late adulthood
(Cases et al, 1995). In addition, 5-HT 1A, 5-HT 2A, and 5-HT 2C receptor binding sites were
decreased in brains of Tg8 mice, perhaps reflecting down-regulation by excess 5-HT (Shih et
al, 1999).
As pups, Tg8 mice displayed numerous behavioral perturbations including hyper-
reactivity and difficulty in righting (discussed in the Appendix), and in adulthood, Tg8 mice
exhibited markedly increased aggression and impulsivity (Cases et al, 1995) and higher
retention of fear-related memories (Kim et al, 1997). Moreover, Tg8 mice featured
dysmorphism of the barrel fields in the somatosensory cortex, an aberration that was
partially restored by administration of the inhibitor of 5-HT synthesis,
parachlorophenylalanine (pCPA), during the early neonatal stage (Cases et al, 1995). In
ad dition to restoring the sensorimotor cortex d eficits in Tg8 mice, neonatal pCPA
administration was also reported to attenuate many of the abnormal behaviors observed in
5
these pups, whereas the same treatment with an inhibitor of catecholamine synthesis had no
effect, reinforcing the serotonergic basis for these perturbations (Cases et al, 1995; Salichon
et al, 2001). Coll ectively, these studies revealed an important role for MAO A during early
stages of brain development with implications for behavior. This notion is supported by the
fact that, although long-term use of MAO A inhibitors does not induce aggression in adults,
chronic injection of the MAO A-selective inhibitor clorgyline in perinatal stages reduced the
latency to attack in adult rats (Mejia et al, 2002).
Recently our group generated a line of hypomorphic MAO A mice (MAO A
neo
)
(Bortolato et al, submitted). This line was generated in a 129S6 colony and harbors a floxed
neomycin-resistance cassette (Neo
R
) in the 12
th
intron of the MAOA gene, creating a
chimeric MAOA transcript (MAO B expression and activity are equivalent to WT
counterparts). In addition to the expression of this aberrant MAOA splice variant, MAO A
neo
mice also express small amounts of WT mRNA (~8%) (Fig. 1.1a-b). Unlike Tg8 mice, which
lack MAO A activity throughout all tissues, MAO A
neo
mice exhibit brain regional-selective
deficits in MAO A activity, supporting the possibility that small amounts of WT MAOA mRNA
in the brain of MAO A
neo
may be translated into functional protein (Bortolato et al,
submitted). Specifically, MAO A activity in MAO A
neo
mice is undetectable in all brain
regions except amygdala and prefrontal cortex, in which levels are low in comparison to WT
mice (Fig. 1.1c-h). However, the effects of this mutation on brain regional levels of 5-HT and
NE have not been determined.
6
FIGURE 1.1
B
C
D
E
F
G
H
A C
D
E
F
G
H
MAOA Expression and Function in MAO A
neo
Mice. a, The employment of Ex12F and Ex 14R primers in brain
samples of MAO A
neo
mice show small amounts of a 200 bp product, representing WT MAOA mRNA in MAO A
neo
mice. b,The intronic insertion of the Neo
R
fragment results in a significant reduction of WT MAOA mRNA in the
prefrontal cortex (F1,14 = 27.70; P < 0.001), amygdala (F1, 14 = 31.77; P < 0.001), hippocampus (U8, 8 = 4.50; P <
0.01), striatum (U8, 8 = 5.50; P < 0.001), midbrain (F1, 14 = 10.50; P < 0.01), and cerebellum (F1, 14 = 27.73; P <
0.001). c-h MAO A
neo
exhibited low, yet detectable enzymatic activity levels in prefrontal cortex and amygdala,
which was ablated by treatment with the selective MAO A inhibitor clorgyline (10 mg/kg, i.p., 40 min before
sacrifice). Abbrev: PFC, prefrontal cortex; AMY, amygdala; HIP, hippocampus; STR, striatum; MID, midbrain;
CER, cerebellum; SAL, saline; CLO, clorgyline (Bortolato et al, submitted).
The unique neurochemical profile exhibited by MAO A
neo
mice may contribute to a
distinctive behavioral phenotype, characterized by low impulsive aggression and greater
anxiety in a novel environment. Notably, MAO A
neo
mice exhibited significantly lower levels
of resident-intruder aggression as compared to both WT and MAO A KO conspecifics
(Bortolato et al, submitted). Whereas the aforementioned Tg8-MAO A KO mice exhibited
similar locomotor activity compared to WT in the novel open field (Agatsuma et al, 2006),
MAO A
neo
show reduced locomotor activity and tend to stay mainly in the periphery of the
7
field, a phenomenon known as thigmotaxis which often reflects anxiety-like behavior
(Bortolato et al, submitted). However, MAO A
neo
and Tg8 are derived from different
background strains (129S6 and C3H/HeJ, respectively) rendering direct behavioral
comparisons subject to a number of confounding factors. Nonetheless, mutations such as
the one harbored in MAO A
neo
mice that reduce, but not eliminate, the function of MAO A be
used to investigate how selective deficits in brain regional monoamine metabolism
influence emotional behaviors.
MAO B: Relevance for Brain and Behavior
In contrast to the early appearance of MAO A, most studies in humans and rodents
indicate that MAO B activity is very low in embryonic brain and increases gradually from
birth to adulthood (Nicotra et al, 2004). In rat brain, MAO B activity was found to be very
low in all regions at postnatal day 5 but then increased markedly (Leung et al, 1993). In
human frontal cortex, MAO B activity is fairly constant during childhood but increases in
late adulthood (Kornhuber et al, 1989). Age-related increases in MAO B in the nigrostriatal
dopaminergic system are of great interest due to the marked degeneration of this region in
Parkinson’s disease (PD) (Fahn and Cohen 1992). Enhanced MAO B in this system is
believed to increase susceptibility to neurodegeneration through production of H 2O 2
generated during DA oxidation, which contributes to the formation of reactive oxygen
species (ROS), triggering mitochondrial damage and neuronal death (Halliwell 1992).
Indeed, MAO B-selective inhibitors have proven highly efficacious in the therapeutic
management of PD. The original rationale for using these inhibitors to treat PD was based
on the concept that DA is preferentially degraded by MAO B in the human nigrostriatal
dopaminergic system, and enhancing DA levels through blockade of MAO B should
8
compensate for the deficits caused by PD (Knoll 2000). Studies of the MAO B-selective
inhibitor, deprenyl, revealed that the effectiveness of this compound is also related to
neuroprotective effects induced by MAO inhibition, namely a reduction in oxidative stress
(Ng et al, 2008).
The role of MAO B in behavior has been greatly elucidated through studies of MAO B
KO mice. This line was generated by targeted disruption of the MAO B gene, resulting in
complete loss of MAO B protein and catalytic activity (MAO A activity was equivalent to WT
counterparts). MAO B KO mice were found to have significantly higher levels of PEA but
normal 5-HT, NE, and DA levels (Grimsby et al, 1997). Although MAO B KO mice did not
show abnormal behaviors as pups nor heightened aggression in adulthood (Grimsby et al,
1997), they exhibited a phenotype characterized by behavioral disinhibition and lower
anxiety-like responses, including shorter latency to engage in risky behaviors and enhanced
novelty-seeking responses (Bortolato et al, 2009).
MAO A/B Knockout Mice
Due to the close proximity of the MAO A and MAO B genes on the X chromosome,
MAO A/B double knockout mice (MAO A/B KO) could not be generated by crossing MAO A
KO with MAO B KO mice. However, within the MAO B KO colony, a line of mice harboring a
spontaneous mutation in MAO A was serendipitously discovered. These double mutants
were initially identified by their markedly reduced body weight and extremely hyper-
reactive behaviors, including panic jumping (Chen et al, 2004).
Subsequent analysis revealed that this line harbors a naturally-occurring nonsense
point mutation in the 8
th
exon of MAOA which generates a stop codon, causing termination
of translation and mRNA decay (Chen et al, 2004). Aberrant monoamine degradation is
9
epitomized in MAO A/B KO, which have levels of 5-HT, NE, DA, and PEA that are increased
8.5-, 2.2, 1.7-, and 15.7-fold, respectively, compared to WT (Chen et al, 2004). Indeed,
although increased 5-HT and PEA levels are present, respectively, in the MAO A KO and
MAO B KO mice, the magnitudes of increase are significantly greater in MAO A/B KO mice
providing in vivo evidence for functional synergy between the two MAO isoenzymes.
Moreover, adult MAO A/B KO mice have significantly elevated levels of DA, a characteristic
not observed in either MAO A KO or MAO B KO mice because DA can be inactivated by
either isoenzyme (Chen et al, 2004).
Preliminary behavioral phenotyping indicated that, akin to Tg8 mice, MAO A/B KO
mice exhibited heightened aggression and acute lack of impulse control (Chen et al, 2004).
However, unlike Tg8 mice, which exhibited normal behavior in a novel open field
(Agatsuma et al, 2006) and the elevated plus maze (Popova et al, 2000), MAO A/B KO mice
exhibited marked reductions in exploratory activity in both these paradigms (Chen et al,
2004), deficits generally reflecting a heightened state of anxiety. However, the limited
number of behavior studies which have been performed in MAO A/B KO mice precludes
definitive understanding of these behavioral alterations. Moreover, the potentially
confounding influence of 5-HT on motor function (Jacobs and Fornal 1997) poses additional
challenges in evaluating behavioral performances in these mutants, as these tests often rely
on measures of locomotor and exploratory responses to contextual stimuli. Studies
designed to elucidate complementary aspects of their emotional reactivity, including tests
with anxiety-related “scores” that do not rely on locomotor or exploratory activity, are
warranted to more fully understand the behavioral organization of this line.
In addition to their unique profile of behavioral perturbations, MAO A/B KO mice
exhibited physiological impairments not observed in MAO A KO or MAO B KO mice,
10
including reduced body weight, a deficit which persisted through adulthood (Chen et al,
2004), and abnormal heart rate dynamics (Holschneider et al, 2002). These reports
suggested that loss of both MAOs may have wide-ranging consequences on physiological
development and provided further evidence for partial functional redundancy between
MAO A and MAO B.
TABLE 1.1
Neurochemical and Behavioral Features of MAO KO mice. *MAO A
neo
mice exhibited low, yet detectable
enzymatic activity levels in amygdala and prefrontal cortex; MAO A activity was not detectable in any other
region in MAO A
neo
mice (See Fig. 1.1).
Human Disorders Resulting from MAO Deficiency
In contrast to the beneficial effects of pharmacological inactivation of MAO in the
treatment of depression and PD, a wide spectrum of physiological and behavioral
dysfunctions – ranging from mild perturbations to detrimental impairments – are
associated with chronic deficiencies in MAO function or genetic ablation of MAO.
Selective loss of MAO A is a rare genetic disorder, termed Brunner syndrome, first
reported in large Dutch kindred (Brunner et al, 1993b). Affected males exhibited a similar
11
spectrum of behavioral abnormalities, featuring prominent episodes of aggressive and often
violent behavior. Urine samples indicated altered monoamine metabolism, including
decreased levels of the 5-HT metabolite generated by MAO A, 5-hydroxyind ol eacetic acid (5-
HIAA). Genetic analysis revealed that loss of MAO A was due to a single point mutation in
the 8
th
exon of MAOA, located only a few base pairs downstream from the site of the MAOA
mutation in MAO A/B KO mice, which substitutes a translation termination codon for a
glutamine codon (Brunner et al, 1993b).
Akin to Tg8-MAO A KO mice, the behavior of affected males was characterized by
acute lack of impulse control and a history of aggressive violence, often triggered by stress
(Brunner et al, 1993a). The intensity of these outbursts was described as disproportionate
to the provocation. Moreover, affected males exhibited mild or borderline mental
retardation and a tendency toward stereotypical hand movements, such as hand wringing.
Despite this characteristic pattern of behavioral abnormalities, the low prevalence of the
syndrome and the elusiveness of its nosographical description (Hebebrand and Klug 1995),
hinder our understanding of its neurobiological underpinnings. Moreover, aggressive and
impulsive tendencies feature prominently in many psychiatric illnesses (McElroy et al,
1992), limiting the ability of clinicians to distinguish Brunner syndrome from other
impulse-control disorders.
Although total loss of MAO A resulting from the Brunner syndrome mutation was
found to be a very rare occurrence in humans (Schuback et al, 1999), far more common are
functional polymorphisms within the MAO A promoter region which may alternatively
confer high or low enzyme expression (Sabol et al, 1998). Subjects with alleles conferring
enhanced transcription exhibited less aggression and impulsivity and more pronounced
serotonergic responsivity than subjects with alleles conferring reduced transcription
12
(Manuck et al, 2000b). Moreover, the genotype conferring higher levels of MAO A
expression has been associated with reduced susceptibility to antisocial behavior in
maltreated children (Caspi et al, 2002) although not all studies show conclusive results
(Huizinga et al, 2006).
By contrast, patients with an X chromosome microdeletion resulting in selective loss
of MAOB and the adjacent Norrie disease gene displayed neither abnormal behavior nor
mental retardation despite a complete lack of MAO B platelet activity and increased urinary
excretion of PEA (Lenders et al, 1996). However, several studies have indicated that low
platelet MAO B activity is highly heritable (Oxenstierna et al, 1986) and is correlated with a
greater vulnerability to psychiatric disorders including poor impulse control (Skondras et
al, 2004; Paaver et al, 2007), sensation-seeking (Fowler et al, 1980; Reist et al, 1990;
Ruchkin et al, 2005), and behavioral disinhibition (Buchsbaum et al, 1976).
Complete loss of both MAOs is a rare yet detrimental condition. Recently, a family
with an inherited partial deletion of MAOA and complete deletion of MAOB was discovered.
The two affected males were brothers born to healthy non-consanguineous parents and
presented with severe developmental delay, intermittent hypotonia, stereotypical hand
movements, and height and weight measurements fell into very low percentiles (Whibley et
al, 2010). MAO A/B-deficiency is also found in a subset of Norrie disease patients with an X
chromosomal deletion including not only the Norrie disease gene but also the adjacent
MAOA and MAOB genes (Collins et al, 1992). In addition to the impairments caused by
Norrie disease, including congenital blindness (Berger et al, 1992), patients also lacking
MAO A and MAO B suffer from severely deficient monoamine metabolism and profound
mental retardation, in addition to having autistic-like behavior (Collins et al, 1992; Sims et
al, 1989; Murphy et al, 1990).
13
Interestingly, the repetitive hang wringing and lip smacking observed in MAO A/B-
d eficient patient s (Whibley et al, 2010) are common features of pervasive d evel opmental
disord ers, such as Rett syndrome and autism (Bodfish et al, 2000). Comparabl e, albeit l ess
severe, stereotypic behaviors were also observed in mal es with mutated MAOA (Brunner et
al, 1993a). This resemblance suggests that a similar spectrum of developmental alterations
leads to certain behavioral abnormalities observed in MAO A- and MAO A/B-deficient
humans that are clearly more severe in the latter genotype. More precise dissection of
these behavioral features of MAO A/B KO mice is warranted in order to facilitate the
identification of causal mechanisms, and such studies may have broader implications for
developmental disorders associated with altered monoamine metabolism (Roux and Villard
2010; Whitaker-Azmitia 2001).
Substrates of Monoamine Oxidase
Serotonin: Serotonin (5-HT) has complex effects on mood and emotion and
influences a wide range of behaviors including aggression, impulsivity, mood, cognition,
memory, sleep, appetite, and sexual behaviors (Berger et al, 2009). The principle route of
metabolic degradation for 5-HT is deamination by MAO A, which generates 5-
hydroxyindoleacetic acid (5-HIAA).
5-HT neurons are located in two brainstem regions, the dorsal raphe nucleus (DRN)
and median raphe nucleus (MRN), which project to distinct but overlapping areas of the
brain. The DRN primarily innervates the amygdala, dorsal hippocampus, and frontal cortex,
while the MRN projects mainly to hippocampus, septum, nucleus accumbens and
hypothalamus (Millan 2003). A receptor family that is larger than any other family of G-
protein coupled (GPCR) neurotransmitter receptors contributes to the diversity of
14
functional effects elicited by 5-HT. Seven distinct families of 5-HT receptors, composed of at
least 15 subtypes, have been characterized to date. All 5-HT receptor subtypes are
metabotropic GPCRs with the exception of 5-HT 3, which is a ligand-gated ion channel
(Nichols and Nichols 2008).
5-HT elicits a variety of behavioral effects in different brain regions. In the limbic
system, several studies have indicated that 5-HT functions to attenuate amygdala activity,
thus decreasing memories of aversive stimuli (LeDoux 1998). This evidence has lead to the
notion that 5-HT enhances hippocampal tolerance to aversive experience (Mongeau et al,
1997). A wide body of research has implicated 5-HT in the regulation of aggressive
behavior (for a review, see Nelson and Chiavegatto 2001). Low CSF concentrations of 5-
HIAA have long been associated with aggression in humans (Brown et al, 1979; Brown et al,
1982; Linnoila et al, 1983). Several brain regions - including the amygdala, prefrontal
cortex and hippocampus - work in concert to regulate emotional responses such as
aggression (Davidson et al, 2000), and deficient 5-HT metabolism in these regions has been
associated with violent and impulsive behavior (Chen et al, 2007).
Norepinephrine: Norepinephrine (NE) is a catecholamine that functions both as a
neurotransmitter and a hormone. NE plays a pivotal role in behavioral organization, with
prominent effects related to arousal, vigilance, activation of responses to fear/stress, and
modulation of memory systems, particularly those formed by exposure to aversive stimuli
(Ressler and Nemeroff 2000). NE is preferentially catabolized by MAO A, which converts it
to 3,4-dihydroxymandelic acid or 3-methoxy-4-hydroxyphenylethylene glycol (MHPG or
MOPEG). NE is alternatively degraded by catechol-O-methyl transferase (COMT) to the
metabolite normetanephrine, which in turn is converted by MAO to vanillylmandelic acid
15
(VMA). Moreover, in the adrenal medulla, NE can also be converted to epinephrine via
phenylethanolamine N-methyltransferase (PNMT) (Wong et al, 2002).
Cell bodies containing NE are located in the locus coeruleus (LC) and within the
lateral tegmental field, and noradrenergic projections heavily innervate the amygdala,
cortex, hippocampus, hypothalamus, and periaqueductal gray (PAG) (Millan 2003). There
are three families of NE receptors: β-adrenergic (βAR), α 1 adrenergic, and α 2 adrenergic.
βAR is the chief excitatory postsynaptic receptor and mediates transmission through
activation of adenylate cyclase. α 1 receptors are excitatory, located postsynaptically, and
linked to calcium channel opening and phospholipase C/IP3 activation. α 2 receptors are
inhibitory, located both pre- and postsynaptically, and mediate their effects by inhibiting
adenylate cyclase activity (Insel 1996).
Acute stress activates the brain noradrenergic system, resulting in NE release in
limbic forebrain target regions that facilitate a range of anxiety-like responses (Morilak et
al, 2005). El evated NE neurotransmission has been shown to intensify amygdala-mediated
fear responses (Onur et al, 2009; Buffalari and Grace 2007). In the amygdala, NE plays a
critical role in the HPA stress response and potentiates formation of fear-related memories
in brain regions such as the hippocampus and nucleus accumbens (Feldman and
Weidenfeld 1998; Davis 1998). In other regions, the LC-NE system has fundamental roles in
organizing the behavioral state, with cortical projections implicated in attention and
thalamic projections believed to coordinate overall activity levels (Ressler and Nemeroff
2000).
There is evidence suggesting that elevated 5-HT levels may lead to the development
of a hyporesponsive 5-HT system concomitant with a hyperresponsive NE system, an
imbalance which is implicated in pathological emotional states, including anxiety disorders
16
(Ressler and Nemeroff 2000). During the euthymic state, activation of the locus
coeruleus/NE system in response to fear or stress is countered by inhibition of these
circuits via the raphe nuclei/5-HT system, and numerous mechanisms of reciprocal
regulation between these systems have been identified. Excess 5-HT may lead to an
imbalance via activation of excitatory 5-HT 3 receptors localized on NE terminals, resulting
in increased NE release, which in turn can act on inhibitory α 2 receptors localized on 5-HT
terminals, resulting in inhibition of 5-HT release (Mongeau et al, 1997). The potential
contribution of such an imbalance to the enhanced anxiety-like behaviors observed in MAO
A/B KO mice has not been investigated.
Dopamine: Dopamine (DA) is a catecholamine neurotransmitter with many diverse
effects on behavior and cognition, influencing motivation, punishment and reward, sleep,
mood, attention, and working memory (Björklund and Dunnett 2007). In most brain
regions, including striatum and basal ganglia, DA is inactivated by both MAO A and MAO B
into 3,4-dihydroxyphenylacetic acid (DOPAC); in other regions, DA is believed to be
preferentially inactivated by COMT into 3-methoxytyramine (Martin-Iverson et al, 1994;
Morón et al, 2002). Likely because of these alternative pathways for degradation, DA levels
were not substantially increased in either MAO A KO or MAO B KO mice but were enhanced
1.7-fold in MAO A/B KO mice (Chen et al, 2004).
The neurotransmitter systems formed by DA neurons originate in the substantia
nigra pars compacta, ventral tegmental area (VTA), and hypothalamus, with the largest
concentration of DA neurons located in two nuclei of the ventral midbrain (Prakash and
Wurst 2006a; Prakash and Wurst 2006b). Axons are projected to the prefrontal cortex,
nucleus accumbens, neostriatum, caudate nucleus, putamen, and pituitary gland (Björklund
and Dunnett 2007). There are at least 5 subtypes of G protein-coupled DA receptors which
17
are expressed widely throughout the brain and mediate a diverse array of behavioral
effects. The D 1 and D 5 receptors belong to the “D 1-like family”, whereas the D 2, D 3 and D 4
receptors are members of the “D 2-like family.” Activation D 1-like receptors is coupled to the
G protein G αs, which subsequently increases the intracellular concentration of the second
messenger (cAMP); by contrast, activation of D 2-like receptors is coupled to the G protein
G αi, which inhibits the formation of cAMP (Civelli et al, 1993).
The DA system includes the mesolimbic and mesocortical pathways, which originate
in the VTA, modulate emotional behavior and are central to the brain’s reward system
(Arias-Carrion et al, 2010). The mesolimbic dopaminergic system projects mainly to the
nucleus accumbens (NAc), which is the primary release site for DA, whereas the
mesocortical dopaminergic system extends fibers in the prefrontal, cingulated and
perirhinal cortices. Nearly every drug that causes addiction increases DA release in the
mesolimbic pathway, and repetitive substance abuse has been shown to alter the normal
circuitry of rewarding and adaptive behaviors (Di Chiara and Imperato 1988).
β-phenylethylamine: β-phenylethylamine (PEA) is a trace amine primarily
degraded by MAO B, implicated in regulation of mood and behavior, including exploratory
activity, arousal, and behavioral reinforcement (Sabelli and Javaid 1995). Although acute
administration of PEA induces anxiety (Lapin 1990; Lapin 1993), chronic exposure to high
PEA levels appears to have the opposite effect, as indicated by the behavioral phenotype of
MAO B KO mice (Bortolato et al, 2009). Indeed, the phenotype exhibited by MAO B KO mice
is in substantial agreement with the behavioral effects of the synthetic PEA analog
amphetamine, which enhances novelty-seeking and reduces impulse control in both rodents
and humans (Evenden and Ryan 1996; Leyton et al, 2002; Williamson et al, 1997).
18
Similar to the effects of amphetamine, PEA has been shown to markedly enhance
catecholaminergic signaling, increasing DA efflux (Parker and Cubeddu 1988) and
potentiating cortical neuron responses to NE (Paterson 1993), but has a much less potent
effect on 5-HT (Nakamura et al, 1998). In addition, recent evidence suggests that PEA-
mediated activation of trace amine-associated receptor 1 (TAAR1) may be involved in
certain behavioral responses (Borowsky et al, 2001; Lindemann and Hoener 2005). This
receptor is believed to regulate DA signaling in the striatum (Lindemann et al, 2008;
Wolinsky et al, 2007; Xie and Miller 2009), complementing previous reports on the effects
of PEA-mediated modulation of DA signaling (Kuroki et al, 1990; Sotnikova et al, 2009).
Indeed, although MAO B KO mice did not feature altered synthesis, uptake, or release of DA,
they did exhibit alterations in DA receptors in the striatum (Chen et al, 1999).
Nitric Oxide and Monoamine System: Evidence for Reciprocal Regulation
Increasing evidence suggests that reciprocal modulating interactions between nitric
oxide (NO) and monoamine neurotransmitters may be essential for maintaining brain
homeostasis. NO is a gaseous free radical, with a half life of mere seconds, that easily
crosses the cell membrane; in addition to being a potent vasodilator, NO serves as a
neuromodulator/retrograde messenger in the brain (Bredt and Snyder 1994; Griffiths et al,
1998; Moncada et al, 1991) and has been implicated in the formation of memory (Shibuki et
al, 1991; Bohme et al, 1991; O'Dell et al, 1991). NO is synthesized from L-arginine by the
nitric oxide synthase (NOS) enzymes, of which there are three isoenymes: one inducible
(iNOS) and two constitutive forms, neuronal NOS (nNOS) and endothelial NOS (eNOS)
(Schuman and Madison 1994).
19
Numerous examples of mutual regulation between the nitrergic and monoaminergic
systems highlight the complexity of this relationship. NO has been shown to inhibit the
activity of the primary monoamine degrading enzymes, MAO A and MAO B, and decrease
outer mitochondrial membrane fluidity (Muriel et al, 2003). Conversely, NO is capable of
converting 5-HT into an inactive form (Fossier et al, 1999) and may facilitate the
degradation of excess DA and NE through nitration (d’Ischia et al, 1995). 5-HT reportedly
activates nNOS in vitro (Breard et al, 2007), while NE has been shown to upregulate
expression of both nNOS and iNOS in rat neurons (Grange-Messent et al, 2004).
Notably, several lines of evidence support the notion that a complex relationship of
reciprocal regulation between 5-HT and NO plays a crucial role in modulating mood and
behavior. Interestingly, the constitutively-active forms of NOS in the brain, eNOS and nNOS,
appear to have complementary effects on 5-HT turnover (Frisch et al, 2000; Chiavegatto et
al, 2001). Such functional differences may stem from their distinctive localization
throughout the brain: expression of eNOS is limited to endothelial tissue while nNOS is
expressed in discrete populations of neurons throughout the limbic system (Bredt et al,
1991; Dawson et al, 1991) and in 5-HT neurons of the DRN (Simpson et al, 2003). Likewise,
NO may alternatively increase or decrease extracellular brain 5-HT levels depending on the
region studied (Prast et al, 2001). nNOS was recently discovered to regulate 5-HT uptake
by physically controlling surface expression and function of the serotonin transporter
(SERT) (Chanrion et al, 2007). Conversely, activation of 5-HT 1A receptor regulates the
expression of nNOS, a relationship implicated in the regulation of anxiety (Zhang et al,
2010).
In accordance with its function as a retrograde messenger, there is evidence that the
effects of NO on behavior are a function of its interactions with other neurotransmitter
20
systems. Pokk and colleagues reported that NOS inhibitors induced differential effects on
locomotor and exploratory behaviors in the elevated plus maze in mice which had been
forced to remain on a small platform (SP) surrounded by water for 24 hours prior to
behavioral testing. Consistent with previous studies (Volke et al, 1997; Czech et al, 2003;
Spiacci Jr et al, 2008; Zhang et al, 2010), in non-stressed control mice, the nNOS-selective
inhibitor 7-nitroindazole (7-Ni) increased exploration, whereas the nonselective inhibitor
N
G
-nitro-L-arginine methyl ester (L-NAME) reduced exploration; by contrast, in SP-stressed
mice, the effects of NOS inhibitors were reversed (Pokk et al, 2002). These results suggest
that the impact of NOS inhibitors on the modulation of behavioral responses may depend
largely on the baseline emotional state.
Considering the numerous mechanisms for mutual regulation between the
monoaminergic and nitrergic pathways, it is likely that MAO A- and MAO A/B-deficiency
and their effects on brain 5-HT induce marked changes in the NO signaling. Behavioral and
biochemical studies are warranted to elucidate the potential impact of such alterations on
behaviors in MAO A KO and MAO A/B KO mice. Such investigations will increase our
understanding of the mechanisms by which MAO and NO regulate monoamine signaling in
the brain, which in turn may facilitate the characterization of novel therapeutic targets for
the treatment of psychiatric disorders resulting from perturbed metabolism of monoamine
neurotransmitters.
Current Gaps in Scientific Literature
Although the evidence summarized here represents substantial advances in our
knowledge of how MAO deficiency affects emotional behavior, a number of areas remain
highly elusive. The work included in this dissertation begins to fill the following gaps.
21
First, cogent evid ence indicates that emotional perturbations associated with MAO A
deficiency stem from deficits in the ability to enact appropriate responses to stressful or
fear-inducing circumstances. However, the low prevalence of the MAOA mutation,
compounded with the fact that the associated behavioral abnormalities feature prominently
in other impulse-control disorders, hinders our ability to dissect the clinical features of this
syndrome. Although the Tg8 line of MAO A KO mice has elucidated several features
underpinning this relationship, the translational value of this line is limited by the
expression of mutated MAOA transcripts, which may serve modulatory functions through
interactions with other molecular targets and potentially alter the expression of aggressive
and anxiety-like responses.
Second, a more comprehensive understanding of the biochemical and behavioral
phenotype associated with total ablation of MAO A and MAO B is required to dissect the
clinical features associated with the recently-discovered dual MAO A/B-deletion. The
severe phenotypic consequences of dual MAO A/B-deletion distinguishes it from selective
MAO A- or MAO B-deficiency, and results in developmental and cognitive impairments
resembling a pervasive developmental disorder. Although MAO A/B KO mice may provide a
useful tool to elucidate the mechanisms underlying this phenotype, the limited number of
studies which have been conducted in this line provides an incomplete picture of the scope
of their behavioral and emotional alterations. Moreover, changes in neurochemical
pathways such as NO which have a relationship of reciprocal regulation with the
monoaminergic system have not been investigated yet may be critical in the behavioral
organization of MAO A- and MAO A/B-deficient mice.
22
Overarching Thesis Hypothesis
Mutations in MAO that cause progressive alterations in 5-HT and NE signaling are
associated with a gradual enhancement in maladaptive behaviors, featuring impulsive
aggression and anxiety-like responses, and subvert the rol e nitric oxid e in the modulation of
emotional reactivity.
Subhypotheses
Chapter 2: Novel Monoamine Oxidase A Knockout Mice With Human-Like
Spontaneous Mutation
Hypothesis: A novel line of mice harboring a human-like MAOA mutation exhibit
impulsive aggression and provide an animal model of MAO A deficiency with enhanced
translational value.
Design: Biochemical (MAO A and MAO B genotyping and catalytic activity) and
behavioral (aggression, anxiety-like and depressive-like behaviors) characterizations of a
novel line of mutant mice (MAOA
A863T
KO) will be performed. Brain-regional metabolism of
MAO A substrates and metabolites in MAOA
A863T
KO will be compared with that of the
hypomorphic MAO A
neo
line, which exhibit low MAO A activity selectively in amygdala and
prefrontal cortex and a unique behavioral phenotype characterized by reduced aggression
and heightened anxiety.
Limitations & Potential Pitfalls: Major limitations include the fact that psychiatric
disorders cannot be fully captured in mouse models, and data from these studies may not be
completely applicable in the clinic. Moreover, the methodology outlined above does not
allow for precise causal relationships between neurochemistry and behavior to be defined,
23
but rather provides a framework which can be used to test hypotheses elucidating these
mechanisms.
Chapter 3: Monoamine Oxidase Deficiency Reverses The Emotional Effects Of
Nitric Oxide Synthesis Inhibition
Hypothesis: MAO A/B-deficiency results in a phenotype characterized by higher
levels of anxiety-related behavior and subverts the role of nitric oxide in the modulation of
emotional reactivity.
Design: Brain regional levels of MAO A substrates and metabolites will be measured
in MAOA
A863T
KO and MAO A/B KO mice; wild type (WT) counterparts will be used as
controls. WT, MAOA
A863T
KO and MAO A/B KO mice will be evaluated in behavioral
paradigms exploring different facets of emotional reactivity, such as anxiety (light-dark
box), reactive aggression (resident-intruder), depression (tail suspension) and fear
(predator urine/emergence task). Moreover, the impact of the inhibitor of NO synthesis N
G
-
nitro-L-arginine methyl ester (L-NAME, 25 mg/kg, i.p.) on the behavioral responses of these
mice in the aforementioned paradigms will be investigated.
Limitations & Potential Pitfalls: Several limitations of this study should be
considered. Notably, dual MAO A/B-deletion may result in more extensive physiological
impairments in humans as compared to mice, for example, the difficulty in ambulation and
hypotonia observed in the former (Whibley et al, 2010).
Additionally, in studying systems with multiple functional redundancies, such as the
MAO/5-HT/NO system, it is important to recognize the inherent limitations of KO mouse
models. In such systems, it is difficult to assign precise causal roles to the observed
perturbations, as numerous mechanisms may be enacted to compensate for the deficient
24
element(s). Ind eed, the aforementioned method ol ogy d oes not includ e analysis of NO l evels in
WT and MAO A/B KO mice, nor d oes it account for the possibl e contribution of MAO B and it s
preferred substrate PEA in the phenotypical anomalies of MAO A/B KO mice.
Lastly, the use of a nonsel ective NOS inhibitor such as L-NAME hind ers our ability to
elucidate the relative contributions of the various NOS isozymes. Although our d ose of L-
NAME was sel ected to elicit substantial bl ockad e of NO synthesis (Moreno-López et al, 2004),
the lack of a full d ose-response analysis restricts our insight into the dynamic rol e of NO in
behavioral regulation across both genotypes.
25
CHAPTER 2:
NOVEL MONOAMINE OXIDASE A KNOCKOUT MICE WITH
HUMAN-LIKE SPONTANEOUS MUTATION
CHAPTER 2 ABSTRACT
A novel line of mutant mice (MAOA
A863T
KO) harboring a spontaneous point
nonsense mutation in exon 8 of the monoamine oxidase A (MAO A) gene was identified in a
129S6 colony. This mutation is analogous to the cause of a rare human disorder, Brunner
syndrome, characterized by complete MAO A deficiency and impulsive aggressiveness.
Concurrent with previous studies of MAO A KO mice generated in a C3H/Hej colony by
insertional mutagenesis (“Tg8”), MAOA
A863T
KO lack MAO A enzyme activity and display
enhanced aggression toward intruder mice. However, MAOA
A863T
KO exhibited lower
locomotor activity in a novel, inescapable open field and similar immobility during tail
suspension compared to wild type (WT), observations that differed from reports of Tg8-
MAO A KO mice. Brain-regional levels of MAO substrates were compared in MAOA
A863T
KO,
WT, and a line of hypomorphic MAO A mutants, generated in a 129S6 colony, which harbor
an intronic neomycin-resistance cassette in the MAO A gene (MAO A
neo
). Previous studies
revealed that MAO A
neo
exhibit low MAO A catalytic activity, reduced aggression and
heightened anxiety-like behavior as compared to both WT and MAOA
A863T
KO. We found
that levels of 5-HT and NE were significantly higher in MAOA
A863T
KO than in WT
counterparts across all brain regions. Furthermore, in MAO A
neo
we found that 5-HT content
in the amygdala and prefrontal cortex was comparable to similar to that featured by WT
and significantly lower than in MAOA
A863T
KO. These findings consolidate evidence linking
MAO A to aggression and suggest that brain-regional variations in 5-HT and NE exert a
profound influence on the regulation of emotional reactivity.
26
INTRODUCTION
In humans, a point nonsense mutation in exon 8 of the MAO A gene (MAOA) causes
Brunner syndrome, a recessive X-linked condition characterized by complete absence of
MAO A activity in association with mild retardation and violent aggressiveness (Brunner et
al, 1993b; Brunner et al, 1993a). The low prevalence of the syndrome and the elusiveness
of its nosographical description (Hebebrand and Klug 1995), however, hinder our
understanding of its specific psychobiological determinants and limit our ability to
characterize its specific features and distinguish it from other impulse-control disorders,
such as the intermittent explosive disorder.
A useful tool to elucidate the phenotypical consequence of MAO A deficiency is
afforded by MAO A knockout (KO) mice. A first line of MAO A KO mice (referred to as the
‘Tg8” line) was generated by the inadvertent insertion of an IFN-β minigene into exon 2 of
the MAOA coding region of one cell embryos of C3H/HeJ mice (Cases et al, 1995). Tg8 mice
displayed several behavioral abnormalities, including increased intermale aggression
(Cases et al, 1995). Nevertheless, the translational value of this line is somewhat tempered
by the different mutation site and the presence of the inserted cassette, which may
influence the epigenetic regulation of MAO A-dependent behaviors.
A line of MAO A hypomorphic mutant mice, harboring a Neo
R
cassette in intron 12 of
MAOA (MAO A
neo
) exhibited a neurochemical profile that is distinct from both Tg8 and WT
mice (Bortolato et al, submitted). Unlike Tg8 mice, which lack MAO A activity throughout
the brain, MAO A
neo
mice exhibit brain regional-selective deficits in MAO A activity;
specifically, MAO A activity is undetectable in all brain regions except amygdala and
prefrontal cortex. This unique neurochemical profile may contribute to their distinctive
27
behavioral phenotype, characterized by reduced aggression and heightened anxiety as
compared to both WT and MAO A KO mice (Bortolato et al, submitted).
Here, we describe the discovery and characterization of a human-like spontaneous
point mutation in the eighth exon of the MAOA gene in 129S6 mice. In addition to studying
the biochemical and behavioral characteristics of this novel line of MAO A-deficient mice,
we also investigated the relationship between emotional reactivity and brain-regional MAO
A activity by comparing monoamine levels in MAOA
A863T
KO mice with WT and
hypomorphic MAO A
neo
mice derived from the same murine strain, 129S6.
MATERIALS AND METHODS
Animals: Mice were housed in cages with free access to food and water on a 12-
hour light cycle, in accordance with the protocol approved by the University of Southern
California Institutional Animal Care and Use Committee. Because our WT and MAOA
A863T
KO are a congenic line on an essentially homogeneous genetic background, we bred WT
mice by WT-WT pairs and MAOA
A863T
KO mice by KO-KO pairs.
Genotyping and Sequencing: Mice were genotyped by polymerase chain reaction
(PCR) using genomic DNA from tail samples as template and Taq polymerase (Invitrogen,
Carlsbad, CA, USA). Each sample was genotyped for MAO A using the following primers:
MAO A Forward: 5’-ACGCGCTCTTCTGGTGCAT-3’, MAO A Reverse: 5’-AGCTTACTTCAGGGC-
3’. MAO A PCR products were then processed with Dra1 (New England Biolabs, Ipswich,
MA, USA), as the loss of this cutting site indicates the A → T point mutation. Undigested
PCR products were cloned into a pCR4-topo cloning vector (Invitrogen, Carlsbad, CA, USA)
and sequencing results analyzed with Sequence Scanner v1.0. Each sample was genotyped
28
for MAO B using the following primers: MAO B Forward: 5’-CTACAAAGCAGATTGCCACGC-
3’; MAO B Reverse: 5’-TACCTGACATCAACTGGTCCC-3’.
MAO A and B Catalytic Activity Assays: One-month old male WT 129S6 (N = 3)
and MAOA
A863T
KO (N = 3) mice were sacrificed by cervical dislocation. Liver and brain
regions (brain stem, cerebellum, frontal cortex, and hippocampus) were removed and
homogenized in assay buffer (50mM sodium phosphate buffer, pH 7.4). To control for the
high level of MAO B in the liver, liver samples were pre-incubated in 10
-6
M deprenyl
(selective MAO B inhibitor) for 20 min. at 37 ˚C prior to measurement of MAO A catalytic
activity. Catalytic activity for MAO A and MAO B was measured as described previously
(Geha et al, 2001).
Behavior Tests: All behavioral experiments were conducted on male MAOA
A863T
KO mice (N = 6) and age-matched male WT 129S6 mice (N = 6), the MAOA
A863T
KO parental
line. Experiments were performed between 1 and 3pm. All mice were at least 3 months of
age and isolated for a minimum of 10 days prior to the initiation of behavioral experiments.
Open field: Locomotor activity was measured in a square arena 40 cm X 40 cm
under low lighting for 5 min. The arena was divided into two virtual zones of equal area,
one central and one peripheral. Data were collected by video camera and scored by
computer interface (Ethovision; Noldus, Inc., Wageningen, Netherlands). The total
distances traveled in the whole arena, center, and periphery (cm), the time spent in the
center and periphery (s), the angular (cm/s) and vector (cm/s
2
) velocities in the whole
arena, center, and periphery, as well as the number of entries into the center zone, were
recorded.
Tail suspension: Mice were suspended vertically by the tail using medical tape for a
total of 6 min. without prior habituation to the test room. The duration of immobility (s)
29
and duration of trunk motion (s) were recorded. Trunk motion was defined as whole-body
upward movement toward the base of the tail, as opposed to paw movement or twitching.
Data were scored using Behavior Tracker software (Greensburg, Pennsylvania, USA).
Resident intruder test: Each mouse was tested in its home
cage following a 30 min.
habituation period in the testing room against an age- and weight-matched WT 129S6 male
for a total of 5 min. A fighting bout was defined as a continuous series of behavioral
interactions including offensive attack, wrestling, and biting. If 2 seconds or more elapsed
between bouts of fighting, these were considered separate events. For each resident, the
number of fighting bouts and latency to first offensive attack (s) were
recorded. Resident
male tail rattling was also recorded. If one second or more elapsed between tail rattles,
these were considered separate events. Data were scored using Behavior Tracker.
HPLC Analyses: Each mouse (N = 10 per genotype) was sacrificed by cervical
dislocation, and each brain region - amygdala, hippocampus, midbrain, and prefrontal
cortex - was quickly removed, immediately placed on ice, and homogenized in a solution
containing 0.1 M trichloroacetic acid, 10 mM sodium acetate, and 0.1 mM EDTA; 1 µM
isoproterenol was used as an internal standard. The homogenates were sonicated and
centrifuged, and the supernatants used for HPLC analysis. 5-HT, 5-hydroxyindoleacetic
acid (5-HIAA), DA, dihydroxyphenylacetic acid (DOPAC) and NE (Sigma, St. Louis, MO, USA)
were used as standards. The protein oncentrations were determined using the pellet using
a bicinchoninic acid kit (Pierce, Rockford, IL, USA) following the manufacturer’s protocol.
The mobile phase was the same as the homogenization buffer (excluding the isoproterenol)
with 7% methanol for detection of 5-HT and 5-HIAA. DA, DOPAC and NE were quantified
separately using trichloroacetic acid mobile phase solution without methanol. The mobile
phases were filtered and deaerated, and the pump speed (Shimadzu LC-6A liquid
30
chromatograph) was set 1.0 ml/min. The reverse-phase column used was a Rexchrom
S50100-ODS C18 column with a length of 25 cm and an internal diameter of 4.6 mm (Regis,
Morton Grove, IL, USA). The compounds were measured at + 0.7 V using a Shimadzu L-ECD-
6A electrochemical detector.
Statistical Procedures: All results are expressed as mean ± S.E.M. Statistical
differences between WT and MAOA
A863T
KO mice with respect to mean enzyme activity
levels, mean duration of immobility and trunk motion during the tail suspension test, mean
latency to first attack and mean numbers of tail rattles and fighting bouts during the
resident intruder test were performed using 2-tailed, unpaired t-tests. Open field data was
analyzed by one-way or two-way ANOVA as indicated. HPLC data was analyzed by ANOVA
followed by Tukey’s post-hoc test when appropriate; nonparametric comparisons were
carried out by the Kruskal-Wallis test, and post-hoc analyses were performed by the Mann–
Whitney U test, with Dunn-Sidak correction for α levels. Statistical significance was
accepted at the probability level of P<0.05.
31
RESULTS
Identification and Biochemical Characterization of MAOA
A863T
KO mice
A line of mice with a spontaneous point mutation in MAOA was discovered through
genotyping in a colony of 129S6 mice. The unexpected PCR products in these mice revealed
the abrogation of a Dra1 restriction enzyme cutting site in the MAO A PCR product,
indicating a single point mutation in exon 8 (Fig. 2.1a); MAO B PCR products for the
MAOA
A863T
KO mice are identical to WT (Fig. 2.1b).
FIGURE 2.1
Genotyping of MAOA
A863T
KO Mice. MAOA
A863T
KO mice were first identified in a mixed colony of MAO B KO
and MAO A/B KO mice. Genotyping revealed that MAOA
A863T
KO mice are WT for MAOB but contain a single
point mutation in MAOA that eliminates a Dra1 restriction enzyme cutting site. a, MAOA genotyping using
primers specific for MAOA exon 8 reveals identical bands for all three genotypes (top). The point mutation is
identified following Dra1 digest (bottom). Lane 1, MAOA
A863T
KO male; lane 2, WT male; lane 3, heterozygous
(+/-) MAOA
A863T
KO female. b, MAOA
A863T
KO mice are WT for MAOB, as revealed by the lack of the 1.2 kb insert
in exon 6 which abrogates this isoenzyme. Lane 1, MAOA
A863T
KO male; lane 2, MAO B KO male; lane 3, MAO B
KO heterozygous (+/-) female.
32
FIGURE 2.2
Sites of Spontaneous Mutation in Mouse and Human MAOA, Exon 8. Arrows indicate sites of mutation
resulting in premature stop codons in mouse (top) corresponding to amino acid 284 (lysine) and in human
(bottom) corresponding to amino acid 296 (glutamine). Nucleic acid numbering is according to mouse MAOA
GenBank entry NM.173740 and human gene bank entry M68840.
Subsequent cloning and sequencing of the MAO A and MAO B genotyping PCR
products from the MAOA
A863T
KO line confirmed these results and revealed that the site of
this point mutation is very closely located to the site of the point mutation in exon 8 of
MAOA harbored by humans with Brunner syndrome (Fig. 2.2). Likewise, MAO A enzymatic
activity is completely abrogated in the mutant MAOA
A863T
KO mice, while MAO B activity is
similar to WT (Table 2.1).
TABLE 2.1
Catalytic Activity of MAO A and MAO B in WT and MAOA
A863T
KO Mice. Mean ± SEM (nmol/20min/mg
protein) in brain and liver samples from one-month-old male pups.
a
Refer to Materials and Methods.
33
Physical and Behavioral Characterization of MAOA
A863T
KO mice
Neonatal MAOA
A863T
KO pups tend to be smaller in body weight than their WT
littermates but do not display any gross physical deformity or ambulatory impairment.
Behavioral studies of the MAOA
A863T
KO line revealed a decrease in locomotor activity as
compared to WT in a novel, inescapable open field (Fig. 2.3a-e). Over three consecutive test
days, MAOA
A863T
KO mice traveled less throughout the entire arena (F 1,10=14.43, P=0.003,
two-way ANOVA) (Fig. 2.3a) and averaged lower vector velocity (F 1,10=13.77, P=0.004, two-
way ANOVA) (Fig. 2.3b). Although MAOA
A863T
KO mice entered the center zone fewer times
than WT on the first day (F 1,10=5.56, P=0.038, one-way ANOVA) (Fig. 2.3c) there was no
difference in the time spent in the central zone between the two genotypes on either Day 1
or over the 3-day test period (Day 1, F 1,10=0.05, P=0.8261, one-way ANOVA; all three days,
F 1,10=0.01, P=0.916, two-way ANOVA) (Fig. 2.3d). MAOA
A863T
KO also showed higher
angular velocity throughout the whole arena on all days (F 1,10=25.92, P=0.0007, two-way
ANOVA) (Fig. 2.3e). Complete immobility time did not differ between the two genotypes
during the tail suspension test (P=0.25) (Fig. 2.3f), although MAOA
A863T
KO mice did spend
significantly more time engaged in trunk motion than WT (P=0.007, data not shown). The
resident intruder test revealed significantly higher aggression in the MAOA
A863T
KO mice, as
indicated by a reduced latency to first attack from 275.5 seconds in WT to 30.5 seconds in
MAOA
A863T
KO (P<0.01) (Fig. 2.3g). In addition, the number of fighting bouts (P<0.05) and
resident mouse tail rattles (P<0.05) were significantly increased in MAOA
A863T
KO (Fig.
2.3h).
34
FIGURE 2.3
Behavioral Alterations in MAOA
A863T
KO Mice. MAOA
A863T
KO mice showed decreased locomotor activity in a
novel, inescapable open field, indicated by reduced distance traveled in the entire arena a, and average vector
velocity (cm/s2) b, during the first 5min in the field. Although MAOA
A863T
KO entered the center zone fewer
times on day 1 c, they did not spend significantly less time in the center on any test day d. MAOA
A863T
KO also
demonstrated consistently higher angular velocity (cm/s) e. MAOA
A863T
KO mice did not spend significantly less
time immobile during 6 min. tail suspension test f. MAOA
A863T
KO showed increased aggression in the resident
intruder paradigm, as measured by their reduced latency to first attack (s) g, and increased number of tail
rattles and bouts of fighting h. *P<0.05, **P<0.01, ***P<0.001
35
Brain-Regional Measurement of MAO A Substrates and Metabolites in WT, MAO A
neo
,
and MAOA
A863T
KO mice
We characterized the levels of monoamines and their metabolites in several brain
regions of WT, MAO A
neo
and MAOA
A863T
KO mice. All mice were male and 60 days of age (N
= 10 for each genotype). This developmental time point was chosen based on studies
performed in the original MAO A KO line, Tg8: between 30-90 days of age, whole brain
levels of 5-HT, 5-HIAA and NE in both WT and Tg8 mice remained consistent, and
consequently, the percent increase or decrease of these monoamines in Tg8 mice compared
to WT was also consistent. By contrast, the amount of brain 5-HT in neonatal Tg8 pups
compared to WT was increased 9-fold at day 1, dropped to 6-fold at day 12, stabilized to an
approximately ~2-fold increase between 30-90 days of age, and eventually returned to a
level equivalent with WT in late adulthood (Cases et al, 1995). These age-dependent
variations are likely due to developmental changes in the function of MAO B, and perhaps
other metabolic enzymes involved in the pathways of monoamine degradation (Eisenhofer
et al, 2004). Thus, in order to detect potentially subtle differences between MAOA
A863T
KO
and MAO A
neo
mice, we opted to analyze mice at a stage of development at which we
expected the levels of MAO A substrates and metabolites to be relatively stable.
Both MAO A
neo
and MAOA
A863T
KO mice displayed significantly higher 5-HT levels in
the hippocampus (H 2,29 =11.21, P<0.01, Kruskal-Wallis) and midbrain (H 2,46 =9.37, P<0.01,
Kruskal-Wallis) compared to WT (Table 2.2). MAOA
A863T
KO mice exhibited significantly
higher 5-HT levels in the amygdala (F 2,24 =9.4, P<0.001, one-way ANOVA) and prefrontal
cortex (F 2,42 =13.15, P<0.001, one-way ANOVA) as compared to WT mice. In agreement
with the low levels of MAO A activity in the amygdala and prefrontal cortex of MAO A
neo
, we
found that 5-HT content in those two regions was comparable to that featured by WT mice
36
and significantly lower than that in MAOA
A863T
KO [amygdala (P<0.01, Tukey’s); prefrontal
cortex (P<0.01, Tukey’s)]. MAO A
neo
and MAOA
A863T
KO mice displayed higher NE levels in
the amygdala (F 2,23 = 19.63, P<0.01, one-way ANOVA), hippocampus (F 2,26 = 8.04, P<0.01,
one-way ANOVA), midbrain (H 2,30 = 11.80, P<0.01, Kruskal-Wallis) and prefrontal cortex
(F 2,26 = 12.61, P<0.001, one-way ANOVA) compared to WT mice. NE levels tended to be
higher in the amygdala of MAOA
A863T
KO as compared to MAO A
neo
mice (P<0.1, Tukey’s
test). DA levels were equivalent across all regions in the three lines (data not shown).
5-HIAA levels were significantly reduced in all regions in MAOA
A863T
KO mice, and in
all regions in MAO A
neo
mice with the exception of midbrain (Table 2.2). Levels of the DA
and NE metabolite DOPAC were significantly reduced in all regions of MAO A
neo
and
MAOA
A863T
KO mice, as compared with WT controls (data not shown).
TABLE 2.2
WT MAO A
neo
MAOA
A863T
Amygdala 5-HT 29.52 (1.42) 28.14 (1.83) 56.09 (1.84)**##
5-HIAA 6.88 (0.32) 1.15 (0.17)** 3.58 (0.24)*
NE 15.85 (0.38) 27.23 (1.26)* 30.40 (0.67)**
a
Hippocampus 5-HT 9.15 (0.23) 15.13 (0.68)* 15.86 (0.51)*
5-HIAA 9.01 (0.34) 4.42 (0.27)** 1.62 (0.11)**
NE 6.55 (0.19) 11.09 (0.30)** 10.33 (0.33)*
Midbrain 5-HT 29.78 (1.22) 58.81 (2.79)* 65.45 (2.21)**
5-HIAA 10.51 (0.72) 4.98 (0.30) 4.62 (0.22)*
NE 13.09 (0.45) 27.11 (1.30)** 23.03 (0.71)*
Prefrontal 5-HT 19.20 (0.47) 20.66 (1.07) 33.76 (0.88)**##
Cortex 5-HIAA 8.42 (0.25) 0.84 (0.20)** 3.53 (0.20)**
NE 9.05 (0.25) 17.67 (0.53)** 17.29 (0.57)**
Data represent mean (SEM).
Units of measurement are PM/mg protein.
*P<0.05, **P<0.01 compared with WT mice (post-hoc tests)
a
P<0.1, ##P<0.01 compared with MAO A
neo
mice (post-hoc tests)
37
DISCUSSION
Different mutations in the same gene have been shown to lead to KO lines with
distinct phenotypes (Simoni 1998; Fleming et al, 2005). Thus, the availability of different
mutant lines for the same gene offers an interesting approach to elucidate the mechanisms
of functional regulation of the targeted genes and proteins. Indeed, it is worth noting that
the nonsense point mutation in MAOA
A863T
KO mice is similar to the human mutation
reported in Brunner syndrome. This resemblance highlights MAOA
A863T
KO mice as a highly
reliable model to study the neurochemical and neurophysiological alterations associated
with Brunner syndrome and related disorders.
Similarly to Tg8-MAO A KO mice, MAOA
A863T
KO resident mice exhibited marked
levels of aggressiveness in the resident–intruder paradigm (Cases et al, 1995). This finding
further confirms that MAO A deletion results in aggressiveness and impulsivity, behaviors
likely mediated by increased levels of 5-HT and NE. Indeed, genetic reduction of NE
function reduces aggression in the resident–intruder paradigm (Marino et al, 2005),
whereas altered 5-HT metabolism has been linked to impulsivity in humans and primates
(Brunner et al, 1993b; Mehlman et al, 1994).
Although similar in their display of aggressive behavior, MAOA
A863T
KO failed to
display significant changes in absolute immobility time in the tail suspension test, a
dependable parameter for the measurement of depressive-like behaviors (Steru et al, 1985;
El Yacoubi et al, 2003). This evidence is at variance with previous findings in Tg8 mice,
which displayed reduced immobility duration in another validated model of depression, the
forced swim test (Cases et al, 1995). In addition, MAOA
A863T
KO exhibited markedly reduced
locomotion in the open field, a paradigm in which Tg8 mice behaved normally (Agatsuma et
al, 2006). As MAOA
A863T
KO mice do not exhibit any impairment in gait, this finding suggests
38
that the reduction of locomotor activity may be a unique phenotype of this line, perhaps
reflecting divergent environmental reactivity. A possible interpretation for these apparent
discrepancies is that Tg8 and MAOA
A863T
KO mice may display a different sensitivity to
environmental stress. Different intrinsic variations between the two mutations or the two
different background strains (129S6 and C3H/HeJ) may account for these divergent
phenotypes.
TABLE 2.3
Neurochemical and Behavioral Features of MAO A-deficient mice. (Bortolato et al, submitted; Cases et al,
1995; Chen et al, 2004; Scott et al, 2008)
Recent studies by our group indicated that the exploratory deficits in MAOA
A863T
KO
mice may result from impaired emotional regulation. Notably, despite a marked reduction
in defensive behaviors, MAOA
A863T
KO mice exhibited neither major dysfunctions of
olfactory or visual perception, nor deficits in object recognition in the absence of light,
ruling out the role of microvibrissae in their lack of exploration (Godar et al, submitted). In
Tg8 mice, excessive 5-HT during the postnatal stage causes the cortical representations of
39
the mystacial vibrissae in the snout (Erzurumlu and Jhaveri 1990) to develop abnormally
(Cases et al, 1995; Cases et al, 1996). This “barrelfield” structure is critical in the
coordination of the mystacial vibrissae in the rodent snout (Luhmann et al, 2005), and its
disorganization can result in profoundly deficient abilities related to perceptual processing,
exploration, and sensory integration of environmental cues (Cases et al, 1996; Dowman and
Ben-Avraham, 2008; Hurwitz et al, 1990; Sanders et al, 2001; Straube et al, 2009).
However, studies in MAOA
A863T
KO suggest that such alterations are not a major
contributing factor to their reduced exploratory drive (Godar et al, submitted), which may
reflect a general lack of inquisitiveness resulting from high levels of anxiety.
Interestingly, the responses exhibited by MAOA
A863T
KO mice in the open field and
tail suspension test are comparable to those previously reported in a line of MAO A/B
double KO mice (MAO A/B KO) which harbor the identical human-like mutation in MAOA
(Table 2.3). MAO A/B KO mice were discovered in a colony of MAO B KO mice (Grimsby et
al, 1997) (also from a 129S6 parental strain) and are deficient in both isoenzymes (Chen et
al, 2004). Akin to both Tg8 and MAOA
A863T
KO mice, MAO A/B KO mice displayed acute lack
of impulse control and heightened aggression but differed from Tg8 in exhibiting marked
deficits in exploratory activity, including reduced locomotor activity in the novel open field
(Fig. 2.4). Moreover, despite having dramatically increased brain 5-HT levels, MAO A/B KO
mice do not differ from WT mice in terms of immobility time in the tail suspension test
(Refer to Chapter 3). These similarities suggest a similar spectrum of behavioral
dysfunctions in both MAOA
A863T
KO and MAO A/B KO mice.
40
FIGURE 2.4
Locomotor Activity in MAOA
A863T
KO and MAO A/B KO mice in a Novel Open Field. Locomotor activity was
compared in MAOA
A863T
KO and MAO A/B KO littermates; age-matched 129S6 WT mice served as controls. Both
mutant lines showed similar reductions in activity. N=10 each genotype. *P<0.05, **P<0.01 compared with WT
mice.
Additionally, we found that in MAO A
neo
mice, low MAO A activity in amygdala and
prefrontal cortex were sufficient to restore 5-HT to levels equivalent with WT mice.
Further, in amygdala - the brain region of MAO A
neo
mice with the highest levels of MAO A
activity - NE levels, albeit higher than in WT mice, were also lower in comparison with
MAOA
A863T
KO. The impact of small amounts of MAO A activity is likely to be greater on 5-
HT than NE, in view of the much lower affinity of this enzyme for the latter
neurotransmitter (Strolin Benedetti et al, 1983), as well as the important role of
41
catecholamine-O-methyl-transferase (COMT) in NE degradation. Indeed, these data suggest
that the presence of small amounts of MAO A activity in amygdala and prefrontal cortex of
MAO A
neo
mice may be sufficient to markedly attenuate the expression of aggressive and
antisocial behaviors (Fig. 2.5), observations which appear to complement our group’s
previous findings that restoration of MAO A in forebrain regions normalized the local
monoamine levels and rescued the behavioral deficits in MAO A KO mice (Chen et al, 2007).
Indeed, the region-specific variations in 5-HT and NE levels may account for the high levels
of anxiety in MAO A
neo
mice, as there is abundant evidence that these monoamines play a
crucial role in the regulation of emotional reactivity (Ressler and Nemeroff 2000). At this
point, however, such contentions are merely speculative, as numerous factors could
potentially contribute to the behavioral abnormalities observed in MAO A
neo
mice,
warranting further studies to elucidate the mechanisms underpinning the behavioral
phenotype of MAO A
neo
mice.
FIGURE 2.5
Aggressive Behaviors in WT, MAO A
neo
, and MAOA
A863T
KO Mice. MAO A
neo
mice exhibited significant
increases in a latency to attack intruders compared to WT and MAOA
A863T
KO mice. Furthermore, MAO A
neo
mice
show a significant reduction in b fighting bouts and c fighting duration compared to MAOA
A863T
KO mice. Values
are represented as mean ± SEM. **P<0.01, ***P<0.001 compared to WT mice and
##
P<0.01,
###
P<0.001
compared to MAO A
neo
mice (Bortolato et al, submitted).
42
Conclusion
MAOA
A863T
KO mice are the first murine line with a naturally occurring nonsense
mutation selectively in the MAO A gene. The similarity of this mutation to the human
condition makes this line of mice a valuable tool for translational research and further
exploration of the role of this enzyme on brain function and behavioral regulation.
Moreover, MAOA
A863T
KO mice exhibit anxiety and depressive-like responses that are
divergent from previous reports of transgenic MAO A KO mice but comparable to MAO A/B
KO mice, which share the identical human-like MAOA mutation but presumably have more
severely deficient metabolism of 5-HT. Collectively these lines may provide useful models
to explore how varying degrees of deficient 5-HT metabolism influence emotional
behaviors.
43
CHAPTER 3:
MONOAMINE OXIDASE DEFICIENCY REVERSES THE EMOTIONAL EFFECTS
OF NITRIC OXIDE SYNTHESIS INHIBITION
CHAPTER 3 ABSTRACT
Monoamine oxidases (MAOs) A and B are key enzymes in the degradation of
monoamine neurotransmitters. We previously showed that, in mice, combined deficiency of
MAO A and MAO B results in maladaptive responses to fear or stress. As several lines of
evidence indicate that the role of monoamines in the regulation of emotional reactivity is
modulated by nitric oxide (NO), we hypothesized that this mediator may be involved in the
alterations observed in MAO A/B knockout (KO) mice. Thus, in the present study we
analyzed the impact of the inhibitor of NO synthesis N
G
-nitro-L-arginine methyl ester (L-
NAME, 25 mg/kg, i.p.) on the behavioral responses of MAO A/B KO and wild-type (WT)
mice in a number of paradigms exploring different facets of emotional reactivity, such as
anxiety, reactive aggression, depression and fear.
In WT mice, L-NAME reduced exploratory responses and aggression, while
increasing freezing and depression-like behaviors; conversely, in MAO A/B KO mice, this
drug exerted opposite properties, inducing an enhancement of exploratory and fighting
behaviors and a decrease of freezing reactions. Furthermore, while L-NAME did not affect
regional MAO A activity or monoamine levels in any brain region of WT mice, it enhanced
the levels of 5-HT in MAO A/B KO mice exposed to predator urine, an innate, fear-inducing
stimulus. Taken together, these findings indicate that MAO deficiency subverts the role of
NO in emotional reactivity, and points to this molecule as an interesting substrate for some
of the behavioral disturbances featured in MAO A/B KO mice.
44
INTRODUCTION
Monoamine oxidases (MAO A and MAO B) are the main enzymes that degrade the
monoamine neurotransmitters serotonin (5-HT), dopamine (DA) and norepinephrine (NE),
and phenylethylamine (PEA) in the brain (Shih and Thompson 1999; Bach et al, 1988). The
two MAO isoenzymes share ~70% homology, exhibit identical exon-intron organization and
follow the same kinetic mechanism (Grimsby et al, 1991; Bortolato et al, 2008; Shih et al,
1998), yet they display significant differences in substrate specificities: MAO A prefers 5-HT
and NE as substrates while MAO B prefers PEA as a substrate (Geha et al, 2001); both
catabolize dopamine (DA) (O'Carroll et al, 1983; Garrick and Murphy 1980). Several studies
have demonstrated a link between MAO deficiency and maladaptive emotional behaviors
(Brunner et al, 1993a; Cases et al, 1995; Bortolato et al, 2009; Scott et al, 2008). Similarly,
reductions in the expression or function of MAO A and/or MAO B have been associated with
abnormal psychological traits, including impulsivity (Skondras et al, 2004; Paaver et al,
2007), sensation-seeking (Fowler et al, 1980; Reist et al, 1990; Ruchkin et al, 2005), as well
as antisocial and aggressive behavior (Caspi et al, 2002; Manuck et al, 2000a). In the
absence of MAO A, MAO B oxidizes MAO A’s preferred substrates, and vice versa; brain
levels of 5-HT, NE, DA and PEA are therefore increased to a much greater extent in MAO A/B
double knockout (MAO A/B KO) mice (Chen et al, 2004) than in either MAO A KO (Cases et
al, 1995) or MAO B KO mice (Grimsby et al, 1997). The behavioral phenotype of MAO A/B
KO mice, while bearing several features similar to MAO A KO mice, including high levels of
impulsive aggression (Cases et al, 1995; Scott et al, 2008), features prominent abnormalities
of emotional reactivity, with hyper-reactivity and increases in anxiety-like behaviors (Chen
et al, 2004).
45
Although monoaminergic systems play a fundamental role in the pathogenesis of
these alterations, the specific neurochemical changes underpinning their dysregulated
function remain poorly understood. Increasing evidence suggests that nitric oxide (NO) is a
key factor in the dynamic modulation of monoaminergic signaling in the mediation of
emotional responses, such as impulsive aggressiveness (Chiavegatto et al, 2001), anxiety
(Zhang et al, 2010), and depression (Chanrion et al, 2007). A diffusible intercellular
modulator in the nervous system (Bredt et al, 1991; O'Dell et al, 1991; Bredt and Snyder
1994; Schuman and Madison 1994; Shibuki and Okada 1991), NO is synthesized from L-
arginine by nitric oxide synthase (NOS) enzymes, of which there are three isoenzymes: one
inducible (iNOS or Type II NOS) (Xie et al, 1992) and two constitutive forms, neuronal NOS
(nNOS, Type I NOS) (Bredt and Snyder 1990; Bredt et al, 1990) and endothelial NOS (eNOS,
Type III NOS) (Lamas et al, 1992; Förstermann et al, 1994).
It appears that the constitutively-active forms of NOS, eNOS and nNOS, work in
concert to modulate emotional reactivity (Volke et al, 1997; Czech et al, 2003; Spiacci Jr et
al, 2008; Zhang et al, 2010) and have contrasting effects 5-HT turnover (Frisch et al, 2000;
Chiavegatto et al, 2001). Such functional differences may stem from their distinctive
localization throughout the brain: expression of eNOS is limited to endothelial tissue while
nNOS is expressed in discrete populations of neurons throughout the limbic system (Bredt
et al, 1991; Dawson et al, 1991) and in 5-HT neurons of the dorsal raphe nucleus (DRN)
(Simpson et al, 2003). Exposure to fear or stressors increases nNOS in the amygdala and
other brain regions (Chiavegatto et al, 1998; De Oliveira et al, 2000; Masood et al, 2003;
Echeverry et al, 2004), and several lines of evidence indicate that NO derived from neuronal
sources may facilitate anxiety (Volke et al, 1997, Zhang et al, 2010). Conversely, NO derived
from eNOS may counteract this effect (Frisch et al, 2000). Indeed, microinjection of a low
46
dose of either the nNOS-selective inhibitor 7-nitroindazole (7-Ni), or the nonselective
inhibitor N
G
-nitro-L-arginine methyl ester (L-NAME), directly into the nNOS-rich DRN was
shown to increase locomotor and exploratory behaviors, suggesting an anxiolytic effect, but
higher doses of either inhibitor produced opposite effects (Spiacci Jr. et al, 2008).
Conversely, systemic injection of 7-Ni induced a dose-dependent anxiolytic effect (Volke et
al, 1997) and L-NAME induced a dose-dependent anxiogenic effect (Czech et al, 2003).
Likewise, administration of 7-Ni enhanced fighting behaviors (Demas et al, 1997), whereas
L-NAME prol onged the latency to attack (Ankarali et al, 2009). Collectively, these studies
suggest that selective inhibition of nNOS – induced by an nNOS-selective inhibitor such as 7-
Ni or low doses of L-NAME targeted to a region such as the DRN – enhanced emotional
reactivity, indicated by increases in locomotor and exploratory activity and heightened
aggression. By contrast, non-selective eNOS/nNOS inhibition – induced by high doses of 7-
Ni or L-NAME injected systemically – induced opposite effects.
Several lines of evidence support the notion that the modulation of emotional
behaviors by NO is bi-directional as well as dynamic. Notably, nNOS KO mice exhibit brain-
regional deficits in 5-HT turnover (Chiavegatto et al, 2001), an alteration that was linked to
their high levels of aggressive behavior (Nelson et al, 1995); by contrast, 5-HT turnover is
accelerated in eNOS KO mice (Frisch et al, 2000), in which aggression is virtually eliminated
(Demas et al, 1999). In addition, Pokk and colleagues reported that NOS inhibitors induced
differential effects on locomotor and exploratory behaviors in mice which had been forced
to remain on a small platform (SP) surrounded by water for 24 hours prior to behavioral
testing. Consistent with the aforementioned studies (Volke et al, 1997; Czech et al, 2003;
Spiacci Jr et al, 2008; Zhang et al, 2010), in non-stressed control mice, 7-Ni increased
exploration whereas L-NAME reduced exploration; by contrast, in SP-stressed mice, the
47
effects of NOS inhibitors were reversed (Pokk et al, 2002). These results suggest that the
impact of NOS inhibitors on the modulation of behavioral responses may depend largely on
the baseline emotional state.
Based on these premises, in the present study we analyzed the effects of the
nonselective eNOS/nNOS inhibitor L-NAME on different facets of emotional reactivity in
MAO-deficient mice and wild type (WT). Although the nNOS-selective inhibitor 7-Ni would
have more clearly elucidated the impact of neuronally-derived NO, in the regulation of these
behaviors, the use of this inhibitor would have introduced an inappropriate confounding
factor, as it has been reported to inhibit the activity of MAO in the brain (Desvignes et al,
1999).
48
MATERIALS AND METHODS
Animal Husbandry: We used 3-5 month old, experimentally naïve male
129S6/SvEvTac mice (N = 99; 42 WT, 20 MAOA
A863T
KO, 37 MAO A/B KO), weighing 25-30 g.
The MAOA
A863T
KO mutation - consisting of an X-linked, single-point nonsense mutation in
exon 8 of MAOA - arose spontaneously in one of our 129S6 in-house colonies (Scott et al,
2008). The MAO A/B KO mice, featuring the same mutation as MAOA
A863T
KO mice, were
generated as previously described (Chen et al, 2004). Animals were housed in group cages
with ad libitum access to food and water. The room was maintained at 22°C, on a 12 h: 12 h
light/dark cycle. Each mouse was tested individually, unless indicated otherwise.
Experimental procedures were in compliance with the National Institute of Health
guidelines and approved by the University of Southern California Animal Use Committees.
Drugs: L-NAME (Sigma Aldrich, St Louis, MO) was dissolved in 0.9% saline (SAL)
and always administered at the dose of 25 mg/kg (i.p.) 30 min prior to behavioral testing.
The selection of this dose was based on previous studies in which similar doses
administered to mice, 25 mg/kg and 30 mg/kg, respectively, were shown to alter behavior
(Czech et al, 2003) without affecting spontaneous locomotor activity (Celik et al, 1999).
Drug stock was prepared fresh every morning.
Light-dark Box Test: The light-dark box test was performed as previously
described (Bourin and Hascoët 2003). The apparatus consisted of a Plexiglas box, divided
into two compartments: a larger (20 x 20 x 20 cm), white compartment, which was
surmounted by a bright light (200 lux); and a smaller (20 x 10 x 20 cm), black, enclosed
compartment. The two compartments were separated by a divider with a 7 x 4 cm opening
at floor level. Mice (WT saline = 15; WT L-NAME = 16; MAOA
A863T
KO saline = 9; MAOA
A863T
KO L-NAME =
8; MAO A/B KO saline = 10; MAO A/B KO L-NAME = 11) were initially habituated for 30 min to the
49
test room, in the absence of the light-dark box apparatus. Subsequently, each mouse was
placed in the white compartment and video-recorded for 10 min. The time spent in each
compartment, the duration of freezing behavior (calculated as total stillness) and the
number of transitions between compartments were measured.
Home Cage Locomotor Activity: Mice (WT saline = 16; WT L-NAME = 15; MAO A/B
KO saline = 10; MAO A/B KO L-NAME = 10) were individually housed for 2 weeks. At the end of
this period, mice were habituated for one hour to the test room in their home cages, with
light equival ent to that of the hol ding facilities during the light cycl e. Animals were then
injected with L-NAME or SAL, and vid eorecord ed for the subsequent 60 min. Locomotor
activity was then scored as the total number of crossings on a grid superimposed onto the
image of each cage in a video monitor.
Resident Intruder Test: Each resident mouse (WT saline = 15; WT L-NAME = 15; MAO
A/B KO saline = 14; MAO A/B KO L-NAME = 14) was isolated for 7 days prior to testing. Following
a 30-min period in the testing room, resident mice were tested against an age-and weight-
matched 129S6 WT intruder male mouse. Environmental light was dim (5 lux) to minimize
freezing behavior. Animals were videorecorded for 5 min. For each resident mouse, the
latency to first attack (s), duration of fighting (s) and number of fighting bouts were scored.
Fighting was defined as a series of behavioral interactions including offensive attack,
wrestling, and biting.
Tail Suspension Test: The tail suspension test was performed as previously
described (Steru et al, 1985). Mice (WT saline = 20; WT L-NAME = 20; MAO A/B KO saline = 16; MAO
A/B KO L-NAME = 16) were individually suspended by the tail using medical tape affixed to a
hook, at 30 cm from the floor. Environmental light was kept at 300 lux. Animals were
50
videorecorded for 5 min, and the duration of immobility (s) and number of fecal boli were
measured.
Emergence/Predator Urine Test: The emergence test was performed as
previously described (Holmes et al, 2003; Liu et al, 2007) with minor variations. The
apparatus consisted of a black Plexiglas box, divided in two compartments separated by a
black Plexiglas partition. One compartment (start chamber; 26 x 10 x 10 cm) was covered
with a ceiling, while the other (36 x 10 x 10 cm) was left open. The experiment was
performed on three consecutive days. On the first and second days, mice (WT = 32; MAO
A/B KO = 23) were individually placed in the start chamber for 10 min of acclimation. The
partition was lifted and the animal was allowed to freely explore the other compartment for
5 min. Mice that did not exit the start chamber on any day of training were excluded from
the last day of testing. During the third day, after a 10-min acclimation to the start chamber,
an object (tennis ball, 7 cm in diameter), impregnated with 10 mL of diluted bobcat urine
(Lexington Outdoors Inc, Robbinston, ME; vol:vol, 1:1) was placed in the open chamber, at
5 cm from the partition. The partition was then lifted, and the mouse was allowed to freely
explore the apparatus for 5 min. Behavioral measures included: latency to emerge from the
start chamber; time spent in each compartment; number of fecal boli; number of head pokes
into the open compartment.
HPLC Determination of Monoamine Levels: Mice (N = 6-10 per group) were
sacrificed by cervical dislocation and the amygdala, hippocampus, midbrain, and prefrontal
cortex were removed. Each region was homogenized in a solution containing 0.1 M
trichloroacetic acid, 10 mM sodium acetate, and 0.1 mM EDTA; 1 µM isoproterenol was used
as an internal standard. The homogenates were sonicated and centrifuged, and the
supernatants were used for high performance liquid chromatography (HPLC) analysis. 5-
51
HT, 5-hydroxyindoleacetic acid (5-HIAA) and NE (Sigma, St. Louis, MO, USA) were used as
standards. The protein concentrations were determined using the pellet using the
bicinchoninic acid kit (Pierce, Rockford, IL, USA) following the manufacturer’s protocol. The
mobile phase was the same as the homogenization buffer (excluding the isoproterenol) with
7% methanol for detection of 5-HT and 5-HIAA. NE was quantified separately using
trichloroacetic acid mobile phase solution without methanol. The mobile phases were
filtered and deaerated, and the pump speed (Shimadzu LC-6A liquid chromatograph) was
1.5 ml/min. The reverse-phase column used was a Rexchrom S50100-ODS C18 column
with a length of 25 cm and an internal diameter of 4.6 mm (Regis, Morton Grove, IL, USA).
The compounds were measured at +0.7 V using a Shimadzu L-ECD-6A electrochemical
detector.
MAO A Catalytic Activity Assay: Mice (WT saline = 10; WT L-NAME = 10; were killed by
cervical dislocation, and brain regions removed and homogenized in assay buffer (50mM
sodium phosphate buffer, pH 7.4). Catalytic activity for MAO A was measured as described
previously (Wu et al, 1993).
Pain Test: To measure nociception, MAO A/B KO (N = 8) and WT (N = 8) mice were
restrained in a plastic cylinder (inner diameter, 4 cm). The tail was put on a hot plate (56°
C), and the onset latency of tail flick was recorded three times with 1 min intervals.
Auditory Test: To assess hearing, animals were anesthetized (ketamine and
xylazine) and placed in a sound-attenuated chamber on an isothermal pad. Platinum
subdermal needles were inserted on the vertex of the skull and ventolateral to a pinna.
Animals were presented with a 2kHz tone (10 msec duration; 1 msec rise-fall; 100 msec
between tones), filtered through a Krohn-Hite filter (model 3700; 1.5 kHz low cutoff; 20 kHz
52
high cutoff; Krohn-Hite, Brockton, MA). Each mouse was tested with 2000 tone-on (85 dB)
trials and followed by 2000 tone-off (0dB) control trials.
The auditory brainstem response (ABR) is widely used to test for hearing in rodents
(Steel and Hardisty, 2000). The signal was amplified (10,000 gain) and filtered (300 Hz low
cutoff; 5kHz high cutoff) through a differential AC amplifier (model 1800; A-M Systems,
Carlsborg, WA) into the sound card. The sound card averaged the ABR signal recorded from
2000 trials and calculated the average amplitude (peak-to-peak microvolt) of the ABR
during the presentation of the tone.
Object Recognition: We used a modified version of the protocol described
previously (Bortolato et al, 2009). Mice (WT = 8, MAO A/B KO = 9) were individually
acclimatized to a dimly lit gray Plexiglas cubic box (20 x 20 x 20 cm) for 15 min. Twenty-
four hours later, the animals were tested in two separate object interaction sessions, each
separated by exactly 90 min. In the first session, two novel black plastic cylinders (8 cm tall
x 3.5 cm in diameter) were affixed to the floor and symmetrically placed at 6 cm from the
two nearest walls. Mice were placed in a corner, facing the center and at equal distance
from the two objects. Their start position was rotated and counterbalanced for each
genotype throughout the test. 90 min later, the animals’ short term memory was tested by
replacing one of the black plastic cylinders with a novel colored block. The animal was
placed in the same start position as the previous test, with the novel object to the right of
the start position. Twenty-four hours following the initial object interaction session, the
animals’ long term memory was tested in a final objection interaction session, with another
novel block different in color and shape from the second object interaction session
replacing one of the black cylinders. The animal was again placed in the same start position,
with the novel object to the right of the start position, which was rotated and
53
counterbalanced for each genotype throughout the test. Each object interaction session was
15 min long. Exploration was defined as sniffing either of the two objects with the snout;
sitting on the object was not considered exploration.
Fear Learning: All behavior was performed and analyzed with the experimenter
blind to the condition and genotype of the animals. Behavior was recorded with a video
camera that was linked to a TV monitor and VCR for online observation and recording. On
day one, mice (WT = 15; MAO A/B KO = 12) were individually placed in a 10.5 × 12 × 12 in.
conditioning chamber with electrifiable flooring (Coulbourne Instruments). To enhance the
salience of the chamber, three walls had broad, 1 in., red and white diagonal stripes at 45°
while one wall of the chamber was Plexiglas to allow video recording. One mL of vanilla
extract was placed in a small Petri dish below the cage flooring and out of the animals reach,
also to enhance the salience of the context.
Mice were allowed to explore the chamber for three minutes. During this time,
activity was scored to control for possible locomotor differences between WT and mutant
mice by counting the number of crossings through the midline of the chamber and the
number of rearings performed. After 3 min, an 80 dB tone (2 kHz) was played for 20 s.
During the last second of the tone, a mild electric current (0.70 mA) was passed through the
flooring with a one second duration. Tone and footshock delivery were controlled by
software (Lab Linc Operant Control Software; Coulbourne Instruments, Whitehall, PA).
Following a 1-min interval, the tone was played again for 20 s accompanied by a one second
current stimulus overlapping the last second of the tone. After another 1-min interval, the
tone-shock sequence was repeated one last time for a total of three pairings, and the mice
were left in the chamber for another 30 s. Freezing behavior was scored by observing the
mouse every 5 s at which time it was either considered freezing or not. The number of
54
observations during which the animal was freezing was divided by the total number of
observations made to determine the percent freezing.
The chamber was cleaned with 70% ethanol between individual sessions. Twenty-
four hours later, on day 2, mice were evaluated for fear conditioning to context by
reintroducing them to the conditioning chamber for 8 min without tone or footshock and
freezing behavior was scored (context test). Forty-eight hours after the initial conditioning,
on day 3, mice were evaluated for cued fear conditioning to the tone (tone test). Mice were
placed in a novel chamber (12 × 8 × 5 in.) that was cleaned between individual sessions
with 70% isopropyl alcohol. Following 3 min of exploration, an 80 dB, 2 Hz tone was played
for 8 min and behavior was observed and recorded every 5 s for the duration of the tone. It
should be noted that, on day 3, a limited number of mice (WT = 11; MAO A/B KO = 9) were
subjected to the tone test, and the remaining mice were used for another study.
Statistical Procedures: Homoscedasticity of data distribution was verified using
Bartlett’s test. Parametric comparisons were performed with one-way or two-way ANOVAs,
as appropriate, followed by Tukey’s test for post-hoc comparisons. Significance threshold
was set at P<0.05. Nonparametric comparisons were carried out by the Kruskal-Wallis test;
post-hoc analyses were performed by the Mann–Whitney U test, with Dunn-Sidak correction
for α levels.
55
RESULTS
Light-dark Box Test and Brain Regional Monoamine Levels in WT, MAOA
A863T
KO and
MAO A/B KO mice
We first evaluated the effects of L-NAME on the behavior of MAOA
A863T
KO and MAO
A/B KO mice as compared to WT in the light -dark box, a well-validated paradigm for
assessment of anxiety- and fear-related responses. Analysis of the time spent in the white
compartment reveal ed significant differences between the groups (H 5,68=11.11; P<0.05,
Kruskal-Wallis) (Fig. 3.1a). Post-hoc tests reveal ed that saline-treated MAO A/B KO, but not
MAOA
A863T
KO mice, spent significantly more time in the light compartment in comparison
with WT controls (U 15,9=115.5, P<0.01 for comparison MAO A/B KO vs WT , Mann-Whitney
test). Administration of L-NAME significantly reduced the time spent in the white
compartment in MAO A/B KO mice (U 9,11=17, P<0.05, Mann-Whitney test) but had no effect
on either WT (U 15,16=156, N.S., Mann-Whitney test) or MAOA
A863T
KO mice (U 8,8=33, N.S.,
Mann-Whitney test).
Significant differences were also found for other behavioral indices, such as freezing
duration (H 5,62=23.73, P<0.001, Kruskal-Wallis) (Fig. 3.1b), inter-compartment transitions
(H 5,67=18.43, P<0.01, Kruskal-Wallis) (Fig. 3.1d) and head pokes (H 5,67=21.22, P<0.001,
Kruskal-Wallis) (Fig. 3.1e). Post-hoc comparisons showed that, in comparison with WT
mice, MAO-d eficient mice exhibited significant increases in freezing (MAOA
A863T
KO vs WT:
U 12,8=77, P<0.05; MAO A/B KO vs WT: U 12,9=105, P<0.001, Mann-Whitney test); and
reductions in transitions (MAOA
A863T
KO vs WT: U 15,9=21.5, P<0.01; MAO A/B KO vs WT:
U 15,8=11.5, P<0.01, Mann-Whitney test) and head pokes (MAOA
A863T
KO vs WT: U 15,9=20,
P<0.01; MAO A/B KO vs WT: U 15,8=11, P<0.01, Mann-Whitney test). Furthermore, freezing
was significantly l onger in MAO A/B KO than MAOA
A863T
KO mice (U 8,9=63, P<0.05, Mann-
56
FIGURE 3.1
Effects of L-NAME on locomotor and exploratory behaviors in WT, MAOA
A863T
KO, and MAO A/B KO mice
in the light-dark box. All values represented as mean ± SEM.
a
P<0.1, *P<0.05, **P<0.01, ***P<0.001 compared
with WT mice administered saline; §P<0.05 compared with MAOA
A863T
KO mice administered saline; #P<0.05
compared with MAO A/B KO mice administered saline
Whitney test). Notably, L-NAME administration induced an attenuation of these differences
among genotypes: in WT mice, this drug increased freezing (saline- vs L-NAME-treated WT:
U 12,14=124, P<0.05, Mann-Whitney test) and reduced transitions (U 15,16=66.5, P<0.05, Mann-
Whitney test) and head pokes (U 15,16=64.5, P<0.05, Mann-Whitney test); in MAOA
A863T
KO
57
mice, it did not significantly affect behavioral performance [freezing (U 8,8=25, N.S., Mann-
Whitney test); transitions (U 9,8=39.5, N.S., Mann-Whitney test); head pokes (U 9,8=53.5,
P<0.10, Mann-Whitney test)]; finally, in MAO A/B KO, it significantly reduced freezing
(U 9,11=16, P<0.05) and increased transitions (U 8,11=74.5, P<0.05, Mann-Whitney test) and
head pokes (U 8,11=77, P<0.01, Mann-Whitney test). There were no statistically significant
differences between the groups with respect to the number of fecal boli produced (data not
shown). The groups did not differ not significantly with regards to latency to enter the dark
compartment (H 5,68=7.03, N.S., Kruskal-Wallis) (Fig. 3.1c).
These results indicated that MAOA
A863T
KO and MAO A/B KO mice exhibited
prol onged freezing and pronounced d eficits in expl oration. The greatest severity of these
d eficits was observed in MAO A/B KO mice. L-NAME attenuated these abnormalities in both
genotypes, but its effects were more robust on MAO A/B KO mice. Therefore, subsequent
behavioral tests were performed only in WT and MAO A/B KO mice.
We also compared brain regional differences in the metabolism of 5-HT and NE in
MAOA
A863T
KO and MAO A/B KO mice. Although preliminary studies indicate that whole
brain levels of these monoamines are higher in MAO A/B KO versus MAO A KO mice (Chen
et al, 2004), the extent to which the levels of these monoamines are increased likely differs
throughout the brain depending on the regional distribution of MAO A and MAO B
(Westlund et al, 1985; Shih and Chen 1999).
58
TABLE 3.1
WT MAOA
A863T
KO MAO A/B KO
Amygdala 5-HT 39.1 (2.4) 64.7 (1.7)* 126.3 (2.7)**##
5-HIAA 10.1 (0.4) 3.8 (0.5)** u/d
NE 14.2 (0.4) 26.2 (0.7)** 28.2 (1.3)**
Hippocampus 5-HT 14.2 (0.4) 23.7 (0.8)** 74.3 (2.3)**##
5-HIAA 8.7 (1.7) 2.5 (0.5)** u/d
NE 5.9 (0.4) 10.4 (0.3)* 15.1 (0.8)**#
Midbrain 5-HT 40.5 (5.3) 74.9 (1.0)* 105.4 (5.3)*##
5-HIAA 16.3 (2.0) 6.8 (0.3)* u/d
NE 10.3 (0.2) 17.7 (3.5)* 26.4 (1.3)**#
Prefrontal Cortex 5-HT 25.2 (0.9) 43.2 (1.6)* 66.8 (5.4)**
a
5-HIAA 12.9 (0.8) 3.0 (0.6)** u/d
NE 6.3 (0.3) 13.0 (0.7)* 12.3 (1.1)*
Data represent mean (SEM).
Units of measurement are PM/mg protein.
u/d=undetectable
*P<0.05, **P<0.01 compared with WT mice
a
P<0.1, #P<0.05, ##P<0.01 compared with MAOA
A863T
KO mice
Results are found in Table 3.1. In amygdala, hippocampus, midbrain, and prefrontal
cortex, we found markedly increased 5-HT and NE levels in both MAOA
A863T
KO and MAO
A/B KO mice as compared to WT, and decreased levels of 5-hydroxyindoleacetic acid (5-
HIAA), the 5-HT metabolite produced by MAO A, in MAOA
A863T
KO as compared to WT mice;
5-HIAA was not detectable in any brain region in MAO A/B KO mice. Moreover, in
MAOA
A863T
KO mice, the presence of MAO B conferred less severely deficient metabolism of
5-HT across all regions measured and attenuated NE levels in hippocampus and midbrain as
compared to MAO A/B KO mice, further supporting the premise that in the absence of one
MAO its preferred substrates may be inactivated by the remaining functional isoenzyme.
59
Moreover, the proportionate increase in 5-HT and NE levels in MAO A/B KO as compared to
MAOA
A863T
KO mice was brain region-specific, likely reflecting the relative distributions of
MAO B and catecholamine-O-methyl-transferase (COMT), which plays an important role in
NE degradation.
Home Cage Locomotor Activity
To d etermine whether the effects of L-NAME observed in the light-dark box was
refl ective of a non-specific reduction in l ocomotor activity, we tested the motor effects of this
drug on the ambulation of WT and MAO A/B KO mice in their home cages. Und er these
conditions, the l ocomotor activity of both genotypes was comparabl e, and not affected by L-
NAME (No. of crossings: WT saline=49.1 ± 4.8; WT L-NAME=32.2 ± 1.7; MAO A/B KO saline=45.6 ±
3.5; MAO A/B KO L-NAME=44.0 ± 5.4; N.S, Kruskal-Wallis) (data not shown).
Resident-intruder Test
We next studied the effects of L-NAME on impulsive aggression in the resident
intruder test. Analysis of latency to first attack indicated significant differences between the
groups (H 3,57=20.16, P<0.001, Kruskal-Wallis) (Fig. 3.2a), and SAL-treated MAO A/B KO
mice displayed a markedly lower latency to the first attack as compared to SAL-treated WT
mice (U 15,14=54.5, P<0.05, Mann-Whitney test). Post-hoc tests revealed that L-NAME did not
have any significant effect on the latencies in either WT (U 15,14=150, P<0.10, Mann-Whitney
test) or MAO A/B KO mice (U 14,14=116, N.S., Mann-Whitney test). The number of attacks was
significantly higher in MAO A/B KO mice (F 1,54=15.15, P<0.001, two-way ANOVA) regardless
of treatment (F 1,54=1.11, N.S., two-way ANOVA). There was no genotype x treatment
interaction (F 1,54= 0.44, N.S., two-way ANOVA) (Fig. 3.2b).
60
FIGURE 3.2
Effects of L-NAME on emotional behaviors in resident-intruder test. All values are represented as mean ±
SEM. *P<0.05, **P<0.01, ***P<0.001 compared with WT mice administered saline; #P<0.05, ##P<0.01
compared with MAO A/B KO mice administered saline; ^^main effect of genotype (P<0.01), ^^^main effect of
genotype (P<0.001)
Analysis of overall fighting duration revealed a significant effect of genotype
(F 1,53=18.12, P<0.001, two-way ANOVA), but not L-NAME (F 1,53=1.39, N.S., two-way ANOVA),
and a significant genotype x L-NAME interaction (F 1,53=7.35, P<0.01, two-way ANOVA) (Fig.
3.2c). L-NAME significantly increased fighting duration in MAO A/B KO mice as compared
to both SAL-injected WT (P<0.01, Tukey’s test) and MAO A/B KO mice (P<0.05, Tukey’s
test), and had no significant effect on WT mice (N.S., Tukey’s test). The analysis of the
average duration of each attack indicated significant differences between the groups
(H 3,57=27.27, P<0.0001, Kruskal-Wallis) (Fig. 3.2d). Post-hoc testing revealed no differences
61
between SAL-treated WT and MAO A/B KO mice (U 14,14=137, N.S., Mann-Whitney test);
however, administration of L-NAME significantly prolonged attack duration in MAO A/B KO
(U 14,14=158, P<0.01, Mann-Whitney test), but not WT mice (U 14,15=63, N.S., Mann-Whitney
test).
Tail rattling was significantly increased in MAO A/B KO mice (F 1,51=8.35, P<0.01,
two-way ANOVA) regardless of treatment (F 1,51=0.04, N.S., two-way ANOVA). A trend for a
genotype x treatment interaction was detected (F 1,51=3.02, P<0.10, two-way ANOVA) (Fig.
3.2e). On the other hand, the durations of sniffing the intruder mouse were comparable
across the groups (N.S., two-way ANOVA) (Fig. 3.2f). These results indicate that L-NAME
exacerbated fighting in MAO A/B KO mice, but had no significant effect in WT counterparts.
Tail Suspension Test
We next examined the effects of L-NAME on d epressive-like behavior in WT and MAO
A/B KO mice in the tail suspension test (Fig. 3.3). Overall, no difference between groups was
found. However, the diachronic analysis of immobility duration (Fig. 3.3a) indicated no
differences between the groups during the first four minutes of the test, but a significant
difference at the fifth minute (H 3,72=9.04, P<0.05, Kruskal-Wallis), which was found to
d epend on L-NAME’s ability to increase immobility in WT (U 20,20=288, P<0.05, Mann-
Whitney test), but not MAO A/B KO mice (U 16,16=130, N.S., Mann-Whitney test). No
difference was found between SAL-treated WT and MAO A/B KO mice (U 20,16=144, N.S.,
Mann-Whitney test). MAO A/B KO mice tend ed to produce more fecal boli than WT mice
(genotype effect: F 1,61=3.81, P<0.10, two-way ANOVA); although there was no significant
effect of L-NAME (F 1,61=0.05, N.S., two-way ANOVA), there was a significant genotype x
treatment interaction (F 1,61=6.07, P<0.05, two-way ANOVA) (Fig. 3.3c). Post-hoc analyses
62
indicated that saline-treated MAO A/B KO mice produced significantly more fecal boli than
saline-treated WT mice (P<0.05, Tukey’s test), and this increase was attenuated by L-NAME,
as the number of fecal boli produced by L-NAME-treated MAO A/B KO mice did not differ
significantly from WT counterpart s (N.S., Tukey’s test). In summary, although baseline
immobility was similar in WT and MAO A/B KO mice, the latter were significantly l ess
sensitive to the subtl e, time-d epend ent ability of L-NAME in increasing d epressive-like
behavior. By contrast, L-NAME reduced d efecation sel ectively in MAO A/B KO, but not WT
mice.
FIGURE 3.3
Effects of L-NAME on emotional behaviors in the tail suspension test. All values are represented as mean ±
SEM. *P<0.05 compared with WT mice administered saline
Emergence/Predator Urine Test
To further elucidate how L-NAME affects emotional reactivity in WT and MAO A/B KO
mice, we tested their emergence in normal conditions and in the presence of an object
impregnated with predator urine (PU) as a natural d eterrent.
63
In the absence of the PU-object, a higher percentage of MAO A/B KO mice fail ed to
emerge (3.3% of WT vs. 27.8% of MAO A/B KO mice, data not shown), consistent with their
reduced expl oratory drive. However, the median latency to emerge was similar in both
genotypes (U 32,23=333.5, N.S., Mann-Whitney test, data not shown).
FIGURE 3.4
Effects of L-NAME on anxiety-like behaviors in WT and MAO A/B KO mice in the emergence test. All
values are represented as mean ± SEM. **P<0.01 compared with WT mice administered saline; #P<0.05
compared with MAO A/B KO mice administered saline
Among the animals tested with PU, analysis of latency to emerge indicated significant
differences (H 3,46=8.34, P<0.05, Kruskal-Wallis) (Fig. 3.4a); however, there was no significant
difference between SAL-treated WT and MAO A/B KO mice (U 16,9=95.5, N.S., Mann-Whitney
64
test). L-NAME significantly increased the latency to emerge in WT mice (U 16,13=168.5,
P<0.01, Mann-Whitney test) but had no effect on MAO A/B KO mice (U 9,8=27, N.S., Mann-
Whitney test). Likewise, analysis of the duration of time and number of entries into the PU
compartment reveal ed significant differences [duration (H 3,47=13.33, P<0.01, Kruskal-Wallis)
(Fig. 4b); entries (H 3,48=9.72, P<0.05, Kruskal-Wallis) (Fig. 3.4c)]; however, post-hoc tests
indicated no difference between SAL-treated WT and MAO A/B KO mice [duration (U 17,9=
87.5, N.S., Mann-Whitney test); entries (U 17,9= 65, N.S., Mann-Whitney test)]. Whil e
administration of L-NAME elicited a marked d ecrease in duration and entries into the PU
area in WT mice [duration (U 17,12=23.5, P<0.001, Mann-Whitney test); entries (U 17,13= 43,
P<0.01, Mann-Whitney test)], this drug did not affect the behavior of MAO A/B KO mice
(U 9,9=42.5, N.S., Mann-Whitney test); entries(U 9,9= 30, N.S., Mann-Whitney test)].
All groups spent a comparabl e time sniffing the PU-imbibed object (N.S., two-way
ANOVA) (Fig. 3.4d). Analysis of the number of expl oratory head pokes (Fig. 3.4e) between
compartment s reveal ed no significant differences overall between genotypes (F 1,44=0.22, N.S.,
two-way ANOVA) or treatment s (F 1,44=1.19, N.S., two-way ANOVA). However, there was a
significant genotype x treatment interaction (F 1,44=4.93, P<0.05, two-way ANOVA). Whereas
post-hoc analyses reveal ed a statistical trend indicating the l ower proclivity of SAL-treated
MAO A/B KO mice to poke their head in comparison to WT counterpart s (P<0.10, Tukey’s
test), MAO A/B KO mice administered L-NAME did not differ from WT mice (N.S., Tukey’s
test). By contrast, in WT mice L-NAME tend ed to reduce the number of head pokes (P<0.10,
Tukey’s test).
Analysis of the number of fecal boli (Fig. 3.4f) showed a trend for the effect of
genotype (F 1,42=2.89, P<0.10, two-way ANOVA), a significant effect of L-NAME (F 1,42=4.25,
P<0.05, two-way ANOVA), and a significant genotype x L-NAME interaction (F 1,42=4.29,
65
P<0.05, two-way ANOVA). Post-hoc tests reveal ed a tend ency for control MAO A/B KO mice to
d efecate more than control WT mice (P<0.10, Tukey’s test), and L-NAME significantly
reduced d efecation in MAO A/B KO (P<0.05, Tukey’s test) but not WT mice (N.S., Tukey’s
test). In summary, MAO A/B KO mice exhibited only subtl e trends indicating enhanced fear
toward PU. Moreover, administration of L-NAME sel ectively increased fear responses in WT
but tend ed to yiel d the opposite effect in MAO A/B KO mice.
Biochemical Studies
We next studied the effects of fear – elicited by PU in the emergence test – on brain
regional MAO A catalytic activity (WT mice) and 5-HT and NE l evels (WT and MAO A/B KO
mice). Immediately foll owing behavioral testing, each mouse was sacrificed and the
amygdala, hippocampus, midbrain, and prefrontal cortex were removed; control mice were
not exposed to PU and were kept in their home cages until sacrificed.
In WT mice, neither PU nor L-NAME significantly affected MAO A activity in the
amygdala (Fig. 3.5a), hippocampus (Fig. 3.5b), midbrain (Fig. 3.5c), or prefrontal cortex (Fig.
3.5d) (for all regions: N.S., two-way ANOVA). However, among SAL-treated WT mice, PU
showed a tend ency to reduce MAO A activity in the amygdala (P=0.07, Stud ent’s t test), but
neither PU nor L-NAME significantly altered levels of 5-HT, 5-HIAA, the 5-HT metabolite
produced by MAO A, or NE in the amygdala of WT mice [5-HT (N.S., Kruskal-Wallis); 5-HIAA
(N.S., two-way ANOVA); NE (N.S., two-way ANOVA)] (Table 3.2).
66
FIGURE 3.5
Effects of fear induced by predator urine (PU) on MAO A catalytic activity (nmole/20min/mg protein) in
the a amygdala (AMY), b hippocampus (HIPP), c midbrain (MID), and d prefrontal cortex (PFC). All values
represented as mean ± SEM. P=0.07 compared to non-stressed control (CON) mice
By contrast, analysis of 5-HT l evels in the amygdala of MAO A/B KO mice indicated
significant differences between the groups (H 3,18=9.77, P<0.05, Kruskal-Wallis) (Tabl e 3.2).
Ind eed, post-hoc analysis reveal ed that L-NAME increased amygdala 5-HT l evels sel ectively in
MAO A/B KO mice exposed to PU (U 5,5=25, P<0.05, Mann-Whitney test). Conversely, PU
exposure significantly increased amygdala NE l evels in MAO A/B KO mice (F 1,15=6.55, P<0.05,
two-way ANOVA) regardl ess of drug administration (F 1,15=0.45, N.S., two-way ANOVA), and
there was no PU x L-NAME interaction (F 1,15=0.08, N.S., two-way ANOVA). 5-HIAA was not
d etectabl e in any brain region in MAO A/B KO mice, and no significant changes in 5-HT or NE
were found in the hippocampus, midbrain, or prefrontal cortex (data not shown).
These results indicated that L-NAME may have a bi-directional, MAO-dependent
effect on the capacity for fear-induced regulation of monoamine turnover in amygdala.
67
TABLE 3.2
Predator Urine: (-) (-) (+) (+)
Saline L-NAME Saline L-NAME
WT 5-HT 56.3 (4.6) 44.3 (1.2) 42.1 (3.6) 51.4 (3.2)
5-HIAA 5.0 (0.9) 5.5 (1.0) 5.4 (0.2) 5.2 (0.8)
NE 12.5 (0.6) 11.4 (1.8) 12.5 (0.3) 11.8 (0.5)
MAO A/B KO 5-HT 128.1 (3.8) 119.2 (3.2) 114.7 (1.1) 151.8 (4.9)*
5-HIAA u/d u/d u/d u/d
NE 28.3 (1.3) 25.9 (1.1) 32.8 (0.8)^ 32.5 (0.9)^
Data represent mean (SEM). Units of measurement are PM/mg protein.
u/d=undetectable
*Significantly different from saline-treated MAO A/B KO mice exposed to predator urine
(P<0.05).
^
Main effect of predator urine (P<0.05).
DISCUSSION
The present study yiel d ed several novel findings in relation to the emotional
reactivity of MAO A/B KO mice and the rol e of NO in their behavioral organization. Our
original characterization of MAO A/B KO mice had shown that these mutants display poor
expl oratory activity, anxiety-like responses in the open fiel d and in the el evated plus maze,
and a reduced latency to attack in the resid ent -intrud er aggression task (Chen et al, 2004). In
the present study, we have extended our understanding of the behavioral characteristics of
these animals, by elucidating complementary aspects of their emotional reactivity.
Specifically, we found that the higher levels of fear- and anxiety-related behaviors in this
genotype are accompanied by deficits in the assessment of environmental cues and the
enactment of appropriate responses. Interestingly, inhibition of NO synthesis mediated by L-
NAME attenuated these d eficits, but elicited opposite effects in WT mice, suggesting a
pl eiomorphic rol e of NO in behavioral organization of both genotypes. Specifically, our data
68
support the possibility that the emotional effects of NO may consid erably vary between
physiol ogical and pathol ogical conditions.
In the light-dark box test, MAO A/B KO mice remained in the light compartment
(where they had been originally placed) for a significantly longer duration than their WT
counterparts. Although this behavior is commonly interpreted as an index of reduced
anxiety (Bourin and Hascoët 2003), we showed that this phenomenon was actually
reflective of their dramatic enhancement in freezing behavior, a well-documented acute fear
response to aversive stimuli (Amorapanth et al, 2000; Blanchard et al, 2003). Ind eed, this
reaction caused a significant reduction in l ocomotor activity, as measured by the d ecrease in
transitions between the brightly lit and dark compartment. In keeping with previous report s
on the anxiogenic properties of L-NAME (Czech et al, 2003; Pokk and Väli 2002), this
compound enhanced freezing in WT mice; conversely, it reduced this response in MAO A/B
KO mice, suggesting that NO may contribute to the high l evels of fear in these mutant s. It is
worth noting that MAOA
A863T
KO mice also exhibited an intermediate response to the light-
dark box setting between WT and MAO A/B KO mice, characterized by d ecreased l ocomotor
activity and increased freezing behavior. Although these variations resulted in an increment
of the time spent in the brightly-lit compartment in this genotype, this phenomenon was not
significant, in keeping with previous reports (Popova et al, 2001). The increase in freezing
behavior in MAOA
A863T
KO and MAO A/B KO mice is in line with previous observations on
the exaggerated fear response and reduced l ocomotor activity of both genotypes in response
to mil d stressors, such as environmental light, particularly in open arenas (Chen et al, 2004;
Scott et al, 2008; Kim et al, 1997).
In contrast, MAO A/B KO mice displayed no major alterations in the responses to
predator urine, an innate fear-inducing stimulus that elicits in mice behavioral and
69
neurochemical alterations consistent with increased anxiety (Belzung et al, 2001; Hayley et
al, 2001), excepting subtle increases, albeit not significant, in the latency to exit and the
number of fecal boli, suggesting an enhancement of their anxiety-like behavior. However,
these mutant s exhibited fewer head pokes into the open compartment in comparison with
their WT counterparts. This phenomenon is evocative of a d ecrement in risk assessment
(Quartermain et al, 1996), suggesting their inability to attune their d efensive responses to
the l evel of environmental danger. Accordingly, we recently found that MAOA
A863T
KO mice
exhibit also l ower l evels of risk assessment than their WT littermates, deficits that were not
accompanied by alterations in locomotor, visual, olfactory and microvibrissal functions
(Godar et al, submitted). Notably, L-NAME induced a marked enhancement of fear-related
behaviors in WT mice, prol onging the latency to emerge from the protected compartment
and reducing the time spent in predator urine compartment. In contrast, bl ockad e of NO
synthesis significantly reduced the number of fecal boli produced by MAO A/B KO mice,
reinforcing the concept that NO may play a rol e in the anxiety-like behaviors of these
transgenic animals.
In line with our previous results on MAOA
A863T
KO mice (Scott et al, 2008), no
difference in baseline immobility was found in the tail suspension test between WT and MAO
A/B KO mice. Interestingly, L-NAME increased the immobility in WT, but not MAO A/B KO
mice, in the last minute of behavioral testing. These data suggest that, whil e NO may
modulate the expression of tail-suspension immobility in WT mice, this behavioral function is
ablated by MAO d eficiency. It shoul d be noted that although the 129S6 background of the
aforementioned MAOA
A863T
KO and MAO A/B KO lines has proven suitabl e for studies of
emotional reactivity (Marques et al, 2008), the performance of this strain in the tail
70
suspension test may not be strictly reliabl e, in view of their “clasping” phenomenon (Dalvi
and Lucki 1999).
In the resid ent -intrud er test, we confirmed that MAO A/B KO mice exhibited a
significant reduction in the latency to attack but no difference in total fighting duration as
compared to WT mice (Chen et al, 2004). Excessive aggression is a manifestation of both
high and low trait anxiety, often reflecting a general impairment of emotional regulation
(Neumann et al, 2010). Indeed, it is likely that the impulsive aggression observed in MAO
A/B KO mice is the consequence of an extremely heightened emotional state, as indicated by
the high levels of anxiety-like behavior reported here and previously. In MAO A/B KO mice,
L-NAME significantly increased fighting duration, but had no effect on the latency to attack,
possibly because of a “fl oor” effect. Conversely, in WT mice this compound increased the
latency to attack (albeit not significantly), but fail ed to affect fighting duration. These result s
are in keeping with previous investigations on the antiaggressive rol e of NO in experimental
animals (Demas et al, 1997). Furthermore, recent reports showed that L-NAME prol onged
the latency to attack without changing fighting duration in maternal aggression (Ankarali et
al, 2009).
The gradual enhancement in fear-related responses in MAOA
A863T
KO and MAO A/B
KO mice suggests that these behaviors may be related by their progressive alterations in 5-
HT and NE signaling, as the l evels of these monoamines in MAO A/B KO mice is significantly
higher than MAOA
A863T
KO mice across all brain regions (Tabl e 3.1). Our group has shown
that both MAO A KO and MAO A/B KO mice feature alterations of the sensorimotor cortex
(Cases et al, 1995; Chen et al, 2004). These alterations are the result of excessive 5-HT l evels
during the early postnatal stage (Cases et al, 1995; Salichon et al, 2001), suggesting a
d evel opmental origin for certain behavioral alterations observed in MAO A/B KO mice. This
71
possibility is also supported by the fact that l ong-term use of MAO inhibitors in adult rod ent s
has not been associated with cognitive d ecline or stereotyped behaviors.
Perturbed utilization of 5-HT and NE has l ong been associated with vulnerability to
mood and anxiety disord ers (Brown et al, 1982; Sevy et al, 1989; Wyatt et al, 1971). In mice,
exposure to a predator (or its od or) l eads to brain regional changes in monoamine
metabolism in the amgydala and other limbic areas (Belzung et al, 2001; Hayl ey et al, 2001).
Accordingly, we found that predator urine reduced MAO A catalytic activity, albeit not
significantly. However, we did not d etect changes in 5-HT , NE, or 5-HIAA in WT mice.
Predator urine also increased NE l evels in the amygdala of MAO A/B KO but not WT mice.
These data highlight the important rol e of amygdala in the integration of emotional responses
(Wallace and Rosen 2001). Fear is associated with the rel ease and metabolism of 5-HT
(Hayl ey et al, 2001; Kawahara et al, 1993) and NE in the amygdala (Onur et al, 2009; Tanaka
et al, 2000), and maladaptive responses may result from imbalance between the activation of
these systems. El evated NE neurotransmission has been shown to intensify amygdala-
mediated fear responses (Onur et al, 2009; Buffalari and Grace 2007) and enhance
contextual-fear learning (Huff et al, 2005). Indeed, an elevated amygdala NE responsemay
contribute to the higher levels of fear conditioning displayed by MAO A/B KO mice in the
context test (Supplementary Results), in addition to the anxiety-like behavior reported
previously (Chen et al, 2004).
The alterations featured by MAO A/B KO mice may be und erpinned by cognitive or
perceptual impairments (Carter et al, 2003; España et al, 2010; McGaugh et al, 2002; Ohl et
al, 2003; Ohl et al, 2002; Sarter and Bruno 1999). Accordingly, profound mental retardation
was d escribed in patient s with d oubl e MAOA and MAOB mutation (Whibl ey et al, 2010),
whereas mil d to bord erline mental impairments were reported in patients with a sel ective
72
MAOA mutation (Brunner et al, 1993b; Brunner et al, 1993a). Both syndromes were
associated with unusual stereotypical hand movements and lip smacking, similar to the
repetitive behaviors observed in Rett syndrome and autism-spectrum disorders (Young et
al, 2008), pervasive developmental disorders also associated with deficient monoamine
metabolism (Roux and Villard 2010; Whitaker-Azmitia 2001). It is possibl e that the rol e of
NO in the behavioral organization of WT and MAO A/B KO mice may partially result from
cognitive effects; this modulator is strongly implicated in a number of cognitive functions,
including spatial performance (Kirchner et al, 2004; Yamada et al, 1995), avoidance l earning
(Huang and Lee 1995) and memory (Frisch et al, 2000). However, MAO A/B KO mice
displayed no impairments in object recognition, a task that measures the animal’s abilty to
recall previously encountered objects (Ennaceur and Delacour 1988) and exhibited normal
pain and heat perception (Suppl ementary Results). Future studies assessing reactivity to a
wid er range of subtl e environmental and contextual stimuli may further our und erstanding of
the rol es of MAO A and MAO B in these processes.
Numerous lines of evid ence suggest a neurochemical link between monoamines and
NO (Breard et al, 2007; d’Ischia et al, 1995; Fossier et al, 1999; Grange-Messent et al, 2004;
Muriel and Pérez-Rojas 2003; Sammut et al, 2006), suggesting that the effects of NO may be
mediated by a compl ex net of region- and system-specific alterations in monoaminergic
signaling. Accordingly, L-NAME induced an increase of 5-HT l evels in the amygdala of MAO
A/B KO mice exposed to predator urine. Alternatively, the rol e of NO in the modulation of
MAO A/B KO mice may involve other systems, such as glutamate. Ind eed, this
neurotransmitter is increased in response to fear or stress (Karreman and Moghad dam 1996;
Lowy et al, 1993; Moghad dam et al, 1994; Saulskaya and Marsd en 1995; Timmerman et al,
1999) and plays an important rol e in the modulation of both NO and monoamine signaling, in
73
particular through its N-methyl-D-aspartate (NMDA)-type receptors. NMDA receptors
stimulate NO synthesis by nNOS (Garthwaite et al, 1989; Garthwaite 2008; Prast and
Philippu 1992), have a marked influence on the activity of monoaminergic pathways (Ad ell
et al, 2002; Babar et al, 2001; Del Arco and Mora 2001; Millan 2002; Takahata and
Moghad dam 1998; Tao and Auerbach 2002) and play an important rol e in the modulation of
fear responses in the amygdala (Bauer et al, 2002; Cratty and Birkl e 1999; Tang et al, 1999;
Walker and Davis 2000) and aggressiveness (Lang et al, 1995; Le Grevès et al, 1997).
Several limitations of this study shoul d be consid ered. First, our neurochemical
analyses did not includ e the analysis of NO l evels in WT and MAO A/B KO mice, thereby
providing a limited picture on the mechanisms und erpinning the effects of L-NAME in WT
and MAO A/B KO mice. Second, although our d ose of L-NAME was sel ected to elicit a near-
total bl ockad e of NO synthesis, the lack of a full d ose-response analysis restricts our insight
into the dynamic rol e of NO in behavioral regulation across both genotypes. Third, the use of
a nonsel ective NOS inhibitor such as L-NAME hind ers our ability to elucidate the relative
contributions of eNOS and nNOS to neurochemical or behavioral regulation. Ind eed, there is
evid ence that eNOS and nNOS have complementary influences on emotional and aggressive
behavior (Chiavegatto et al, 2001; Frisch et al, 2000; Demas et al, 1999). Nevertheless, it
should be noted that empl oyment of the sel ective nNOS inhibitor 7-Ni was preclud ed in this
study by its reported ability to inhibit MAO in the brain (Desvignes et al, 1999). Fourth, we
did not analyze the possibl e contribution of MAO B and its preferred substrate PEA in the
phenotypical anomalies of MAO A/B KO mice. However, PEA is unlikely to play a substantial
rol e in their abnormal reactivity, in line with its more subtl e rol e in behavioral regulation in
comparison with 5-HT and NE (Grimsby et al, 1997). Accordingly, our group recently
showed that the behavioral profil e of MAO B KO mice is profoundly divergent from that of
74
MAO A/B KO mice, in that the former animals exhibit attenuation of anxiety-like behaviors
and increased behavioral disinhibition (Bortolato et al, 2009).
Conclusion
Irrespective of this limitation, these data enrich our und erstanding of the mechanisms
und erlying the maladaptive emotional reactivity observed in MAO-d eficient humans and
mice. Consid ering that the efficacy of certain psychiatric drugs is evid ent only und er
pathol ogical conditions, MAO A/B KO mice may be a useful translational tool to evaluate the
therapeutic value of novel compound s for the treatment of mood and affect disord ers in
subjects with perturbed monoamine metabolism.
SUPPLEMENTARY RESULTS
To further extend their behavioral phenotyping, we studied emotional learning in
MAO A/B KO mice using a fear conditioning task. Previous studies in Tg8-MAO A KO mice
found that this line exhibited significantly enhanced classical fear conditioning, as indicated
by freezing in response to contextual stimuli and tone (Kim et al, 1997). This alteration was
presumed to result from the elevation of NE in the amygdala of MAO A KO mice, as
treatments that increase noradrenergic transmission in this brain region are known to
increase fear learning (Liang et al, 1990). In addition, we evaluated MAO A/B KO mice for
impairments in sensory perception and memory that could alter their performance in a fear
conditioning task.
75
Hearing and Nociception†
We first evaluated MAO A/B KO mice for sensory impairments that may affect their
hearing and pain perception. Tone is one of the conditional stimuli used in the behavioral
paradigms, and MAO A/B KO mice were tested with auditory brainstem recordings. Our
results show that MAO A/B KO mice have comparable hearing to their WT littermates. No
significant difference in brainstem auditory responses was observed between the two
genotypes (data not shown). During the initial training sessions involving tone-footshock
pairings, MAO A/B KO mice jumped and vocalized in response to the electric footshock to a
similar degree as the WT, suggesting normal pain perception in these KO mice. To further
assess nociception in MAO A/B KO mice, we measured the heat-induced tail-flick response
in these animals. Both MAO A/B KO and WT mice exhibited characteristic tail-flick
responses in this test, and no significant difference was found in the average latencies to tail
flick between the two genotypes, indicating normal pain and heat perception in MAO A/B
KO mice (data not shown) (Singh et al, submitted).
Object Recognition
An object recognition task was used to evaluate working memory in MAO A/B KO
mice as compared to the WT mice. Their behavior was assessed to investigate both short-
term memory (STM) - 90 min. - and long-term memory (LTM) - 24 hours - for a familiar
object. As shown in Fig. 3.6a, MAO A/B KO mice displayed similar levels of % novel sniffing
bouts when tested for both STM (F 1,15 = 0.09, N.S., one-way ANOVA) and LTM (F 1, 15 = 1.49,
N.S., one-way ANVOA).
†The work described in this section of results was performed by Dr. Chanpreet Singh during his graduate
studies in the Neuroscience Department at the University of Southern California. These studies were conducted
prior to the experiments outlined in the following sections entitled “Object Recognition” and “Fear Learning”,
which were conducted either solely by A. Scott, or by A. Scott in collaboration with Dr. Singh.
76
Total sniffing bouts were also similar in MAO A/B KO and WT mice (data not shown).
Furthermore, no difference was detected in the time spent exploring a novel object
compared to a familiar object in the two strains for both short-term (U 8,9 = 33.5 and N.S.,
Mann-Whitney test) and long-term (U 8,8 = 22, N.S., Mann-Whitney test) memory (Fig. 3.6b).
FIGURE 3.6
Object Recognition in WT and MAO A/B KO mice. WT and MAO A/B KO mice exhibited similar exploration of
novel objects and recognition of familiar objects 90 min (STM) and 24 hrs (LTM) following initial exposure.
Values are represented as mean ± SEM.
Fear Learning
On the first day, basic motor activity patterns were compared in MAO A/B KO and
WT mice by measuring the number of cage crossings during the first 3 min. after placing
them in the fear conditioning chamber. No significant difference was observed between
77
MAO A/B KO and WT mice in the number of crossings (data not shown) and baseline
freezing during this time period (Fig. 3.7). During paired (footshock-tone) training, the %
freezing was significantly elevated in the MAO A/B KO mice, in comparison to WT
[immediate postshock freezing during min. 4, 5, and 6 (F 1, 79 = 6.609; P <0.01)] (Fig. 3.7).
FIGURE 3.7
Fear conditioning in WT and MAO A/B KO mice. On day 1 (left panel), freezing was increased in MAO A/B
KO mice as compared to WT during paired tone-foot shock training (min 4, 5, 6). On day 2 (middle panel),
contextual fear was enhanced in MAO A/B KO mice. On day 3 (right panel), MAO A/B KO mice showed higher
freezing, both during the baseline and in response to the tone. All values are represented as mean ± SEM.
**P<0.01, †P<0.0001, ††P<0.00001 compared with WT mice
Mice were exposed to the context that was used during the paired training 24 hours
later. MAO A/B KO mice displayed significantly enhanced contextual learning as compared
to WT mice (Fig. 3.7). Although the starting levels of freezing were identical for both
genotypes (F 1,28 = 0.726, N.S., one-way ANOVA), by the 2
nd
min. of the test, the % freezing in
MAO A/B KO mice was significantly increased as compared to WT mice (F 1,28 = 8.40, P<0.01,
one-way ANOVA). By contrast, the freezing of WT mice increased gradually to a maximum
level during the 5
th
min. of exposure and then decreased significantly, equal in levels to the
78
baseline at the start of the contextual test. The % freezing between the 1
st
and the 5
th
min.
for WT was significantly different (F 1,33 = 18.9, P<0.0001, one-way ANOVA). Apart from this
significant increase in freezing of the WT, there was also a significant decrease in their
freezing from 5
th
to the 8
th
min. (F 1,33 = 4.8, P<0.05, one-way ANOVA), and their freezing
during the 8
th
and final min. was not significantly different from that during the 1
st
min.
(F 1,33 = 1.74, N.S., one-way ANOVA). These observations suggest that WT mice processed
the contextual information in a gradual manner, with the feared behavior being elicited at a
slower/normal rate. On the other hand, the freezing response in MAO A/B KO mice was
signficantly more robust. Their freezing increased significantly from the 1
st
to the 2
nd
min.
(F 1,23 = 9.87, P<0.01, one-way ANOVA) and persisted for the remaining duration of the
context test. In MAO A/B KO mice, % freezing in the 8
th
and final min. of the test tended to
be higher than the 1
st
min. (F 1,23 = 3.79, P<0.1, one-way ANOVA).
On day 3, the mice were exposed to a novel environment for 3 min., after which time
they were tested for cue (tone)-associated fear learning for 8 min. Fear conditioning (%
freezing) was significantly elevated in MAO A/B KO mice as compared to WT in the novel
context, including the 3
rd
min. before exposure to the tone (F 1,19 = 23.44, P<0.0001, one-way
ANOVA). However, it should be noted that the lighting used in this context was significantly
brighter than days 1 and 2. Since MAO A/B KO mice showed significantly higher freezing
during the baseline, we compared the change in freezing during the first min. of tone
exposure. As was observed during the context test, MAO A/B KO mice exhibited a more
robust fear response as compared to WT mice (Fig. 3.7). During the first min. of the tone,
the freezing of MAO A/B KO mice increased significantly as compared to that during the 3
rd
min. of the baseline (F 1,17 = 27.81, P<0.00001, one-way ANOVA) and persisted after that for
the entire duration of the tone. Freezing in WT mice did not differ significantly between the
79
3
rd
min. of baseline and the 1
st
min. of tone (F 1,21 = 2.31, N.S., one-way ANOVA) and was not
increased significantly until the 2
nd
min. of tone exposure (F 1,21 = 7.08, P<0.05, one-way
ANOVA) and persisted after that. The levels of freezing during the 8
th
and final min. of tone
exposure were significantly higher in MAO A/B KO mice as compared to WT (F 1,19 = 24.28,
P<0.00001, one-way ANOVA).
80
CHAPTER 4:
OVERALL DISCUSSION AND CONCLUSIONS
Summary of Overall Findings
This dissertation includ es the discovery and initial biochemical and behavioral
characterizations of the first murine line with a spontaneous mutation sel ectively in MAOA,
MAOA
A863T
KO (Scott et al, 2008). The equival ence of this mutation to the cause of Brunner
syndrome (Brunner et al, 1993a; Brunner et al, 1993b) provid es a novel translational tool to
distinguish the clinical features of this disord er from psychiatric illnesses with similar
manifestations of emotional and cognitive impairments.
Akin to previous studies of Tg8-MAO A KO mice, which were generated by insertional
mutagenesis, MAOA
A863T
KO mice exhibited heightened aggression in the resident-intruder
paradigm but differed from Tg8 in their marked reduction in open field exploration. In
addition, MAOA
A863T
KO mice showed no change in depressive-like behaviors, again
diverging from reports of Tg8 mice, which exhibited less immobility in a classical paradigm
of depression. These discrepancies may reflect altered environmental reactivity stemming
from characteristic perceptual abilities inherent in the different background strains of these
lines. Notably, whereas abnormal retinal projections have been reported in Tg8 mice
(Upton et al, 1999), recent studies performed by our group indicated that MAOA
A863T
KO
mice exhibited no overt impairments in visual discrimination (Godar et al, submitted).
Alternatively, emotional processing may be differentially altered in these lines as a result of
molecular interactions between the mutated MAOA transcripts and putative cellular targets.
Indeed, different mutations in the same gene have been shown to produce mutant lines with
varying phenotypic alterations (Simoni 1998; Fleming et al, 2005).
81
The present work substantially extend ed the behavioral phenotyping of MAO A/B KO
mice through studies that elucidated compl ementary aspects of their emotional reactivity.
Specifically, we provid e evid ence that an inability to adapt their emotional responses based
on cues from the environment as well as enhanced fear l earning likely contribute to the high
l evels of fear- and anxiety-related behaviors featured by this line (Scott et al, submitted; Singh
et al, submitted). Ind eed, although MAO A/B KO mice exhibited a marked enhancement in
freezing in response to light, which likely represents a potential threat to rod ent s, they
displayed only subtl e increases in fear-related behavior in response to predator urine, which
likely represent s an actual threat. This finding is compl emented by recent studies performed
by our group demonstrating that MAOA
A863T
KO mice exhibited a distinct inability to adapt
their responses based on cues from the environment; interestingly, MAOA
A863T
KO mice
displayed higher levels of fear-related behaviors in response to inanimate objects but lower
fear-related behaviors in response to predator cues (Godar et al, submitted). On the other
hand, the marked enhancement in freezing displayed by MAO A/B KO mice in the light-dark
box could reflect an abnormally aversive response to the particular threat of bright light.
Indeed, we show that fear learning is enhanced in MAO A/B KO mice, and the mice are
typically handled by researchers and staff members in rooms that are brightly-lit when they
are moved to different areas for behavioral testing or transferred to clean cages.
These findings were the first to show that MAO d eficiency reversed the emotional
effects of NO synthase inhibition and strongly implicated NO in the behavioral organization
of MAO A KO and MAO A/B KO mice (Scott et al, submitted). In general, L-NAME-mediated
inhibition of NO synthesis differentially affected emotional reactivity in WT and MAO A/B KO
mice, reducing it in the former and attenuating it in the latter. These observations hint at
functional changes induced by MAO A/B-d eficiency in eNOS and/or nNOS, which have been
82
shown to have complementary influences on emotional and aggressive behavior
(Chiavegatto et al, 2001; Frisch et al, 2000; Demas et al, 1999). Moreover, recent studies
have indicated that altered gluatamatergic signaling contributes to fear- and anxiety-related
responses in MAO A/B KO mice (unpublished). Thus, the observed alterations in NO
signaling may be the result of “upstream” changes in N-methyl-D-aspartate (NMDA)-type
receptors in MAO A/B KO mice which impact nNOS (Garthwaite et al, 1988; Prast and
Philippu 1992). Ad ditional studies are currently und erway to elucidate the mol ecular basis
for these observations.
Relationship of the Findings to the Overarching Hypothesis
In general, this dissertation has proven the overarching thesis hypothesis; namely,
mutations in MAO that cause progressive alterations in 5-HT and NE signaling are associated
with a gradual enhancement in maladaptive behaviors, featuring impulsive aggression and
anxiety-like responses, and subvert the rol e nitric oxid e in the modulation of emotional
reactivity. These findings are largely in agreement with the state of the art, including the
well-d ocumented relationship between chronic MAO A-deficiency and impulsive aggression
(Brunner et al, 1993b; Brunner et al, 1993a; Cases et al, 1995; Chen et al, 2004; Chen et al,
2007). In ad dition, our extension of the phenotypic anxiety-like responses in MAO A/B KO
mice compl ements previous studies in MAO A KO mice, which also exhibited reduced
investigative activity (Popova et al, 2001), enhanced fear l earning (Kim et al, 1997), and poor
ability to evaluate contextual risk (Godar et al, submitted).
Major obstacl es in this fiel d includ e the wid e distributions of MAO A and MAO B in the
brain and the extensive, diverse nature of functional effects elicited by monoamine
neurotransmitters. To overcome this, several compl ementary approaches – including
83
neurochemical, mol ecular, cellular, and behavioral techniques – must be utilized to expl ore
different facets of emotional responsiveness and elucidate mechanisms und erpinning the
emotional impairments resulting from MAO d eficiency. Although knock out mice have
served as valuabl e tools in the id entification of mol ecular mechanisms and causal
relationships in this fiel d (Cases et al, 1998; Grimsby et al, 1997; Evrard et al, 2002), the
exclusive utilization of these mod els presents a major overall limitation to this work. Al ong
these lines, the foll owing caveats shoul d be ad dressed.
Regarding the characterizations of MAOA
A863T
KO and MAO A/B KO mice, although
the progressive deficiencies in brain regional metabolism of 5-HT and NE are likely major
contributions to the exacerbation of emotional impairments in the latter genotype, the
potentially important impact of PEA was not addressed by these studies. This is
particularly relevant considering the fact that brain regional increases in this trace amine in
MAO B KO mice were associated with reduced anxiety-like responses (Bortolato et al, 2009)
and altered DA receptor functioning in the striatum (Chen et al, 1999). Likewise, unlike
MAO A KO mice, which showed reduced preference for nicotine (Agatsuma et al, 2006),
MAO B KO mice responded normally to nicotine (Lee et al, 2004). Considering that nicotine
exerts potent control over striatal DA release (Zhou et al, 2001), future studies evaluating
behaviors in the various lines of MAO-deficient mice following administration of DA
receptor agonists and antagonists may shed light on the functional significance of PEA in the
modulation of catecholamine signaling.
In most tests, L-NAME-mediated inhibition of NO synthesis induced differential
effects on emotional reactivity in WT and MAO A/B KO mice; however, in MAO A/B KO
mice, L-NAME had no measurable effect on depressive-like behavior and only subtle effects
on fear-related responses to predator urine (Scott et al, submitted). The fact that baseline
84
emotional responses were similar in WT and MAO A/B KO mice in these tests renders it
unlikely that so-called “floor” or “ceiling” effects are responsible for these phenomena.
These observations suggest that MAO deficiency ablates certain behavioral functions
modulated by NO but fails to elucidate causal relationships. Nonetheless, these
observations may highlight distinct pathways by which NO modulates emotional reactivity
in response to different stressors. Future studies correlating behavioral responses to
various fear- and stress-inducing stimuli with functional analyses of eNOS and nNOS and
measurement of brain NO levels are warranted to elucidate these mechanisms.
Preclinical and Clinical Rel evance
The availability of different MAOA-mutated lines affords opportunities to dissect
mechanisms of functional regulation of the mutant gene, its related proteins and resulting
behavioral effects. Notably, future studies shoul d be carried out to evaluate the potential
mechanisms which enabl e l ow amounts of WT mRNA in the brain of hypomorphic MAO A
neo
mutants to be translated into functional MAO A proteins sel ectively in regions such as the
amygdala, resulting in normal 5-HT metabolism (Bortolato et al, submitted).
Consid ering the wid e of range of psychiatric illness associated with altered
monoamine metabolism, MAO-d eficient mice may prove to be a useful tool in evaluating the
efficacy of novel therapeutic compound s with potentially divergent effects in physiol ogical
and pathol ogical states. The recent discovery of an inherited d el etion of MAOA and MAOB,
which was associated with profound mental retardation, d evel opmental d elay, and stereotypic
behaviors, is a paradigmatic example (Whibley et al, 2010). The severe impairment s
associated with this d el etion are reminiscent of those seen in pervasive d evel opmental
disord ers, such as Rett syndrome and autism, which are also associated with d eficient 5-HT
85
metabolism (Roux and Villard 2010; Whitaker-Azmitia 2001; Young et al, 2008). Although
increased 5-HT is the most frequent neurochemical alteration observed in autistic subject s
(Anderson et al, 1987; Minderaa et al, 1987; Whitaker-Azmitia 2001), its causal rol e in this
disord er remains mysterious. Ind eed, the d evel opmental studies includ ed in the Appendix of
this dissertation suggest that MAO A/B KO mice may be utilized to investigate mechanisms
by which perturbed monoamine metabolism influences brain d evel opment.
In summary, a wid e spectrum of neuropsychiatric disord ers – ranging from subtl e
behavioral perturbations to d etrimental emotional and neurol ogical impairment s – is
associated with chronic d eficiencies in MAO A and/or MAO B. The studies included in this
work significantly extend our understanding of the mechanisms by which mutations in MAO
lead to dysregulated emotional behaviors. The new models and molecular substrates
defined in this dissertation can be utilized in future investigations to address critical gaps in
the field.
86
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APPENDIX:
CONSEQUENCES OF MONOAMINE OXIDASE DEFICIENCY
DURING EARLY DEVELOPMENT
APPENDIX ABSTRACT
Monoamine neurotransmitters play crucial roles in brain development. We
examined the consequences of MAO A and MAO A/B ablation during early developmental
stages using neurochemical, cellular, and behavioral techniques. Comparison of monoamine
levels in whole brain from neonatal pups revealed that 5-HT levels were increased to a
similar extent in both MAOA
A863T
KO and MAO A/B KO as compared to WT. By contrast,
distinct changes in catecholamine (DA and NE) levels were found in MAOA
A863T
KO and MAO
A/B KO. We found that neural stem cell (NSC) self-renewal ability is impaired in
neurosphere (NS) cultures derived from MAO A/B KO mice as compared to WT during late
embryonic and early postnatal developmental stages. In NS from WT mice, MAO A activity,
but not MAO B activity, was detected at embryonic (E) day 13 NS and its levels were
increased at E16, with a further increase at postnatal (P) day 2. However, treatment of P2
NS from WT mice with an MAO A inhibitor at a concentration that completely blocked MAO
A activity had no effect on NS, suggesting that MAO A activity per se is not required for NSC
self-renewal. We next examined the production of ultrasonic vocalizations (USVs) in pups
following administration of the 5-HT synthesis inhibitor parachlorophenylalanine (pCPA)
during embryogenesis, using a treatment regimen previously shown to normalize 5-HT
levels in MAO A/B KO neonates. In male neonates, either MAO A/B KO or administration of
pCPA was sufficient to significantly reduce USVs, whereas in females both MAO A/B KO and
pCPA were required. These findings reveal novel outcomes of MAO deficiency during early
brain development.
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INTRODUCTION
Even prior to the maturation of conventional synapses, monoamines are important
signaling molecules, influencing a wide range of events critical during early brain
development. Indeed, several lines of evidence strongly suggest a developmental origin for
certain dysfunctions present in MAO-deficient humans and mice (Whibley et al, 2010; Cases
et al, 1998; Cheng et al, 2010). In this sense, MAO KO mice provide important models which
can be used to investigate how aberrant control of monoamine metabolism impacts specific
developmental outcomes.
Abnormal behaviors were evident during early postnatal stages in both Tg8-MAO A
KO and MAO A/B KO mice (Cases et al, 1995; Chen et al, 2004); by contrast, no significant
abnormalities were reported in MAO B KO mice during early development (Grimsby et al,
1997). The major neurochemical distinction between neonatal MAO A-deficient (MAO A KO
and MAO A/B KO) mice as compared to MAO B KO mice is the marked increase in 5-HT in
the MAO A-deficient genotypes. Indeed, levels of 5-HT were increased approximately 9-fold
and 11-fold in Tg8 and MAO A/B KO neonates (aged P1-2), respectively (Cases et al, 1995;
Cheng et al, 2010), whereas no changes in 5-HT levels or metabolism were detected in MAO
B KO mice during early development or adulthood (Grimsby et al, 1997). However, levels of
5-HT in neonatal MAO A-deficient mice harboring a spontaneous, human-like mutation
(MAOA
A863T
KO) have not been determined.
Excess 5-HT during early development has been implicated in both neuroanatomical
and behavioral abnormalities in Tg8-MAO A KO mice, the most well-characterized line of
MAO-deficient mice. Tg8 pups at age P7 showed dramatically reduced levels of the 5-HT 1A
receptor (Lanoir et al, 2006), a sub-type prominently involved in the regulation of anxiety
(Zhang et al, 2010; Gross et al, 2002). Moreover, this line features dysmorphism of the
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barrel fields in the somatosensory cortex, an aberration that was partially restored by
administration of the inhibitor of 5-HT synthesis, parachlorophenylalanine (pCPA), during
the early neonatal stage (Cases et al, 1995). In ad dition to restoring the sensorimotor cortex
d eficits in Tg8 mice, neonatal pCPA administration was also reported to attenuate many of the
abnormal behaviors observed in these pups, whereas the same treatment with an inhibitor
of catecholamine synthesis had no effect, reinforcing the serotonergic basis for these
perturbations (Cases et al, 1995).
Monoamines are believed to play an important role in the fate of neural stem cells
(NSC) in developing brain; indeed, neurogenesis peaks during mid-gestation, which is also
when monoaminergic neurons from the midbrain and brainstem begin to innervate cortical
areas (Berger-Sweeney and Hohmann 1997; Coyle and Molliver 1977; Lauder 1993; Levitt
et al, 1997; Levitt and Rakic 1982; Lidov et al, 1980; Schlumpf et al, 1980). 5-HT-releasing
axonal growth cones are among the earliest to reach the developing cortex and make
contact with dividing cells (Dori et al, 1996; Ivgy-May et al, 1994; Vergé and Calas 2000;
Wallace and Lauder 1983). In the developing brain, 5-HT regulates many important
functions, including neuronal proliferation, migration and differentiation (Vitalis and
Parnavelas 2003). Several complementary models have demonstrated that developmental-
stage specific changes in 5-HT synthesis, uptake, and receptor expression are critical in the
pathogenesis of psychiatric disorders (Gaspar et al, 2003). However, the impact of excess 5-
HT on progenitor cell proliferation is poorly understood.
5-HT has been implicated in the etiology of several pervasive developmental
disorders, including Rett syndrome and autism (Roux and Villard 2010; Whitaker-Azmitia
2001), but its relationship to the severity or progression of both these disord ers remains
highly elusive. Interestingly, rat pups developed as a “hyperserotonemic model” of autism,
111
produced by administration of a nonselective serotonin agonist during neonatal
development (Whitaker-Azmitia 2001), did not produce ultrasonic vocalizations (USV)
when separated from their mothers (Kahne et al, 2002). This deficit may be analogous to
the paucity of communication and social interaction often observed in humans with this
disorder (Whitaker-Azmitia 2001; Whitaker-Azmitia 2005). However, no studies to date
have investigated the impact of MAO ablation on USVs in neonatal pups.
Stereotypical behaviors such as handwringing and lip smacking are common in
patient s with Rett syndrome and autism (Bodfish et al, 2000). Recently, a comparabl e
spectrum of unusual repetitive behaviors featured prominently in the d escription of patients
with d oubl e MAOA and MAOB mutation, who also exhibited severe d evel opmental d elay and
profound mental retardation (Whibl ey et al, 2010). Moreover, height and weight
measurements for these patients fell into very low percentiles, a characteristic that was not
observed in subjects lacking only MAO A or MAO B (Brunner et al, 1993b; Brunner et al,
1993a; Lend ers et al, 1996). Likewise, MAO A/B KO pups were considerably smaller than
their WT littermates, a deficit which persists throughout adulthood (Chen et al, 2004).
Indeed, this reduction is so marked that, in a mixed litter of WT and MAO A/B KO pups,
mutant neonates can generally be distinguished on this factor alone (Fig. 4.1). By contrast,
both MAO A KO and MAO B KO mice exhibited normal growth and body weight (Cases et al,
1995; Grimsby et al, 1997). Together these studies suggest that total ablation of MAOs has
marked and enduring effects on physical development, the mechanisms for which remain
highly elusive.
Here we investigated the developmental impact of MAO ablation using a range of
neurochemical, cellular, and behavioral techniques, and report novel roles for these
enzymes in several key processes during embryonic and early postnatal stages.
112
FIGURE 4.1
MAO A/B KO Pups Exhibit Diminished Body Weight. Approximately 3 days postnatal, this MAO A/B KO pup
(left) is noticeably smaller than its WT littermate (right).
MATERIALS AND METHODS
Animals and Genotyping: Mice were housed in cages with free access to food and
water on a 12-hour light cycle, in accordance with the protocol approved by the University
of Southern California Institutional Animal Care and Use Committee.
For genotyping, two complementary PCRs were performed on the genomic DNA
from mouse tail biopsies. The first reaction used primers for MAO A (5’-
GCTTCACAGTGGATTGAT-3’ and 5’-CACAAATACGAGCAACCTAC-3’) and second reaction
used primers for MAO B (5’-CTACAAAGCAGATTGCCACGC-3’ and 5’-
TACCTGACATCAACTGGTCCC-3’). The PCR product of the first reaction using MAO A
primers (approximately 300 bp) was digested with the restriction enzyme Dra1 (New
England Biolabs, Ipswich, MA, USA), as the loss of this cutting site indicates the A → T point
mutation.
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HPLC Analyses: Each pup was decapitated and the whole brain was quickly
removed, immediately placed on ice, and homogenized in a solution containing 0.1 M
trichloroacetic acid, 10 mM sodium acetate, and 0.1 mM EDTA; 1 µM isoproterenol was used
as an internal standard. The homogenates were sonicated and centrifuged, and the
supernatants used for HPLC analysis. 5-HT, 5-HIAA, DA and NE (Sigma, St. Louis, MO, USA)
were used as standards. The protein concentrations were determined using the pellet using
a bicinchoninic acid kit (Pierce, Rockford, IL, USA) following the manufacturer’s protocol.
The mobile phase was the same as the homogenization buffer (excluding the isoproterenol)
with 7% methanol for detection of 5-HT and 5-HIAA. DA and NE were quantified separately
using trichloroacetic acid mobile phase solution without methanol. The mobile phases were
filtered and deaerated, and the pump speed (Shimadzu LC-6A liquid chromatograph) was
set 1.0 ml/min. The reverse-phase column used was a Rexchrom S50100-ODS C18 column
with a length of 25 cm and an internal diameter of 4.6 mm (Regis, Morton Grove, IL, USA).
The compounds were measured at + 0.7 V using a Shimadzu L-ECD-6A electrochemical
detector.
Generation of WT and MAO A/B KO Littermates For NSC and NS Studies: Since
both MAO A and MAO B are on the X chromosome and closely linked, WT and MAO A/B KO
littermates can be generated by breeding heterozygous MAO A/B KO female mice with
hemizygous (-/y) male MAO A/B KO or WT (+/y) mice. Littermates of WT and MAO A/B KO
mice were obtained from timed-pregnant females. The separation day was designated as
embryonic day 0.5 (E0.5).
Neurosphere Culture and Analysis: Neurosphere (NS) cultures were prepared
from embryonic and postnatal mouse dorsal telencephalon as described previously (Cheng
et al, 2007; Lathia et al, 2008). Briefly, pregnant mice were euthanized and embryo brains
114
were removed and the cortical cerebral wall was dissected in sterile Hanks' balanced saline
solution (HBSS). For neonatal mice, the brains were removed from skulls and then washed
in sterile HBSS. Then brains were placed dorsal side up on sterilized filter paper and sliced
in the coronal plane with a razor into 5 sections. The brain slices were transferred to a
sterile cell culture dish with a small volume of HBSS for specific removal of the region
surrounding the lateral ventricles. The corresponding tail of each individual embryo or
neonatal mouse was collected for genomic DNA extraction and genotyping. The brain tissue
from each individual embryo or neonatal mouse was incubated in 0.05% trypsin-EDTA for
15 min. at 37 °C and then transferred to NS culture medium consisting of Dulbecco's
modified Eagle's medium (DMEM)/F12 (1:1) supplemented with B-27 and 30 ng/ml basic
fibroblast growth factor (bFGF; Becton Dickson, Bedford, MA, USA) and 30 ng/ml epidermal
growth factor (EGF; Invitrogen, Carlsbad, CA, USA) and were dissociated by titration using a
fire-polished pasteur pipette. The cells were cultured at a density of 10,000 cells/ml in
uncoated plastic culture flasks for embryonic tissue, and at a density of 50,000 cells/ml for
neonatal tissue. After NS had grown for 4-5 days in culture, 10-20 random images were
captured on a Leica inverted microscope with a 10X objective lens for primary NS diameter
measurements. Approximately 150-200 NS were analyzed for each culture. For NS clonal
analysis, primary NS were dissociated using the NeuroCult cell dissociation kit (StemCell
Technologies, Vancouver, BC, Canada) and plated into 96 well plates (Nunc, Rochester, NY,
USA) at 2000 cells per well in 200 μl NS culture medium. After 5-7 days in culture, pictures
of NS in 8 wells per experimental condition were taken using a digital camera. Images were
then analyzed using Adobe Photoshop for both NS number and diameter. NS prepared from
at least three littermates were analyzed at each developmental stage.
115
RNA Isolation and Quantitative Real-time RT-PCR: Total DNA-free RNA was
purified with Trizol (Invitrogen) following the manufacturer’s instructions. Two
micrograms of total RNA was used for reverse transcription (RT) by M-MLV reverse
transcriptase (Promega, Madison, WI, USA), following the manufacturer’s instructions. The
RT products were used as the template for quantitative real-time PCR. Quantification of the
PCR products was determined by SYBR Green reagent (Maxima SYBR Green qPCR Master
Mix 2X; Fermentas, Glen Burnie, MD, USA) using the iCycler optical system (Bio-Rad,
Hercules, CA, USA). The forward primer for both EGFR Variant 1 and Variant 2 was 5’-
GACCAGACAACTGCATCCAGTGTG-3’. For quantification of EGFR Variant 2, the reverse
primer 5’-CACTTCACATCCTTGAAGACCTGG-3’ (fragment length, x bp) was used; for
quantification of EGFR Variant 1, the reverse primer 5’-GGAGATGTGGCTTCTCTTAACTCC-3’
(fragment length, x bp) was used. The primers for 18SrRNA were forward 5’-
CGCCGCTAGAGGTGAAATTC-3’ (fragment length, x bp) and reverse 5’-
CGAACCTCCGACTTTCGTTC-3’ (fragment length, x bp). PCR conditions included an initial
denaturation step of 3 min at 94 °C, followed by 40 cycles of PCR consisting of 30 s at 94 °C,
30 s at 60 °C and 30 s at 72 °C. The PCR data were analyzed by 2
-ΔΔCT
method (Livak and
Schmittgen 2001).
MAO Catalytic Activity Assays: Neurospheres were collected by centrifugation
and homogenized in assay buffer (50 mM sodium phosphate buffer, pH 7.4). Samples were
incubated with 100 μM
14
C-labeled serotonin for measurement of MAO A activity or 10 μM
14
C-labeled phenylethylamine (PEA) (Perkin Elmer Life and Analytical Sciences, Boston, MA,
USA) for measurement of MAO B activity, for 20 min at 37 °C. Reactions were terminated by
addition of 100 μL of 6 N HCl, and reaction products were extracted with
ethylacetate/benzene 1:1 for MAO A or toluene for MAO B and centrifuged for 7 min. The
116
organic phase containing the reaction product was extracted and its radioactivity measured
by liquid scintillation spectroscopy as reported previously (Geha et al, 2001). Protein
concentrations were measured to calculate the specific catalytic activity.
Ultrasonic Vocalization Tests: Recently our group demonstrated that
administration of pCPA during embryonic development (E14-E19) restored 5-HT in MAO
A/B KO neonatal mice to levels equivalent with WT mice without affecting DA or NE (Cheng
et al, 2010). Briefly, timed-pregnant dams received a single injection each day from E14.5
to E19.5 at 300 mg/kg (E14.5), 200 mg/kg (E15.5, E16.5), 100 mg/kg (E7.5, E18.5) pCPA or
vehicle. The pups’ date of birth was considered “P0” and the litter was tested 6 days later, at
“P6.” USVs produced in two bands during 5 min. of isolation from the dam were recorded as
follows: “Band 1” captured ultrasonic events occurring between 20 KHz and 60 KHz, and
above 40 dB, and “Band 2” captured ultrasonic events occurring between 50 KHz and 90
KHz, and above 40 dB. Immediately following USV testing and before being returned to its
dam, a small piece of tail was snipped from each pup for genomic DNA isolation and
genotyping.
Statistical Procedures: All results are expressed as mean ± S.E.M. Statistical
analyses were performed with one-way or two-way ANOVAs, as appropriate, followed by
Tukey’s test for post-hoc comparisons. Significance threshold was set at P<0.05.
Homoscedasticity of data distribution was verified using Bartl ett’s test. Nonparametric
comparisons were carried out by the Kruskal-Wallis test; post-hoc analyses were performed
by the Mann–Whitney U test, with Dunn-Sidak correction for α levels.
117
RESULTS
Brain Monoamine Levels in Neonatal WT, MAOA
A863T
KO, MAO A/B KO Mice
Neonatal pups (P1-6) were sacrificed and whole brains analyzed by HPLC for
quantification of 5-HT, 5-HIAA, DA and NE (analysis of DOPAC is not included here due to
the very low levels of this metabolite in neonatal brain).
As expected, levels of 5-HT were significantly higher in both MAOA
A863T
KO (P<0.01,
Tukey’s test) and MAO A/B KO (P<0.01, Tukey’s test) as compared to WT (1-way ANOVA,
F 2,20=87.28, P<0.0001) (Fig. 4.2a). 5-HIAA levels were significantly reduced in MAOA
A863T
KO (P<0.01, Tukey’s test) as compared to WT (1-way ANOVA, F 2,20=28.77, P<0.0001); 5-
HIAA was not detectable in MAO A/B KO (Fig. 4.2b).
FIGURE 4.2
Brain Monoamine Levels in Neonatal WT, MAOA
A863T
KO, MAO A/B KO Mice: All values represented as
mean ± SEM. **P<0.01 compared with WT mice; ##P<0.01 compared with MAO A/B KO mice. For more details,
see Results section.
118
DA levels were equivalent in WT and MAOA
A863T
KO but tended to be lower in MAO
A/B KO as compared to WT (U 9,9=18, P=0.052) (Fig. 4.2c). Conversely, NE l evels were
equival ent in WT and MAO A/B KO but increased significantly in MAOA
A863T
KO as compared
to both WT (P<0.01, Tukey’s test) and MAO A/B KO (P<0.01, Tukey’s test; 1-way ANOVA,
F 2,20=11.02, P<0.001) (Fig. 4.2d).
Effect of MAO Deficiency on Neurosphere Cultures Derived from Embryonic and
Neonatal Neural Stem Cells
To study the proliferative capacity of neural stem cells (NSC) in vitro, the
neurosphere (NS) assay was performed. In this assay, cells explanted from the proliferative
subventricular zone (SVZ) are dissociated into single cells and cultured in serum-free media
containing EGF and bFGF. Under these conditions, only mitogen-responsive cells
proliferate, forming clusters referred to NS. Both the size and frequency of NS formation is
used to estimate the proliferative capacity of NSC (Reynolds and Rietze 2005).
We found that NSC self-renewal ability is disturbed in a developmental stage-
specific manner in NS cultures derived from MAO A/B KO mice as compared to WT mice
(Fig. 4.3). The size of NS at E17 and P2 (Fig. 4.3a-b) from MAO A/B KO mice was
significantly smaller than age-matched NS from WT mice; no difference was found at E14
(data not shown). NS forming assays showed that significantly fewer NS formed from P2
MAO A/B KO mice (~30% decrease) compared with P2 WT controls (Fig. 4.3c).
119
FIGURE 4.3
Proliferative Capacity of MAO A/B KO NSC from the Developing Telencephalon in vitro. a, Images of
neurospheres in cultures prepared from the VZ/SVZ of the dorsal telencephalon of E14, E17, and P2 MAO A/B
KO and WT littermate mice. Scale bar = 1.0 mm. b, Size and c number of neurospheres established from
telencephalic tissues of P2 WT and MAO A/B KO mice. Values are the mean ± SEM (N = 3-4 mice). *P<0.05,
***P<0.001
Moreover, we found that expression of the full-length, functional EGFR (Fig. 4.4a-b)
was significantly reduced in P2 MAO A/B KO NS as compared to WT controls (Fig. 4.4d); by
contrast, no difference in EGFR expression was found in E14 MAO A/B KO NS (data not
shown). Together these data suggested that NSC self-renewal ability is impaired in vitro in
NS cultures from MAO A/B KO mice in late embryonic and early postnatal stages.
120
FIGURE 4.4
Expression of Epidermal Growth Factor Receptor (EGFR) Isoforms in WT and MAO A/B KO Neurosphere
Cultures. SVZ-derived neural stem cells generate neurospheres in vitro when stimulated by epidermal growth
factor (EGF). Two EGFR receptor isoforms are expressed in human and mice: Variant 1 is the full-length,
functional isoform and Variant 2 is the truncated isoform containing only the extracellular ligand-binding
domain. a, Only activation of Variant 1 stimulates NSC proliferation, whereas Variant 2 is believed to serve a
regulatory role (Reiter et al., 2001). b, PCR primers (blue arrows) were designed to amplify the 5’ domain
common to both Variant 1 and Variant 2 (yellow) and the 3’ domain found only in Variant 1 (purple). c, Total
expression of both variants is similar in P2 WT and MAO A/B KO neurospheres but d, Variant 1 expression is
significantly reduced in P2 MAO A/B KO. Values are the mean ± SEM (N = 3-4 mice). ***P<0.001
We also found that MAO A activity, but not MAO B activity, can be detected in WT
E13 NS, and its levels were increased at E16, with a further increase at P2 (Fig. 4.5a).
However, MAO A activity per se is not required for the self-renewal of NSC in NS because
treatment of P2 NS from WT mice with the MAO A inhibitor clorgyline, at a concentration
that completely blocked MAO A activity (Fig. 4.5b), did not affect NS size (Fig. 4.5c) or the
frequency of NS formation (Fig. 4.5d). Thus, the decreased size and number of NS from
MAO A/B KO mice may not be the direct effect of MAO A deficiency.
121
FIGURE 4.5
MAO A and MAO B Activity in NSC from the Developing Telencephalon. a, Levels of MAO A and MAO B
activity in neurospheres cultured from WT mice of the indicated ages. Note that cultured NS exhibit MAO A, but
not MAO B activity, and MAO A activity significantly increases as development proceeds. b, Levels of MAO A
activity in E17 neurospheres measured 24 h after treatment with the indicated concentrations of clorgyline (an
inhibitor of MAO A). Clorgyline administration did not significantly alter the diameter c or number d of
neurospheres in cultures established from P2 cortical tissue. Values are the mean ± SEM (N = 3-4 mice).
**P<0.01
Ultrasonic Vocalizations in Neonatal WT and MAO A/B KO Mice
We compared the production of isolation-induced USVs in P6 WT and MAO A/B KO
mice; we also investigated the effects 5-HT synthesis inhibition during embryogenesis on
this behavior by administrating pCPA to pregnant dams according to the protocol that was
recently shown to reduce 5-HT in MAO A/B KO neonatal mice to levels equivalent with WT
mice (Cheng et al, 2010).
122
Analysis was stratified by gender, as USV production has been shown to differ in
male and female pups (Naito and Tonoue 1987). Analysis of male (+/y) and (-/y) pups
revealed that the total number of USVs differed between the groups (H 3,52=14.37; P<0.01,
Kruskal-Wallis) (Fig. 4.6). Among males administered saline, total USVs were lower in (-/y)
pups as compared to (+/y) pups (U 20,12=50, P<0.01). pCPA reduced total USVs in (+/y) pups
(U 20,10=44, P<0.05) but had no effect on total USVs in (-/y) pups (U 12,10=52, N.S.).
FIGURE 4.6
USVs in Male WT and MAO A/B KO Neonates. Band 1 (Bd 1), Band 2 (Bd 2), and Band 1 + Band 2 (Total) USVs
in male mice administered pCPA or saline vehicle during embryonic development. Values are the mean ± SEM.
*P<0.05 compared to vehicle mice of the same genotype; #P<0.05, ##P<0.01 compared to vehicle mice of other
genotype
Analysis of femal e (+/+) and (-/-) pups reveal ed that the total number of USVs
differed between the groups (H 3,39=8.84; P<0.05, Kruskal-Wallis) (Fig. 4.7). Among females
administered saline, total USVs did not differ between (+/+) and (-/-) pups (U 12,7=42, N.S.).
pCPA had no effect on total USVs in (+/+) pups (U 12,9=44, N.S.) but reduced total USVs in (-/-)
pups (U 7,11=15, P<0.05).
123
Analysis of femal e (+/+) and (+/-) pups reveal ed that Band 1 USVs tended to differ
between the groups (H 3,43=7.1; P=0.06, Kruskal-Wallis) (Fig. 4.7). Among femal es
administered saline, Band 1 USVs did not differ between (+/+) and (+/-) pups (U 12,13=98.5,
N.S.). pCPA had no effect on Band 1 USVs in (+/+) pups (U 12,9= 45, N.S.) but reduced Band 1
USVs in (+/-) pups (U 13,9=19.5, P<0.05).
FIGURE 4.7
USVs in Female WT and MAO A/B KO Neonates. Band 1 (Bd 1), Band 2 (Bd 2), and Band 1 + Band 2 (Total)
USVs in female mice administered pCPA or saline vehicle during embryonic development. Values are the mean ±
SEM. *P<0.05 compared to vehicle mice of the same genotype
124
DISCUSSION
In this chapter, we studied the impact of MAO-deficiency during early stages of
development, examining effects on brain monoamine levels, neural progenitor cell
proliferation, and USV production in neonates.
The first major result of this study is that MAOA
A863T
KO and MAO A/B KO neonates
have comparable marked increases in 5-HT but distinctive alterations in DA and NE as
compared to age-matched WT mice. We confirmed previous reports in neonatal MAO A KO
mice (Cases et al, 1995) by showing that, in comparison with age-matched WT mice,
MAOA
A863T
KO mice have increased NE levels but no change in DA levels. Interestingly, MAO
A/B KO mice showed no change in NE levels, whereas DA levels tended to be lower in
comparison to WT mice.
Although the mechanism underpinning the differences in catecholamine levels in
MAOA
A863T
KO and MAO A/B KO mice was not investigated in this study, a wealth of
literature has suggested that excessive 5-HT levels may alter catecholamine synthesis and
signaling. Abundant extracellular 5-HT may be taken up by the DA or NE plasma membrane
transporter and can accumulate in catecholamine neurons (Daws 2009); indeed, this
phenomena was reported in developing brain of Tg8-MAO A KO mice, and results in 5-HT
accumulation in atypical locations during crucial periods of embryonic and early postnatal
development (Cases et al, 1998). With this in mind, excess 5-HT, located “unconventionally”
in DA neurons in MAOA
A863T
KO and MAO A/B KO mice, may be sequestered and co-released
in DA vesicles, leading to reduced DA tissue content (Zhou et al, 2005). This reduction could
be exacerbated by 5-HT receptor activation, which has also been shown to reduce DA levels
in nucleus accumbens and striatum (De Deurwaerdère and Spampinato 1999; Spampinato
et al, 1985).
125
Excess 5-HT likely accumulates in catecholamine neurons in both neonatal
MAOA
A863T
KO and MAO A/B KO mice; however, this effect may be attenuated in MAOA
A863T
KO mice due to the presence of very low levels of MAO B. MAO B expression is abundant in
5-HT neurons and astrocytes, and its localization in these cell types is believed to contribute
to the degradation of 5-HT within these neurons and after being released from their
terminals, respectively (Levitt et al, 1982). However, unlike MAO A, which attains adult
levels during early development, MAO B activity is very low during embryogenesis and
throughout the neonatal stage, and gradually increases throughout adulthood (Nicotra et al,
2004). Nonetheless, we did detect low amounts of 5-HIAA in neonatal MAOA
A863T
KO (but
not MAO A/B KO) mice, suggesting that MAO B does exert limited control of 5-HT levels in
the absence of MAO A during the early postnatal stage. The impact of MAO B on 5-HT levels
may be sufficient to reduce the uptake of 5-HT in catecholamine neurons in MAOA
A863T
KO
mice as compared to MAO A/B KO, especially considering the low affinity of DA and NE
transporters for 5-HT (Eshleman et al, 1999). Thus, increased 5-HT uptake in DA neurons
could compensate for the lack of MAO B in MAO A/B KO mice, resulting in comparable
extracellular 5-HT levels in MAOA
A863T
KO and MAO A/B KO mice. Consequently, enhanced
5-HT-mediated regulation of catecholamine signaling via the aforementioned mechanisms
could also account for attenuated levels of DA and NE in MAO A/B KO mice.
Alternatively, the attenuation of DA levels in MAO A/B KO mice may indicate a delay
in the maturation of the catecholamine system, an intriguing possibility suggested by
evidence that the 5-HT system exerts an inhibitory trophic effect on neonatal in-growth of
DA fibers (Benes et al, 2000). Moreover, the 5-HT system matures more rapidly in
comparison to catecholamine systems. Brain 5-HT content exhibits the least fluctuation
throughout development and attains adult levels more quickly than either DA or NE
126
(Goldman-Rakic and Brown 1982). Likewise, in primate brain the density of catecholamine
appositions on cortical neurons matures slowly, gradually reaching adult levels by 2 years
of age; the density of 5-HT appositions in cortical areas reaches the adult level before the
second week after birth (Lambe et al, 2000).
The possibility that ablation of MAO A and MAO B may result in delayed or altered
development of certain systems is supported by the second major finding presented here,
that neurospheres generated from neural stem cells of MAO A/B KO mice show reduced
proliferative capacity. Indeed, recent studies performed by our group indicate that this
alteration can also be detected in vivo in developing brains of MAO A/B KO mice and
appears selective for intermediate progenitor cells (IPC) based on the location of these cells
relative to the ventricular wall (Cheng et al, 2010) (Fig. 4.8). NSCs are located in a
proliferative region surrounding the lateral ventricles called the ventricular zone (VZ) and
the subventricular zone (SVZ) (Angevine et al, 1970). Neurogenesis is believed to originate
from VZ neuroepithelial precursors (NEP), which in turn generate specialized cells called
radial glial cells (RGC). Around mid-gestation, RGC transition into another specialized cell,
the IPC (Götz and Huttner 2005; Haubensak et al, 2004; Miyata et al, 2004; Noctor et al,
2004). IPC can be distinguished from NEP and RGC partly on the basis that IPC divide away
from the ventricle. Intrinsic as well as extracellular signals from differentiated cells
regulate NSC fate during brain development (Lathia et al, 2007; Temple 2001), and these
studies lend further support to the notion that monoamine neurotransmitters play an
integral role in the regulation of NSC proliferation in the developing brain (Cameron et al,
1998).
127
FIGURE 4.8
Proliferation of MAO A/B KO Neural Progenitor Cells in the Subventricular zone (SVZ) During Embryonic
Development. Littermates of WT and MAO A/B KO mice at E12.5, 14.5 and 17.5, and P2 and 7 were obtained
from timed-pregnant females. Pregnant dams or individual neonatal mice received bromodeoxyuridine (BrdU)
by intraperitoneal injection (50 mg/kg) 1 h before euthanization. A, Brain sections were immunostained with
BrdU (green) and counterstained with PI (red). Analysis was performed on a sector of the dorsomedial cerebral
wall, overlying the medial region of the lateral ventricle and corresponding to the location of the future primary
frontal cortex. This sector has as its base a segment of the ventricular zone (VZ) that is 100 μm in its
mediolateral dimension. The sector was divided into bins, parallel to the ventricular surface, 10 μm in height,
and the bins were numbered 1, 2, 3 and so on from the ventricular surface outward (Takahashi et al, 1992).
BrdU-labeled and unlabeled nuclei were scored with respect to their bin location. Nuclei on the boundary
between two bins were assigned to the bin closer to the ventricle. Nuclei touching the medial margin, but not
those touching the lateral margin, of each bin were included in the analysis. A labeling index (LI: BrdU+ cells as
a proportion of total cells) was calculated for each bin. The plots of LIs for each bin for each time point is
referred as the LI profile for that time point. The average LI for each bin was derived from three nonadjacent
sections in each brain (every sixth coronal section from the front to the back of the brain). B-D, Representative
confocal images of a slab of the middle telencephalon wall of WT and MAO A/B KO mice at E12.5, E14.5 and
E17.5, respectively. Scale bar = 50 μm. E-G, The LI was plotted for each of the bins within the analysis areas at
E12.5, E14.5 and E17.5, respectively. At E17.5, BrdU+ cells were significantly reduced in the area corresponding
to SVZ area, but not in the VZ. *P<0.05 (N = 4 mice) (Cheng et al, 2010).
128
In cultures of WT neural progenitor cells, MAO A activity, but not MAO B activity,
was detected at E12.5, and its levels were increased at E15.5 with a further increase at P2,
indicating a rapid increase in the ability for these cells to degrade 5-HT coinciding with the
time when 5-HT neurons initially reach the developing cortex during mid-gestation (Ivgy-
May et al, 1994). Indeed, previous studies performed by our group revealed that MAO A/B
KO mice have increased 5-HT levels beginning around E14.5, with a further large increase
during the neonatal stage (no significant changes in DA or NE were detected at these stages)
(Cheng et al, 2010). This time course parallels the alterations in NSC proliferation we
observed in MAO A/B KO mice, which become evident during mid-embryonic development
and persist during the early postnatal stage, and strongly implicates 5-HT in these changes.
There is abundant evidence from the literature that 5-HT acts a morphogenetic
signal during development. However, most studies indicate that 5-HT stimulates NSC
proliferation (Banasr et al, 2001; Banasr et al, 2004), whereas depletion of 5-HT with pCPA
produces the opposite effect, inhibiting proliferation (Brezun and Daszuta 1999; Gould
1999). However, the regulatory effects of 5-HT on cellular growth are receptor-dependent;
indeed, specific 5-HT receptor subtypes have been linked to numerous signal transduction
pathways that can alternatively promote or inhibit cell proliferation (Lauder 1993;
Buznikov et al, 2001). Moreover, akin to Tg8-MAO A KO mice (Lanoir et al, 2006), it is likely
that changes in the expression and/or function of various 5-HT receptor subtypes have
been induced in MAO A/B KO mice to compensate for the lack of regulation by MAOs, which
could in turn lead to reduced cellular proliferation.
We demonstrate that MAO A/B KO NSCs are reduced in number and proliferative
capacity using evidence not only from in vivo studies of the proliferative SVZ (Cheng et al,
2010), but also by the in vitro cell culture methods described here. Indeed, the fact that
129
MAO A/B KO NSCs form fewer, smaller NS than WT counterparts in culture suggests that
the reduction in proliferative capacity is not a direct consequence of factors present in the
stem cell niche in vivo but rather a long-lasting alteration that persists in the absence of
many environmental cues. Indeed, we show that expression of the full-length, functional
EGFR is reduced in neonatal, but not embryonic MAO A/B KO NS in culture, an alteration
could contribute to the reduction of NSC proliferation in mutant mice. Activation of the
EGFR is requisite for NSC division, and expression of truncated EGFR is believed to exert
inhibitory control on cell division (Reiter et al, 2001). Moreover, the NS alterations in MAO
A/B KO cells cannot be recapitulated by pharmacological inhibition of MAO A in WT cells,
reinforcing the contention that these alterations are the result of enduring changes in
proliferation capacity.
Indeed, cogent evidence has indicated that many of the dysfunctions present in
MAO-deficient humans and mice have a developmental origin (Whibley et al, 2010; Cases et
al, 1998; Cheng et al, 2010). In contrast to MAO-deficient humans and mice, long-term use
of MAO inhibitors does not cause aggression, impulsivity, or cognitive decline,
characteristics observed in subjects with chronic MAO A-deficiency. Moreover, there is
evidence that excess 5-HT plays an important role in some of these dysfunctions; the
sensorimotor cortex d eficits in Tg8-MAO A KO mice are a key exampl e, which result from
excessive 5-HT 1B receptor activation during the early postnatal stage and can be partly
restored by neonatal administration of pCPA (Cases et al, 1995; Salichon et al, 2001).
In ad dition to restoring the sensorimotor cortex d eficits in Tg8-MAO A KO mice,
neonatal pCPA administration was also reported to attenuate many of the abnormal
behaviors observed in these pups, whereas the same treatment with an inhibitor of
catecholamine synthesis had no effect, again reinforcing the serotonergic basis for these
130
perturbations (Cases et al, 1995). Altered behaviors were evident even in neonates, which
exhibited intense head nodding; at P5, Tg8 pups displayed trembling upon locomotion,
prolonged righting, and exaggerated reactions to pinching (Holschneider et al, 2001). From
P11-P16, the behavior of Tg8 pups was characterized by hyperactive, frenzied responses to
mild stimuli, including chaotic escape attempts, efforts to hide in the nest material, and
increased likelihood to bite the experimenter; postural traits including hunched back were
also observed at this stage (Cases et al, 1995). Although the entire spectrum of behavioral
perturbations in MAO A/B KO mice during these early stages of development have not been
elucidated, preliminary studies revealed that, akin to Tg8-MAO A KO, these double mutant
pups also exhibit hyper-reactive, exaggerated responses to mild stimuli, such as panic
jumping and uncontrolled escape behaviors (Chen et al, 2004).
We further extended the behavioral phenotyping of MAO A/B KO mice during early
developmental stages and report that USVs are significantly reduced in male MAO A/B KO (-
/y) neonates as compared to male WT (+/y) counterparts, whereas USVs were similar in
female WT (+/+), MAO A/B KO (+/-), and (-/-) pups. USVs are typically viewed as a
communicative behavior (Branchi et al, 2001) induced by exposure to anxiogenic
conditions (Hofer 1996), although USV production has been shown to differ markedly in
male and female pups (Naito and Tonoue 1987). Indeed, the selective reduction in male (-
/y) mice suggests that the female gender confers protection against development of USV
perturbations induced by MAO A/B-deficiency.
However, embryonic administration of pCPA using a treatment regimen previously
shown to normalize 5-HT levels in MAO A/B KO neonates (Cheng et al, 2010) did not
restore USVs in male (-/y) pups, suggesting that the reduction in USVs is not a direct
consequence of elevated 5-HT levels. Conversely, pCPA markedly reduced USVs in male
131
(+/y) and female (-/-) pups, whereas female (+/+) and male (-/y) pups were not affected by
pCPA. Again we find that female gender conferred protection against USV attenuation, in
this case induced by embryonic 5-HT depletion. However, the female gender was
insufficient to prevent this decrement when MAO A/B ablation and 5-HT depletion were
combined, as evidenced by the attenuation of USVs in female (-/-) pups administered pCPA.
The USV studies described here highlight two key points. First, these results
indicate that bi-directional perturbations in 5-HT levels lead to diminution of USVs. Indeed,
the 5-HT system has been shown to exert a bidirectional, receptor-dependent effect on USVs
(Branchi et al, 2001; Olivier et al, 1998), suggesting that changes in 5-HT receptor
expression or function may be responsible for observed reductions (Lanoir et al, 2006;
Evrard et al, 2002; Shih et al, 1999b). Notably, activation of 5-HT 1A receptor reduces
neonatal USVs (Branchi et al, 2001; Olivier et al, 1998), and in Tg8-MAO A KO pups aged P7,
the same stage at which USVs were measured in this study, the overall density of
postsynaptic 5-HT 1A receptors was decreased ~30-60%, whereas the density of 5-HT 1A
autoreceptors was decreased even more significantly, by at least 79% (Lanoir et al, 2006).
These observations concur with studies demonstrating that chronic elevations in
extracellular 5-HT lead to 5-HT 1A receptor desensitization (Rossi et al, 2008), rendering it
highly likely that this receptor is also markedly downregulated in MAO A/B KO neonates as
well. Moreover, 5-HT 1A receptor transcripts were significantly reduced in neonatal pups by
either pCPA or a nonselective 5-HT agonist administered during the same period of
embryonic development reported here (Lauder et al, 2000), indicating that both under-
stimulation and over-stimulation of 5-HT 1A during this crucial period attenuates the
expression of this receptor. Thus, 5-HT 1A downregulation, induced by either 5-HT depletion
132
or 5-HT excess, could result in comparable USV decrements in both pCPA-treated WT and
saline-treated MAO A/B KO neonates.
Secondly, these results suggest that interactions between the 5-HT system and sex
hormones may play an important role in the regulation of behavior during early stages of
development. Considering the numerous gender-related differences in the 5-HT system, it
is likely that MAO deficiency differentially affects brain development in males and females.
Notably, changes in 5-HT receptor expression or function are likely to differ in male and
female MAO A/B KO mice, and the most compelling evidence for this premise stems from
studies of the 5-HT 1A receptor. Adaptive changes in 5-HT 1A expression in response to excess
5-HT differ in male and females (Li et al, 2000), coinciding with evidence that testosterone
regulates 5-HT 1A transcription, resulting in gender-related differences in brain regional
expression and density (Zhang et al, 1999). Thus it is possible that testosterone modulates
the excess 5-HT-mediated downregulation of pre- and/or postsynaptic 5-HT 1A receptors,
leading to differential expression and/or function in male and female MAO AB KO mice,
which in turn results in differential USV rates. Moreover, there is evidence that females
have higher 5-HT levels throughout several brain regions (Carlsson and Carlsson 1988)
which could render female (+/+) pups less susceptible than their male (+/y) counterparts
to the developmental consequences of 5-HT synthesis inhibition. These hypotheses should
be addressed in future studies quantifying the brain regional levels of 5-HT and expression
of 5-HT receptors in WT and MAO AB KO pups of both genders.
Akin to male (-/y) pups, rat pups developed as a “hyperserotonemic model” of
autism, produced by administration of a nonselective 5-HT agonist during neonatal
development (Whitaker-Azmitia 2001) did not produce USVs when separated from their
mothers (Kahne et al, 2002). This deficit may be analogous to the paucity of communication
133
and social interaction often observed in humans with this disorder, as these rats displayed
other behaviors common in autistic subjects, including hyper-responsiveness to sound and
difficulty righting themselves (Whitaker-Azmitia 2001; Whitaker-Azmitia 2005). Indeed,
autism is generally diagnosed in children around 2 years of age when deficient
communication skills become evident. Although the underlying cause (or more likely,
causes) of autism are unknown, excessive 5-HT levels during development have been
implicated its etiology (Whitaker-Azmitia 2001). Autistic subjects often have high levels of
5-HT in the bloodstream (Anderson et al, 1987; Minderaa et al, 1987), and a high frequency
of autism was reported in children exposed in utero to cocaine, a drug known to elevate 5-
HT levels throughout the brain (Davis et al, 1992). Moreover, autism is about 4 times more
common in boys than in girls (Newschaffer et al, 2007), a prevalence which lends support to
the theory that high levels of testosterone confer increased susceptibility to this disorder
(Knickmeyer and Baron-Cohen 2006) and provides an interesting translational direction to
explore the basis for the selective reduction of USVs in male (-/y) pups.
Finally, the potential impact of elevated levels of β-phenylethylamine (PEA) on
neurodevelopmental perturbations in MAO A/B KO mice should be not be overlooked. This
trace amine is structurally related to amphetamine and is believed to have similar effects on
catecholamine neurotransmission (Paterson et al, 1990). PEA has been linked to neurotoxic
effects in developing embryos (Denno and Sadler 1990) but its levels are presumably very
low during early stages of development based on evidence that it is deaminated by both
MAO A and MAO B in newborn mouse brain (Koide and Kobayashi 1984). However, PEA
levels are dramatically increased in MAO B KO mice (Grimsby et al, 1997) and are even
higher in MAO A/B KO mice (Chen et al, 2004) (no change was found in MAO A KO mice
(Cases et al, 1995). Although PEA levels in MAO-deficient mice during early stages of
134
development have not been determined, the primary role of MAOs in the inactivation of this
amine renders it highly likely that they are markedly elevated in neonatal MAO A/B KO
brain. Indeed, certain outcomes observed in MAO A/B KO mice are reminiscent of
developmental and behavioral perturbations resulting from neonatal exposure to
amphetamine, including reduced body weight (Smith and Chen 2010) and similar
decrements in open field locomotor activity as adults (Smith and Chen 2009).
Conclusion
These studies reveal novel and important rol es for MAOs during early stages of brain
d evel opment. Notably, l oss of MAOs has prominent effects during early stages of
d evel opment, resulting in markedly increased brain 5-HT l evels, reduced proliferation in
neural progenitor cells, and attenuation of ultrasonic vocalizations in neonates. These data
implicate excessive 5-HT l evels in several l ong-lasting d evel opmental impairments, rend ering
MAO A/B KO mice a potentially important mod el for elucidating the und erlying mechanisms
of pervasive d evel opmental disord ers such as autism that are believed to stem partly from
altered 5-HT signaling during embryonic and/or neonatal stages of growth.
Abstract (if available)
Abstract
Monoamine oxidases (MAOs) serve a crucial function in the regulation of mood and behavior. The two isoenzymes, MAO A and MAO B, are expressed in a variety of brain and peripheral tissues where they catalyze the oxidative deamination of neurotransmitter and dietary monoamines. MAO A preferentially catabolizes serotonin (5-HT) and norepinephrine (NE), MAO B prefers the trace amine phenylethylamine (PEA), and both catabolize dopamine (DA). In the absence of MAO A, MAO B oxidizes MAO A’s preferred substrates and vice versa, indicating partial functional redundancy.
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Scott, Anna Louise
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Monoamine oxidase deficiency and emotional reactivity: neurochemical and developmental studies
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Doctor of Philosophy
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Molecular Pharmacology
Publication Date
11/17/2010
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committee member
), Holschneider, Daniel P. (
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)
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